Faculty Bibliography

During the past century, several biologists have studied the mental processes involved in creativity. In recent years psychologists have approached the subject experimentally. In one such study (Carson, Peterson, and Higgins 2003), creativity has been shown to originate in the subconscious mind and to be transmitted to the conscious mind as a result of a decrease in latent inhibition, an ordinarily strong cognitive barrier between the conscious mind and the subconscious. In my scientific work I have found evidence for creativity in the design of experiments, in which the addition of apparently superfluous controls has led to important discoveries

The present account spans the history of arginine regulation from its discovery in 1955 until the present. In 1957 I demonstrated that not only added arginine but also internally produced arginine represses enzyme formation and that the potential for enzyme synthesis is in excess of what is required for growth. In 1959 I located the regulatory gene argR encoding the arginine repressor. An unusual feature of this research was the finding that in E. coli B, in contrast to E. coli K12, arginine synthesis is permanently repressed, independent of arginine. This was due to a single amino acid difference between the two repressors. Recent studies showed that, in natural populations of E. coli, K12-type regulation is much more frequent than B-type regulation, and that E. coli B evolved from a strain with K12-type regulation. In competition experiments, E. coli K12 was found to be favored in the presence of arginine and E. coli B in its absence, showing that contrary to expectations permanently turned off regulation is favored over negative regulation in some environments.

Protein and mRNA levels of heat-labile enterotoxin (LT) of Escherichia coli are highest at 37 degrees C, and they decrease gradually as temperature is decreased. This temperature effect is eliminated in an Hns- mutant. Deletion of portions of DNA coding for the LT A subunit also results in an increase in LT expression at low temperatures, suggesting that the H-NS protein causes inhibition of transcription at low temperatures by interacting with the LT A-subunit DNA. The region that interacts with H-NS is referred to as the downstream regulatory element (DRE). Plasmids in an hns strain from which the DRE has been deleted still produce elevated levels of LT at 18 degrees C, suggesting that intact DRE is not required for transcription from the LT promoter

Arginine biosynthesis in Escherichia coli is negatively regulated by the hexameric repressor protein ArgR and the corepressor L-arginine. L-Arginine binds to ArgR in the C-terminal domain of the repressor. Binding to operator DNA occurs in the N-terminal domain. The molecular structures of both domains have recently been elucidated. The known stereochemistry of the arginine binding pocket was used for the rational design of a mutant ArgR with altered ligand specificity. Our prediction was that a replacement of Asp128 by asparagine would preferentially lead to the binding of L-citrulline, rather than L-arginine. The D128N mutant was constructed and was shown to fulfill our expectation by several experimental approaches. By isothermal titration calorimetry it was found to bind L-citrulline much more strongly than L-arginine, in contrast to wild-type ArgR. Exchange between the mutant trimers of the hexamer was inhibited by L-citrulline, as it is by L-arginine in the wild-type. The mutant protein was precipitated by L-citrulline but not by L-arginine, whereas the reverse is true for the wild-type protein. Demonstration of a corepressor action was, however, precluded by the superrepressor effect of the D128N mutation by itself. The mutant protein, in the absence of L-citrulline or L-arginine is as strong a repressor as the wild-type protein in the presence of L-arginine. We discuss two possible mechanisms, in terms of the known domain structures that could explain our observations

By studying the interaction of derivatives of RepFIC miniplasmids, we were able to demonstrate that under certain conditions the RepA1 initiator protein inhibits plasmid replication. An analysis of cloned derivatives whose replication is inhibited by the RepA1 protein revealed the existence of two areas of the RepFIC genome that interact with RepA1 in the inhibition reaction. One of these areas, which occurs in the origin region, was explored by in vivo methylation protection footprinting studies. The protected area was 200 bp long and showed a definite periodicity of protected and hypersensitive sites, suggesting that RepA1 promotes a topological change in the RepFIC genome. The significance of our results is discussed in the context of plasmid replication control