Proc. Nat. Acad. Sci. USA Vol. 71, No. 8, pp. 3082-3086, August 1974 A Small Number of Cistrons for Ribosomal RNA in the Germinal Nucleus of a Eukaryote, Tetrahymena pyriformis (evolution of repeated genes/amplification/DNA RNA hybridization/maero- and micronuclei) MENG-CHAO YAO, ALAN R. KIMMEL, AND MARTIN A. GOROVSKY Department of Biology, University of Rochester, Rochester, New York 14627 Communicated by Joseph G. Gall, May 31, 1974 ABSTRACT The percentage of DNA complementary repeated sequences (5, 8). To our knowledge, little evidence to 25S and 17S rRNA has been determined for both the exists to support any of these hypotheses. macro- and micronucleus of the ciliated protozoan, Tetra- hymena pyriformis. Saturation levels obtained by DNA - The micro- and macronuclei of the ciliated protozoan, RNA hybridization indicate that approximately 200 Tetrdhymena pyriformis are analogous to the germinal and copies of the gene for rRNA per haploid genome were somatic nuclei of higher eukaryotes. While both nuclei are present in macronuclei. The saturation level obtained derived from the same zygotic nucleus during conjugation, with DNA extracted from isolated micronuclei was only the genetic continuity of 5-10% of the level obtained with DNA from macronuclei. only the micronucleus maintains After correction for contamination of micronuclear DNA this organism. The macronucleus is responsible for virtually by DNA from macronuclei, only a few copies (possibly all of the transcriptional activity during growth and division only 1) of the gene for rRNA are estimated to be present in and is capable of dividing an indefinite number of times during micronuclei. Micronuclei are germinal nuclei. Macro- vegetative growth. However, if Tetrahymena are allowed to nuclei serve as somatic nuclei during vegetative growth but are destroyed every sexual generation and are re- undergo a sexual cycle (conjugation), the macronucleus formed from products of meiosis, fertilization, and breaks down. It is then formed anew from a division product division of the micronuclei. Thus, the hybridization data of the zygotic nucleus which results from the fusion of gametic suggest that the gene for rRNA must be amplified during nuclei derived from the micronucleus (9). macronuclear formation with each sexual generation. of Tetrahymena has been shown to con- These observations also demonstrate that the multiple The macronucleus copies of a repeated gene in a somatic nucleus of a eu- tain approximately 170-200 copies, per haploid DNA amount, karyote can be generated from a small number of copies of the genes coding for 25S and 17S rRNA (ref. 10, and this of that gene in a germinal nucleus. report). Recent studies show that the repeated copies of the gene for rRNA in the macronucleus are not physically linked The cistrons coding for ribosomal RNA (rRNA) have been to the rest of the genome (11). In this study, we have com- widely studied. It is now well established that these genes pared the number of genes for rRNA in macro- and micro- are present in multiple copies in eukaryotic organisms and nuclear DNA by hybridization with homologous rRNA. DNA it is likely that each of the copies in a particular organism extracted from isolated micronuclei was found to contain ap- is virtually identical (1, 2). In contrast to the homogeneity proximately one-tenth to one-twentieth the number of genes of the multiple copies of the rRNA gene within an organism, for rRNA as did macronuclear DNA. Since most of the hy- a portion of this gene, the transcribed and nontranscribed bridization of rRNA to micronuclear DNA can be accounted spacer, differs markedly in two closely related species (3). for by macronuclear DNA which is known to contaminate The portion of the gene which actually codes for rRNA also the micronuclear preparation, we conclude that micronuclei is demonstrably different in different species, although it ap- contain a small number of genes (possibly only one) for ribo- pears to evolve more slowly than the spacer region (4). Similar somal RNA. These observations suggest that, in at least one intra-species homogeneity coupled with inter-species vari- eukaryotic organism, the repeated copies of a gene found in ability has been shown to occur for other repeated DNA se- a somatic nucleus are formed by expansion from a small quences (5-7). number of copies of that gene which are present in the germi- The identity of repeated copies of a gene within a species nal nucleus. coupled with variability of the same gene between species the raises important questions regarding mechanism(s) by MATERIALS AND METHODS which these genes evolve, since mutation