Physical mapping and characterization of Chlamydomonas mitochondrial DNA molecules: Their unique ends, sequence homogeneity, and conservation

Abstract

Sixteen site-specific endonucleases were used to characterize the mitochondrial (mt)-DNA of Chlamydomonas reinhardtii. Recognition sites for SmaI, XhoI, and BglII were absent in the mtDNA. mtDNA fragments appeared in stoichiometric proportions in every nuclease digest indicating that C. reinhardtii mtDNA consists of a homogenous population of molecules devoid of either inter- or intramolecular heterogeneity. Six DNA fragment maps were derived for those endonucleases that produced discrete and readily measurable DNA fragments. These maps, which exhibited marked internal consistency, also suggested that the linear mtDNA molecules possessed unique ends. This was subsequently confirmed by in vitro 5′-end labeling of mtDNA molecules prior to endonuclease digestion. These results indicate that (1) the linear mtDNA isolated under our experimental conditions possessed not only unique ends but also a nonpermuted gene sequence and (2) such mtDNA molecules were generated by a site-specific cleavage of the closed circular mtDNA molecules shown to exist in vivo. mtDNA sequence conservation in Chlamydomonas is quite striking. No difference in endonuclease cleavage pattern has yet been detected among a number of C. reinhardtii strains or between mating types.