The regulation of genes governing restriction enzymes, including both DNA methylases and endonucleases, will be examined in Streptococcus pneumoniae. Different strains of this bacterium have different patterns of DNA methylation and harbor one of two complementary restriction enzyme systems. Strains that contain the methylated sequence 5'-G-meA-T-C-3' produce DpnII, which cleaves 5'-G-A-T-C-3'. Strains not methylated in this sequence produce DpnI, which is an unusual restriction endonuclease in that it cleaves only the methylated sequence, 5'-G-meA-T-C-3'. The chromosomal genes encoding the DpnII methylase and the DpnI and DpnII endonucleases have been cloned in a pneumococcal host/vector system. The location of these genes in the cloned segments and their DNA sequence will be determined. Two additional genes related to the restriction enzyme phenotype have been tentatively identified, and their presumptive roles in regulation of restriction gene expression will be investigated. The proteins corresponding to the various structural and regulatory genes in the restriction systems will be identified, purified, and characterized with respect to biochemical function. The possible presence and location of unexpressed genes of the complementary phenotype in strains of a particular phenotype will be examined by DNA hybridization techniques. Preliminary results indicate that the restriction phenotypes of S. pneumoniae are determined by an intercellular genetic cassette system. The mechanism and regulation of transitions between the phenotypes will be determined. It has been shown that transitions from DpnI to DpnII production can occur via a null state, in which no restriction endonuclease is produced. The genetic basis of the null phenotype will be examined. Similar cassette mechanisms may govern other pneumococcal traits, such as chromosomally located multiple drug resistance and capsular type. These traits are important in the control and treatment of pneumonia as a disease. Analysis of the regulation of restriction gene expression, including possible functions of DNA methylation, should extend knowledge of genetic regulation in general. It may lead to a greater understanding of those diseases, including cancer, in which developmental mechanisms of gene regulation appear to have gone awry.
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