The role of DNA methylation in the regulation of gene function 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 the corresponding methylase and Dpn II, which cleaves 5'-G-A-T-C-3'. Strains in which this sequence is not methylated produce Dpn I, which cleaves only the methylated sequence, 5'-G-meA-T-C-3'. Mutants lacking Dpn I, Dpn II and the methylase will be obtained using susceptibility or resistance to a restrictable bacteriophage as an indicator or phenotype. Transformation of these mutants with DNA from both types of strains will show whether each carries the genetic information for both restriction systems, and whether methylation of the DNA determines which system is expressed. The pneumococcal genes will also be cloned in dam+ and dam- strains of Escherichia coli. These manipulations should determine how the restriction system enzymes are coordinately regulated, whether DNA methylation directly affects mRNA transcription, and whether the DNA methylation pattern defines two alternative states or cell differentiation in this procaryotic species. Current evidence from a number of eucaryotic genes indicates that DNA methylation interferes with their expression. Information derived from this project could provide, therefore, a well defined molecular model also for the regulation of eucaryotic genes. Methylation of DNA may represent an important means for the control of cellular development. The elucidation of the mechanism of such control could contribute to an understanding of pathological cellular development in diseases such as cancer.
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