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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 cellfree synthesis of a complete polypeptide. Full translational activity of the mRNA is recovered upon the heat melting of the hybrid. Utilizing the rabbit P globin clone PPG1, 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. We have developed a method with which to analyze the relationship between DNA sequences, their corresponding messenger RNAs, and the proteins for which they code. This approach is based upon the observation that mRNA in hybrid form with its complementary DNA (cDNA) is not translated in eukaryotic cell-free systems, while heat dissociation of the hybrid reinstates complete translational activity (1). This observation has been extended to double-stranded DNAs by carrying out the hybridization reactions in a concentration of formamide that favors the formation of DNA-RNA hybrids but essentially prevents DNA-DNA reannealing (2)(3)(4)

The structural organization of intracisternal A-particle genes has been studied, using isolates from a mouse gene library in X phage Charon 4A. The predominant gene form among the isolates was 7.3 kilobases (kb) in length. R-loops between the 7-kb (35S) A-particle genomic ribonucleic acid and several of these genes were colinear, with no visible evidence of intervening deoxyribonucleic acid sequences. One recombinant was found with an A-particle gene that contained a 1.7-kb deletion. Using the deletion as a reference, the deoxyribonucleic acid and ribonucleic acid homology regions were localized with respect to one another and to the restriction map: the 5' terminus of the ribonucleic acid was several hundred base pairs within the 5' end of the deoxyribonucleic acid homology region. Restriction endonuclease fragments encompassing the 5' and 3' regions of one 7.3-kb gene were separately subcloned into pBR322. Heteroduplexes between the two subclones revealed an approximately 300-base pair segment of terminally redundant sequences. The cloned 3' fragment hybridized with restriction fragments from the 5' end of several other A-particle genes, demonstrating the presence of common (though not necessarily identical) terminally repeated sequences. A-particle genes varied in the occurrence of specific restriction sites at characteristic internal loci. However, heteroduplexes between several variant 7.3-kb genes showed continuous homology regions even when spread under stringent hybridization conditions. The relative abundance of restriction site variants was highly conserved in 12 laboratory strains of Mus musculus, in embryonic and adult tissues of a single inbred strain, and in the SC-1 cell line of feral mouse origin, but appeared to differ in a feral Japanese substrain, Mus musculus molossinus. Some evidence suggests that subsets of A-particle genes may have similar flanking sequences. The results are discussed in terms of the evolution of this multigene family.

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