Dispersive labelling of Chlamydomonas chloroplast DNA in ¹⁵N-¹⁴N density transfer experiments

Summary

¹⁵N-¹⁴N density transfer experiments with synchronized vegetative cultures of Chlamydomonas reinhardtii revealed a dispersive labelling of chloroplast DNA (cpDNA) while the labelling of nuclear DNA was consistent with semiconservative replication. The dispersive labelling of cpDNA was progressive and extensive as after less than two net doublings of this DNA in ¹⁴N-medium no significant amount of fully heavy, ¹⁵N-strands could be detected in denatured cpDNA preparations; the average size of DNA in these preparations corresponded to 6% of the intact chloroplast genome or about 12 kbp. The density shifts of native cpDNA samples were found to be consistent with the net amounts of cpDNA synthesized. This observation indicates that essentially all ¹⁵N atoms incorporated prior to the transfer were conserved and that metabolic turnover of cpDNA was probably absent. Our results are best explained by the exchange of homologous single-stranded segments between cpDNA molecules to form heteroduplex regions and by each DNA molecule undergoing several rounds of heteroduplex formation.