From protein synthesis to genetic insertion

In 1946, (14)C-cyanide made its appearance as an offshoot of the Atomic Energy Program. Our colleague Robert Loftfield built it into (14)C-alanine by the Strecker synthesis, and a lusty program directed toward uncovering the unknown mechanism of protein synthesis grew out of this beginning. The necessity for an undiscovered series of steps and enzymes was soon evident. A cell free system was developed, and a succession of components necessary for this new pathway tumbled out. ATP dependence, amino acid activation, the ribosome as the site of polypeptide formation, discovery of tRNA as the translation molecule linking the gene and protein sequence, and GTP as the essential energy ingredient in peptide chain extension all appeared from our laboratory within the next decade. A little later the AP(4)N family, whose functions remain imperfectly defined, of intracellular molecules was discovered. Isolation of specific species of RNA became a high priority, and we sequenced a small segment of the 3' end of the Rous sarcoma virus, just inside the poly(A) tail, at the same time the Gilbert group at Harvard was sequencing the 5' end. The sequence identity and polarity of the two ends suggested a circular intermediate in replication and predicted correctly that a synthetic antisense oligonucleotide targeted against this sequence might be a specific inhibitor of replication. More recently, we have evolved a technique that appears to achieve a trinucleotide insertion into tissue culture cells bearing a specific Delta508 mRNA triplet deletion, resulting in phenotypic reversion in the tissue culture.