FRAGMENTS OF ALKALINE PHOSPHATASE FROM NONSENSE MUTANTS .I. ISOLATION AND CHARACTERIZATION OF FRAGMENTS FROM AMBER AND OCHRE MUTANTS

Small amino-terminal fragments of the alkaline phosphatase molecule have been isolated from two amber and two ochre phosphatase nonsense mutants of Escherichia coli. Fingerprint analyses of the tryptic peptides of the fragments indicate that the structural relationships of the fragments to each other, and to the complete phosphatase molecule, are consistent with the relative genetic map positions of the mutants. These results confirm that the nonsense triplets UAG and UAA can cause premature chain-termination of protein synthesis. The yield of fragment protein from two of the mutants, one amber and one ochre, which are closely linked on the genetic map and which produce fragments of almost equal size, was compared. The ochre mutant produced about twice as much fragment protein as the amber mutant, suggesting that UAA is a more effective chain-terminating signal than UAG. A surprising result from these studies is that a nonsense mutant, containing a single nonsense mutation, can produce more than one species of phosphatase fragment. Multiple fragments were obtained from all of the mutants analyzed; three mutants each produced two separable fragments, and the fourth mutant produced three fragments. The fragments produced by one mutant are very similar to each other in size, chromatographic behavior and peptide composition. Individual fingerprints of two separated fragments from the same mutant revealed a difference in one tryptic peptide, which appeared at a slightly different position in each fingerprint. The chemical basis for this minor difference between multiple species of fragment derived from the same mutant is not known. © 1969.