Template activity of f1 RFI cleaved with endonucleases R·Hind, R·EcoP1 or R·EcoB

Abstract

ENDONUCLEASE R·Hind makes a single, unique, double-stranded break in the double-stranded circular DNA (RFI) which can be isolated from bacteria infected with bacteriophage M13 (ref. 1), fd (refs 2 and 3), or f1 (Horiuchi and Zinder, unpublished observations). We wish to report that f1 RFI cleaved by endo R·Hind acts as a template for protein synthesis in a coupled in vitro transcription-translation system⁴, and that with the exception of the product of gene II of f1, the normal complement⁵ of in vitro phage-specific proteins is made. No prominent new polypeptide is observed (Fig. 1); this suggests that a substantial fragment of the gene II product is not coded for by RFI cleaved with endo R·Hind. In addition, neither the size nor the relative yield of the product of the proximal gene^5,6, gene IV, is affected. This implies that the break is likely to be in a region extending from the C-terminus of gene IV to the N-terminal portion of gene II. A similar location has been assigned to this break with the use of entirely different methods by Seeburg and Schaller⁷.