J. Turk. Phytopath., Vol. 41 No. 1-3, 19-26, 2012 ISSN 0378 - 8024 19 Cloning and Sequencing of Virus Inhibiting Gene Encoding an Antiviral Protein From the Leaves of Pokeweed (Phytolacca Americana L.) Hikmet Murat SİPAHİOĞLU* İlhan KAYA* Mustafa USTA * Sibel SAMSUN ** * Department of Plant Protection, Faculty of Agriculture, YüzüncüYıl University, 65080 Van- Turkey ** Department of Biology, Faculty of Science, YüzüncüYıl University, 65080 Van - Turkey Corresponding author e-mail: hmsipahi@yyu.edu.tr Accepted for publication October 10, 2013 ABSTRACT Pokeweed antiviral protein (PAP) is single-chain ribosome-inactivating protein (RIP) of Phytolacca americana L. (Pokeweed) that is characterized by its ability to depurinate ribosomes. In the present study, we cloned and sequenced complete gene of pokeweed antiviral protein type 1 (PAP-I) from the summer leaves of pokeweed collected from Trabzon (Turkey) using a pair of gene specific primers based on the known N- and C-terminal nucleotide sequences of PAP gene. A product of 942 base pair was purified and inserted into pGEM-T Easy vector (Promega), downstream of the T7 promoter, and transformed into E. coli strain, JM 109. The PAP-I cistron of P. americana contained 313 amino acid residues. The DNA sequence of 942 base pairs included an open reading frame (ORF). The nucleotide sequences of PAP-I gene contained no introns and the comparison with the PAP-I sequence with the PAP isoform, showed an identity of 80-99%. Sequence analysis of PAP-I revealed that it contains a single point mutation, changing the Leucine (L) at position 273 to Phenylalanine (F) (L273F) at the putative active site. Key words: antiviral proteins, characterization, cloning, PAP-I gene, Phytolacca americana INTRODUCTION Pokeweed (Phytolacca Americana L.) antiviral protein (PAP) is a single chain ribosome inactivating protein (RIP) which inactivates both eukaryotic and prokaryotic ribosomes isolated from the leaves of pokeweed plants. The PAP removes specific adenine and guanine residues from the highly conserved alpha-sarcin/ricin (S/R) loop in the large rRNA (Endo et al. 1988; Hartley et al. 1991; Hudak et al. 2000). RIPs have been considered as efficient protein toxins produced by organisms ranging from some prokaryotes to plants. Because of its inhibitory potent against animal and plant viral pathogens, including human immunodeficiency virus, poliovirus, herpes simplex virus, influenza, potato virus X, and brome mosaic virus, PAP is thought to be a defense protein (Aron and Irvin1980; Lodge et al.1993; Tomlinson et al. 1974; Ussery et al.1977; Zarling et al.1990). PAP is currently under clinical trials against cancer, because of its cytotoxicity to dividing cells (Waurzyniak et al.1997; Bijal et al. 2002). PAP has also been shown to efficiently depurinate single stranded DNA (Nicolas et al. 1998), double-stranded DNA (Wang and Tumer, 1999) and adenine-containing polynucleotides (Barbieri et al. 1997). Ribosome inactivating proteins of pokeweed, PAP-I, PAP-II, and PAP-III, are different isoforms of PAP isolated from the spring leaves, early summer leaves and late summer leaves of the pokeweed plant (Irvin and Uckun 1992; Rajamohan et al. 1999). Kataoka et al., (1991) stated that the most of the amino acid residues composing the N-terminal signal sequences are hydrophobic, which is a common feature of signal sequences of eukaryotes and prokaryotes. Today