Early attempts for isolation of ribosomal RNA genes in Xenopus
As we knew, ribosomes consist of ribosomal RNA (rRNA) and proteins. Ribosomal RNA (rRNA) can be of four sizes namely, 28S, 16S, 5.8S and 5S. This ribosomal RNA makes 80 per cent of cellular RNA and is synthesized on ribosomal genes, which could be isolated. Isolation of ribosomal genes was considered convenient due to their following three characteristic features : (i) availability of homogeneous rRNA; (ii) differences between ribosomal genes and other genes, due to relatively high G+C content in rRNA (rRNA has 45-60% G+C, while the remaining RNA has only 40% G+C); (iii) ribosomal genes are present in multiple copies, their number within a cell sometimes approaching several thousands.
In view of the above, ribosomal RNA genes were isolated for the first time (as early as 1965, by H. Wallace and M.L. Birnstiel) in an amphibian named Xenopus. Following steps were involved in this isolation : (i) isolate rRNA from ribosomes of Xenopus and make it radioactively labelled, due to its replication in a medium containing tritiated uridine; (ii) isolate ribosomal DNA by density gradient centifugation followed by its denaturation (G+C content of rDNA differs from that of bulk DNA and helps in its separation by centifugation); (iii) fix single stranded DNA on a filter paper; (iv) add labelled rRNA on filter paper carrying single stranded DNA; (v) allow DNA-RNA hybridization to take place; (vi) wash excess labelled RNA; (vii) measure radioactivity and isolate duplex hybrids, which on denaturation, will give single stranded DNA which can then be made double stranded. For a detailed account of isolation of ribosomal genes in Xenopus as originally done, readers are referred to an article 'Isolation of Genes' written by Donald D. Brown in Scientific American (August, 1973).