The long-term goal of this research is to develop a unified theory of genetic control that incorporates both nuclear and cytoplasmic genetic systems. For this purpose we are using (1) the unicellular eukaryotic microorganisms Chlamydomonas and (2) various mammalian cell lines in culture. With Chlamydomonas, we are examining the role of methylation of chloroplast DNA in the regulation of its expression and destruction by DNA methylation and restriction enzymes, as well as the enzymatic properties of a restriction endonuclease and a series of methyltransferase enzymes isolated from different stages of the Chlamydomonas life cycle. We have developed a new and powerful method for detecting the presence of 5-methylcytosine in DNA fragments bound to nitrocellulose or to DBM paper. This method is now being applied to DNAs isolated from a number of systems in which regulation of transcription by DNA methylation is suspected. With mammalian cells, we are examining the mitochondrial location of mutations such as that responsible for chloramphenicol-resistance and the pyruvate requirement for expression of chloramphenicol-resistance, recently discovered in this laboratory. We plan to examine the mitochondrial DNAs of mouse and Chinese hamster cell lines, newly transformed by chemical mutagens and carcinogens and by viruses. Molecular methods of DNA analysis by restriction fragment patterns, hybridization of highly radioactive nick translated DNA probes to various DNA preparations by the Southern blot technique, and sequencing of fragments will be applied to the search for alterations in the cytoplasmic DNA of mutagenized mutants and transformed cells.
| Sklar, R; Altman, D; Sager, R (1986) An endonuclease from Chlamydomonas reinhardtii that cleaves the sequence TATA. J Biol Chem 261:6806-10 |
