Our major interest is in understanding the uptake and integration of DNA during transformation, however, we are now concerned, as well, with the role of DNA-protein interaction and DNA structure in these processes. Fragments of DNA that bind to DNA have palindromic sequences associated with them. These presumed uptake structures will be examined by physical rearrangement and alterations to demonstrate the role of palindromic sequences in DNA uptake. In addition the uptake of denatured DNA will be followed to determine if its uptake is a function of preferential binding that may be related to its ability to perform palindromic sequences. Studies of the interaction of protein and DNA will be directed toward establishing mechanisms for uptake, entry, and integration of transforming DNA. Proteins involved in DNA uptake will be analyzed by isolating proteins that specifically bind to fragments of DNA containing uptake signals. Furthermore, a system has been developed for cloning genes that correct competence-defective mutants (com+ genes) and it is anticipated that transferring com+ genes to high-level expression vectors will make available their products in high yield for direct biochemical and physical experiments. We have postulated the existence of specific recognition signals for integration that are analogous to """"""""chi"""""""" sequences and would like to identify the sequences or structures important for efficient integration. It appears likely that specific proteins are associated with this process, and attempts will be made to identify these interactions. Further progress is expected on the study of the fate donor DNA during uptake and integration and the interaction between DNA and outer-membrane particles, termed DNA binding vesicles. Evidence from these experiments should be helpful in elucidating the mechanism of uptake and entry. A series of experiments with directed mutagenesis and hybridization techniques are designed to provide a direct test for the role of the postulated 11-bp uptake sequence in uptake or integration.
