the structural sequence of the mt gene the 1 35 kb human hbsag gene containing bam hi fragment from plasmid suam115 a pbr sv40 hbsag plasmid described in moriarty et al 1981 p n a s usa 78 2605 is then cut out and inserted at the bgl ii site of cl28 to form prf300 the sv40 and some pbr sequences in prf300 are then removed by bam hi digestion further digestion with sal i enzyme yields a linear molecule with bam hi and sal i ends plasmid b2 2 not shown containing the entire bpv genome is digested with bam hi and sal i enzymes any source of the bpv genome can be used bpv is available for example from new england biolabs b2 2 was used here only because it has a convenient sal i site the excised bpv containing fragment which also contains pbr dna is ligated to the hbsag mt hybrid gene containing linear bam hi sal i fragment the resulting plasmid prb 1 consists of the entire bpv genome and the hbsag structural gene inserted within the mt gene between the promoter and the remainder of the mt gene prb 1 plasmid dna is introduced into mouse c127 cells using a modification of the transfection technique of wigler et al 1977 cell 11 223 as follows 5 ug of prb 1 dna is added to 0 5 ml of a 240 mm cacl 2 solution containing 10 ug of carrier salmon sperm dna this solution is bubbled into an equal volume of 2 u00d7 hbs.