Cloning of E6 and E7 genes of human papilloma virus type 18 and transformation potential of E7 gene and its mutants

E6 and E7 genes of human papilloma virus type 18 have been subcloned from plasmid pC7, carrying an insert of DNA from squamous cell carcinoma of cervix. Both genes in comparison to prototype variant contain one mutation that changes asparagine to leucine. In the case of E6 gene this mutation is mapped in codon 129, in the case of E7 the same change AAC to AAA mapped in codon 92. In addition both genes contain few point mutations that do not change the aminoacid sequences of the protein. Two mutants of E7 gene have been constructed by site directed mutagenesis based on PCR technology - one in codon 10 (change Asp to Asn) and one in codon 24 (change Asp to Gly). The first type of mutation did not influence the transformation potential of the E7 gene in comparison to the parental one with mutation in codon 92. The mutation in codon 24 (region responsible for the interaction with Rb protein) eliminate the transformation potential of the gene. The cells transformed with E7 mutants in codons 10 and 92 were tumorigenic for syngenic rats.