Induction of Nucleolar and Mitochondrial DNA Replication in Tetrahymena pyriformis

Abstract

Selective induction of replication of nucleolar and mitochondrial DNA has been demonstrated in starved-refed cultures of Tetrahymena pyriformis by different techniques.Labeling of starved cells with [3Hithymidine during a nutritional shift-up and analysis of the DNA in isopycnic CsCJ gradients shows that the two initially labeled species of DNA are two species banding on the heavy and light side of the bulk macronuclear DNA. In isolated macronuclei the radioactivity is found only in the high density fraction, which has been shown to be of nucleolar origin. In sucrose gradients the newly replicated mitochondrial and nucleolar DNAs sediment considerably slower than the bulk DNA, as one discrete band corresponding to a molecular weight of about 3 to 4 X 107.Electron microscope autoradiography of cells labeledwith [3Hjthymidine as above shows that the peripheral nucleoli of the macronucleus as well as the mitochondria are labeled before any radioactivity is found in the chromatin granules of the macronucleus. The results clearly indicate that nucleolar and mitochondrial DNA replication are under a control independent of that for the replication of bulk DNA.In Tetrahymena pyriformis the 500-1000 nucleoli are located peripherally in the nucleus (1) and form ribosomes at a rate per nucleolus which is equal to that of HeLa cell nucleoli (2). During starvation the nucleoli fuse into aggregates that again dissociate into multiple peripheral nucleoli during a nutritional shift-up which concomitantly induces rapid acceleration of rRNA synthesis (1).Since morphological studies show that a rearrangement of the nucleolar material occurs during a nutritional shift-up (1), we undertook this study since we felt that nucleolar DNA might show unusual replicative properties during the dissociation of nucleolar aggregates.We have previously studied the replication of the rRNA genes during the nutritional shift-up and have presented evidence from DNA * RNA hybridization experiments (3) suggesting that early during the refeeding period the rRNA genes are replicated preferentially. In the study reported here, the same starvation-refeeding system has been employed and it is shown by EM-autoradiography and labeling with [3H]thymidine that the nucleolar DNA as well as mitochondrial DNA are preferentially replicated during the early times of refeeding. The two species of DNA band in isopycnic CsCl density gradients as two discrete radioactivity peaks on the heavy and light side, respectively, of the bulk DNA. In sucrose density gradients the newly replicated nucleolar and mitochondrial DNAs sediment as one discrete peak with a molecular weight of about 3 to 4 X 107. MATERIALS AND METHODSCultures of Tetrahymena pyriformis, strain GL, were grown in a complex proteose peptone medium as described earlier (4). Starvation for 24 hr was carried out as described by Cameron and Jeter (5)

DNase I sensitivity of ribosomal genes in isolated nucleosome core particles

“…Our results clearly indicate that the preferential digestion by DNase I of DNA which has been labeled during refeeding of starved Tetrahymena is similar in intact nuclei, isolated nuclesomes and purified nucleosome cores. It has been demonstrated that this defined starvation-refeeding scheme preferentially labels the ribosomal genes (10,11,29,42; see Fig. 1).…”

“…Tetrahymena ribosomal genes (rDNA) can be preferentially labeled utilizing a defined starvation-refeeding scheme modified slightly from that originally developed by Engberg et al (42). This procedure yields nuclei in which the bulk DNA has been labeled with 14C-thymidine during exponential growth while rDNA is preferentially labeled with 3H-thymidine during a 2-hour refeeding period.…”

“…Cells were differentially labeled using a previously described modification (10) of the defined starvation-refeeding protocol of Engberg et al (42). Bulk cellular DNA was labeled during exponential growth with [methyl-14C]-thymidine (0.17 iCi/ml of culture).…”

Macronuclear DNA in Stentor coeruleus: a first approach to its characterization

“…In ciliates, satellite DNA has been found in some strains of P. aurelia (Soldo & Godoy, 1972) and in T. pyriformis strain GL (Sueoka, 1961). Gall (1974) and Engberg et al (1974) have shown that macronuclear satellite DNA of T. pyriformis GL corresponds to ribosomal cistrons. In another strain, T. pyriformis WH-6, no detectable satellites were found by Yao & Gorovsky (1974) and no marked differences between micro-and macronuclear DNA base composition was observed.…”

Mechanisms of action of 5-bromodeoxyuridine based on studies with tetrahymena pyriformis

“…]5 -0.58M sucrose gradient gives three peaks of RNA, the (irst containing 25 S ribas~ma~ RNA, the second 17 S ribosomal RNA and the third a mixture of 5 S ribosomal RNA, tRNA and mRNA. The genes which code for the two large ribosomal RNAs (25S and 17S) are located in the nucleoli (18) and are often referred to as ribosomal genes. These genes replicate autonomously (2), The 5 S RNA genes are not nucleolar and replicate with the bulk DNA (55), In Tetrahymena tRNAs have sedimentation constants around 4 S and they are not separable form 5 S ribosomal RNA by the method here used.…”