SEQUENCING STRATEGY FOR A PROTEIN-LINKED GENOME - SPONTANEOUS REVERSIONS OF OCHRE TRIPLET IN THE PHAGE PHI-29 GENE-17

A direct method using T7 gene 6 exonuclease to prepare single-stranded templates was adapted far the sequencing of the phage phi 29 genome c arrying a covalently linked terminal protein at the 5' ends of both DN A chains. The terminal protein was found to block completely the actio n of the exonuclease. The treatment of the phage DNA by proteinase K d id not restore the accessibility of the 5' ends tct the exonuclease to the full extent bur only partially. The digestion of proteinase K-tre ated terminal fragments of the genome by 5' exonuclease could proceed now From both 5' ends, however, at different rates. The delay of exonu cleolytic digestion apparently due to residual amino acid(s) was calcu lated to be about 800 nucleotides. The relative rates of exonucleolyti c splitting of both chains were of decisive significance for the choic e of suitable primers for the sequencing of desired regions. In all Su s(+) revertants sequenced by the described method the TAA(ochre) codon in the gene 17 was found changed either to the original CAA(Gln) or t o TAT(Tyr) with the same frequencies.