Regulation of gene activity in bacteriophage M13 DNA: Coupled transcription and translation of purified genes and gene-fragments

Abstract

Specific DNA fragments, obtained after cleavage of M13 replicative form DNA with restriction endonucleases from Haemophilus aphirophilus (endo R. Hap.II), H. parainfluenzae (endo R. Hpa.II) and H. aegyptius (endo R. Hae.III), have been used to direct transcription and translation in the cell-free system of Escherichia coli. Analyses of the in vitro products on sodium dodecyl sulfate-polyacrylamide gels revealed that several restriction fragments directed the synthesis of authentic phage specific proteins. 1) Fragment Hap.A directs the synthesis of gene IV protein, 2) fragment Hap.B 2, of gene-VIII protein, and 3) fragment Hae.B, of gene-V protein. In addition, fragments Hap.A and Hae.A direct the synthesis of prematurely terminated translation products of gene IV. It is suggested that the polypeptide designated X-protein (MW, 12,000) and encoded by fragments Hap.C and Hae.B is the product of a hitherto unidentified gene. Furthermore, it is concluded that replicative form DNA is transcribed in a counterclockwise direction along the genetic map published by C. A. van den Hondel, A. Weijers, R. N. H. Konings, and J. G. G. Schoenmakers ((1975) Eur. J. Biochem., 53, 559–567). It is proposed that promoter sites are located on the genetic map immediately proximal to the 5′-ends of genes IV and VII and also immediately proximal to the 5′-end of the “gene” coding for X-protein.