R - Looping and Structural Gene Identification

The advent of recombinant DNA technology has made possible studies on the organization and expression of eukaryotic genes, which were previously restricted to prokaryotic and viral genes. In large measure, these studies exploit and are dependent upon the availability-through recombinant DNA molecules--of large amounts of individual eukaryotic DNA sequences. They are also dependent upon an accurate definition of the structural gene sequence or sequences present in any recombinant DNA molecule of interest. In other words, a recombinant DNA molecule must first be positively identified as containing the structural gene sequence or sequences of interest before more detailed studies on or with it can be pursued. It is often advisable as well to define and localize all other structural gene sequences contained in the same recombinant molecule. In some cases, these preliminary characterizations are fairly straightforward. This is particularly true when the complementary RNA sequences are abundant in the tissue or cells of interest. Since rRNA or an abundant mRNA species can often be extensively purified by size fractionation alone, complementarity between this RNA and a recombinant DNA molecule can usually be detected by hybridization of the RNA and DNA followed by the protection of one labeled species from singlestrand-specific nuclease digestion. In the case of abundant mRNAs, complementarity can also be detected by hybrid-arrested translation. 1,2 The vast majority of eukaryotic mRNAs are, however, not abundant; most mRNA species are relatively rare in that each one constitutes approximately 0.01% of the total mRNA or less. Even a moderately abundant mRNA, such as a ribosomal protein mRNA of yeast, is present in the cell at a "concentration" of approximately 0.1% of the total mRNA. a The detection of complementarity between low-abundance mRNAs and re-