Direct determination of DNA nucleotide sequences. Structure of large specific fragments of bacteriophage φX174 DNA

Abstract

Direct methods for DNA nucleotide sequence analysis have been used to determine the structure of several fragments of the single-stranded DNA genome of bacteriophage φ X174. These fragments were produced by limited digestion of φ X174 DNA with the T4 bacteriophage-induced endonuclease IV, an enzyme which is both sequence and single-strand-specific for DNA cleavage ( Sadowski & Bakyta, 1972 ). The complete structure of one of these fragments, band 6, has already been reported ( Ziff et al. , 1973 ; Galibert et al. , 1974 ). Analysis of other DNA digestion products (bands 5A, 5B, 3 and 2) indicated that many of these fragments were derived from the same region (∼500 residues long) of the φ X DNA molecule as band 6. The nucleotide sequences reported here are shown to be contiguous with the original structure. The sequencing methods employed utilize DNA labelled with 32 P in vivo , and are similar to those previously described ( Galibert et al. , 1974 ). One exception is the extensive use of inspection of two-dimensional fractionations of partial exonuclease digests of endonuclease IV oligonucleotides to deduce sequences. The sequence data presented here, together with information obtained by other sequencing procedures ( Sanger & Coulson, 1975 ; Air, 1976 ; Blackburn, 1976 ) have established the structure of an extensive region of the φ X174 genome which codes for the N-terminal portion of the φ X gene F capsid protein ( Air et al. , 1976 ).