Estimating bacterial DNA synthesis from [3H] thymidine incorporation : discrepancies among macromolecular extraction procedures

The estimation of bacterial DNA synthesis in trophic studies with [3H] thymidine requires quantitative extraction of labeled DNA. To determine the DNA contribution to total macromolecular labeling in a eutrophic ecosystem, we tested three extraction procedures currently used to estimate DNA labeling from thymidine incorportation : enzymatic fractionation, acid-base hydrolysis, and phenol-chloroform extraction. Because labeled macromolecular fractions are generally defined as DNA, RNA, and proteins, we used incorporation of tritiated thymidine, uridine, and leucine to preferentially label DNA, RNA, and proteins. Our data showed that each fractionation method yielded different apparent macromolecular distributions of the same radiolabeled substrates. Enzymatic digestions of the fractions obtained by acid-base hydrolysis and phenol-chloroform extraction showed that these two procedures are inadequate for estimating bacterial DNA labeling in our ecosystem. Finally, using the enzymatic procedure at different sites, DNA labeling appeared to represent a nearly constant proportion of the labeled macromolecules (20,1%, r=0.952, n=101) over a wide range of incorporations rates. (Résumé d'auteur