and selection alone seem inadequate to explain these observations. Several hy- 1, have been put forward to explain the evolution of Culturing of Cells. Tetrahymena pyriformis (Syngen potheses strain WH-6 or strain B-VII) was cultured axenically in en- riched proteose peptone as described previously (12). Foi labeling nucleic acids with radioactive isotope, yeast extract SSC, stan- Abbreviations: EDTA, ethylenediaminetetraacetate; was omitted from the culture medium and 1 ml of an anti dard saline-citrate solution (0.15 M sodium chloride-0.015 M mixture Island Co. 0.1 SSC means that the concentration biotic-antimycotic (Grand Biological sodium citrate, pH 7); X at the time c of the solution used is 0.1 times that of the standard saline-citrate New York) was added per 100 ml of culture solution. addition of isotope. 3082 Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Small Number of rRNA Cistrons in a Germinal Nucleus 3083 25S 17S 4S it) I I 0 it, E I0 0- I a- (. it) x Pe) SLICE NUMBER 25SS 17S 4S F R ACliO N NUMBER FIG. 1. Sucrose gradient profile of whole cell RNA labeled with I I I ['H]uridine. The fractions which were pooled for purification of Fe) 25S and 17S rRNA are indicated. 0 x Isolation of Nuclei. One liter cultures containing 4 to 6 X a- 105 cells per ml were harvested, and macro- and micronuclei were isolated as described previously (13). Contamination of the micronuclear preparation by recognizable macronuclei it) was determined by counting the nuclei under the light micro- scope. One or less than one macronucleus was found for every 200 micronuclei. Labeling ofDNA. Cells were grown in proteose peptone con- taining 0.025 ,uCi/ml of 14C-labeled thymidine ([methyl-14C]- SLICE NUMBER thymidine, 52.7 mCi/mM, 100 ACi/ml; New England Nu- FIG. 2. Polyacrylamide gel electrophoresis of purified (A) 25S clear Corp., Boston, Mass.) for 20 hr. The specific activity of rRNA and (B) 17S rRNA. The positions of the major RNA isolated DNA was about 500 dpm/,ug. species of carrier whole cell RNA run in the same gels are indi- cated. Labeling of RNA. Cells in the log phase of growth (250,000 cells per ml) were harvested and resuspended in an equal crease in AM0 after complete denaturation, there appeared to volume of sterile 10 mM Tris HCl at pH 7.4 containing 50 be little or no contamination by RNA. ,MCi/ml 3H-labeled uridine ([5-3H]uridine, 27.88 Ci/mM, 1.0 In one experiment, DNA was isolated from strain B-VII mCi/ml, New England Nuclear Corp.). In one experiment, by lysing nuclei in a solution containing 1% sodium dodecyl cells were labeled for 1 hr and then were chased by the addition sulfate, 0.5 M ethylenediaminetetraacetate and 10 mM- Tris- of an equal volume of 2X concentrated culture medium con- HCl, pH 9.5, digesting in Pronase (1 mg/ml), and deproteiniz- taining yeast extract. In a second experiment, cells were ing with phenol (11). labeled for 30 min-and chased for 30 min as described above. Extraction and Purification of rRNA. Labeled cells were Extraction of DNA. Freshly prepared nuclei or nuclei which pelleted and fixed in acetic acid-ethanol (1: 3). The cells were had been stored frozen (-20°) for less than 2 days were in- pelleted again and resuspended in NET buffer (0.1 M NaCl, cubated in 0.5% Sarkosyl, 0.1 M disodium EDTA (ethylene- 0.01 M Tris HCl, pH 7.4, 0.001 M EDTA) containing 0.5% diaminetetraacetate) and 0.05 M Tris-HCl pH 8.4, at room sodium dodecylsulfate. The RNA was deproteinized by shak- temperature until lysed. Saturated CsCl was added to ing at 40 with an equal volume of chloroform: isoamyl alcohol give a final density of 1.685 g/cm3 and the lysate was centri- (24: 1) which had been saturated with NET buffer. The fuged at 42,000 rpm for 20 hr at 200 in the 50 Ti rotor of a phases were separated by centrifugation. The aqueous phase Spinco L3-50 ultracentrifuge. Fractions were collected and was deproteinized again, collected, and the RNA was precipi- the A260 was measured for each fraction. Fractions containing tated with 2 volumes of ethanol overnight at -20°. DNA were pooled, diluted with 2 volumes of H20, and were The precipitated RNA was resuspended in NET containing pelleted by centrifugation at 42,000 rpm for 12 hr in the 50 0.5% sodium dodecylsulfate, layered on a sucrose gradient Ti rotor. Pellets were dissolved in 0.1 X SSC (SSC = 0.15 (35 ml of 0.45 M-1.0 M sucrose in NET buffer), and centri- M NaCl + 0.015 M Na-citrate) and were banded in a CsCl fuged in the SW-27 rotor on a Spinco L3-50 ultracentrifuge gradient as before. The DNA-containing fractions were at 26,000 rpm for 24 hr at 20. Fractions (1-ml) were collected pooled and dialyzed against 0.1 X SSC.