Mapping of ribosomal protein genes by in vitro protein synthesis using DNA fragments of lambdafus3 transducing phage DNA as templates.

Abstract

The presence of the structural genes for these proteins on the hfus3 genome was proven by demonstrating the synthesis of these proteins in a DNA-dependent protein-synthesizing system using Afus3 DNA, or its restriction enzyme fragments, or both, as template.Proteins synthesized in the system were identified by a combination of techniques.including gel electrophoresis, immunochemical reactions, and tryptic peptide analysis.Restriction endonuclease-sensitive sites on kfus3 DNA were mapped, and genes were located using restriction endonuclease fragments of Afus3 DNA, isolated directly or cloned in phage or plasmid vectors, as templates for protein synthesis.Additional information about the locations of the genes was obtained using DNA from insertion and deletion mutants of hfux3.The results obtained in these in oilro experiments (rf.Fig. 2), and those obtained in the in uiuo experiments from preceding papers in this series, are in excellent agreement and map the essential genes on Afun and their promoters as follows (going counter-clockwise on the E. coli chromosome): R,,.