Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation.

TLDR

mRNA hybridized with complementary DNA blocks cell‑free synthesis of a complete polypeptide. The study introduces a simple method to directly link DNA structural gene sequences to their corresponding mRNAs. The method employs hybrid‑arrested cell‑free translation, where mRNA–DNA hybrids block synthesis until heat melting restores full translational activity. Using the rabbit beta globin clone PbetaG1, the method identified structural gene sequences in recombinant DNA and precisely located and ordered several adenovirus 2 polypeptides within the viral genome.

Abstract

We present a simple method for directly correlating structural gene sequences in DNA with their corresponding mRNAs. This is based upon the fact that mRNA hybridized with its complementary DNA will not direct the cell-free synthesis of a complete polypeptide. Full translational activity of the mRNA is recovered upon the heat melting of the hybrid. Utilizing the rabbit beta globin clone PbetaG1, we demonstrate the application of hybrid-arrested translation for the identification of structural gene sequences within recombinant DNA molecules. In addition, the method is used to locate and order precisely several adenovirus 2 polypeptides within the viral genome.