Identification and characterization of the in vitro synthesized gene products of bacteriophage M13

Abstract

Bacteriophage M13 replicative form (RF) DNA was used to direct coupled transcription and translation in cell-free extracts prepared from Escherichia coli. By using RF DNA, isolated from cells infected with appropriate amber mutants of this phage, it has been possible to identify the products of genes I through IV. By using the same methods no gene-product relationship could be demonstrated for genes VI and VII. Coupled in vitro protein synthesis studies on RF-III DNA, a linear double-stranded DNA molecule, obtained after cleavage of either RF-I or RF-II DNA with the restriction endonuclease R.Hin11 from Haemophilus influenzae, indicated that the cleavage site for this enzyme is located in gene II. The in vitro products of both gene III and gene VIII are about 30 and six amino acids longer, respectively, than their native counterparts present within the virion. These results suggest that the latter proteins arise in vivo by cleavage of precursor molecules. Coupled transcription and translation studies on a DNA fragment which only contained the genetic information coding for gene IV protein, obtained after cleavage of RF DNA with the restriction endonuclease R.Hap11 from Haemophilus aphirophilus, indicated that a large number of the in vitro synthesized polypeptides are the result of premature chain termination.