Molecular cloning and a stable amplification of the DNA molecules heavily methylated at CpG sequences using a new E....

Fish lymphocystis disease virus (FLDV) DNA is heavily methylated in CpG sequences and therefore the amplification of recombinant DNA of FLDV inserted into a bacterial plasmid vector and/or a bacteriophage system poses severe problems. The problem was solved using a newly constructed and developed bacterial system (E. coli GC-3 system), which allowed the amplification of foreign DNA material heavily methylated at CpG sequences. E. coli GC-3 (Gc-1, rifd) is derived from E. coli GC-1 (K 12, hsd-, MDU) after transformation with phage lambda drifd 18 Hind III-B-fragment. A defined and complete gene library of the FLDV DNA sequences was established by insertion of FLDV DNA fragments (Eco RI, Eco RI/Bam HI, Eco RI/Hind III) into the corresponding restriction sites of bacterial plasmid vector pAT 153. Bacterial colonies harbouring recombinant plasmids were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridizing plasmid DNA to viral DNA. The analyses revealed that sequences representative for the complete viral genome were cloned.