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Proc. Nat. Acad. Sci. USA Vol. 71, No. 5, pp. 1819-1823, May 1974 The Amplified Ribosomal DNA of Dytiscid Beetles [DNA replication/Dytiscidae (Coleoptera)/gene amplification/repetitive DNA/electron microscopy of DNA] JOSEPH G. GALL AND JEAN-DAVID ROCHAIX Department of Biology, Yale University, New Haven, Connecticut 06520 Contributed by Joseph G. GaU, February 21, 1974 ABSTRACT During oogenesis in many animals there 35,000 rpm, 65 hr, 180). The Colymbetes ovary tips were lysed is a massive extrachromosomal synthesis of the genes in the same solution, but were then mixed directly with CsC I for ribosomal RNA. In Dytiscid beetles, as in the toad Xenopus, the amplified ribosomal DNA occurs as circular without further purification. DNA from the CsCl gradient molecules of different sizes. The circles fall into size was precipitated with three volumes of 70% ethanol, washed classes that are integral multiples of a unit circle. It is with 70% ethanol, and redissolved in 0.1 ml of 15 mM NaCl- probable that the unit circle contains the coding sequence 1.5 mM Na3 citrate (pH 7.0). for one precursor ribosomal RNA molecule plus accom- in one of panying spacer sequences. The DNA was spread for electron microscopy several ways. For measurements of contour length the DNA In many animals the genes coding for ribosomal RNA (rRNA) was prepared either by the "aqueous" or "formamide" modi- undergo an extensive extrachromosomal replication or ampli- fications of the Kleinschmidt method (8). Spreading in 50% fication during oogenesis (1, 2). The amplified ribosomal DNA formamide gave more fully extended molecules of somewhat (rDNA) is usually associated with multiple nucleoli whose longer contour length. As an internal molecular weight stan- size, shape, and disposition within the nucleus vary from dard, double-stranded qX174 DNA was included in most species to species. Because the nucleoli in some amphibian preparations. For denaturation mapping, the DNA molecules oocytes occur as circular necklaces, easily broken by treat- were spread in high concentrations of formamide (over 90%) ment with DNase (3, 4), it has been assumed for some years in 40-80 mM C03, pH 9.3. Molecules were picked up on that the amplified rDNA molecules exist as covalently-closed parlodion-coated grids, treated briefly with 50 pM aqueous circles. Direct evidence for the circularity of the amplified uranyl acetate, and then dried. The grids were rotary shad- rDNA molecules in the toad, Xenopus, was provided by the owed with palladium-platinum (1:4) at an angle of about 80. recent electron microscQpical studies of Hourcade, Dressler, Micrographs were taken at an initial magnification of 5,000- and Wolfson (5, 6). These workers showed that rDNA circles 12,000 X in the Philips 200 or 300 electron microscope. The fall into integral size classes whose unit is about 7.5 X 106 plates were projected by a photographic enlarger onto tracing daltons, equivalent to the coding unit for one 18S and one paper, and the contours of individual molecules were drawn 28S rRNA molecule plus accompanying spacer sequences. at a magnification of 1 to 3 X 105X. Lengths were measured We show here that the amplified rDNA in two species of on the drawings with a "map measurer." Lengths were con- Dytiscid water beetles likewise contains circular molecules verted to daltons on the assumption that the 4OX174 molecular of defined circumference. mass is 3.40 X 106. MATERIALS AND METHODS RESULTS Amplified rDNA was obtained from the ovaries of two The ovary tips of Dytiscid beetles contain several dozen Dytiscid water beetles, Colymbetes fuscus and Dytiscus mar- pachytene oocytes, each with a cap of extrachromosomal ginalis. Ovaries were removed from recently killed animals rDNA (Fig. 1). These oocytes are morphologically similar and placed in 70% ethanol for storage. Most of the DNA we to pachytene cells in the toad, Xenopus, except that the beetle have examined came from ovaries held 2 years at -100. The oocytes contain more rDNA (up to 43 pg in Colymbetes fuscus tips of the ovariolps, which contain a high proportion of and 67 pg in Dytiscus marginalis). When the DNA from ovary amplified rDNA (7), were dissected in 70% ethanol and then tips is centrifuged in a CsCl gradient, two peaks are seen lysed in a solution containing 50 mM Tris (pH 8.4), 0.1 M (Fig. 2). The heavier of these contains the amplified rDNA EDTA, 0.5% Sarkosyl (Geigy), and 100 yg/ml of self-digested (7). In Colymbetes the heavier peak (p = 1.721 g/cm') con- Pronase. The Dytiscus DNA was extracted with an equal sists entirely of rDNA, but in Dytiscus approximately half of volume of water-saturated phenol, precipitated from the the heavier peak is a highly repetitive satellite DNA that aqueous phase with two volumes of ethanol, redissolved in happens to have nearly the same buoyant density as the 15 mM NaCl-1.5 mM Na3 citrate (pH 7) and digested with rDNA (p = 1.717 g/cm3). The satellite DNA in the ovary-tip 100 ug/ml each of pancreatic and T1 RNase. It was then preparations is derived primarily from the nurse cells and centrifuged to equilibrium on a neutral CsCl gradient having follicle cells, which accompany the oocytes. Since the satellite an initial density of about 1.70 g/cm' (Spinco Ti 50 rotor, DNA molecules are linear, they do not interfere with the observations on circular rDNA molecules in the same sample. Abbreviations: rRNA, ribosomal RNA; rDNA, DNA that codes When the amplified rDNA of Colymbetes was examined in for rRNA. the electron microscope, approximately 13.5% (n = 282) of 1819 Downloaded by guest on September 27, 2021 1820 Genetics: Gall and Rochaix Proc. Nat. Acad. Sci. USA 71 (1974) FIG. 1. A pachytene oocyte of Dytiscus marginalis showing the large cap of extrachromosomal, amplified rDNA (left) and the synapsed chromosomes (right). Approximately 90% of the nuclear DNA at this stage is rDNA. This is an unlabeled nucleus in an autoradiograph of an ovary taken from an animal injected with [3H]thymidine 6 hr before it was killed. Some follicle cells, oogonia, and nurse cells show label, but most pachytene oocytes from adult beetles have completed amplification and fail to FIG. 3. Electron micrograph of a circular rDNA molecule incorporate thymidine. Giemsa stain, X 1200. from the beetle Dytiscus marginal-is. The molecular mass of this molecule is approximately 60 X 106 daltons, and it therefore the molecules were found to be circular, the remainder being contains three rDNA repeat units. It was spread in 92% forma- linear (Fig. 3). The smallest circles made up a homogeneous mide and shows both native (double-stranded) and denatured population with mean molecular mass of about 10 X 106 (single-stranded) regions. X21,600. daltons. Larger circular molecules were integral multiples of this smallest size, i.e., 20, 30, 40 X 106 daltons. A few circular unit length for all molecules was 10.04 X 106 daltons (Figs. molecules larger than 40 X 106 daltons were seen. The average 4 and 6). The heavy peak of Dytiacus DNA also contained circular molecules, accounting for approximately 19% (n = 526) of the total. In contrast to Coiymbetes, the smallest circles of Dyti~cus had an average molecular mass of about 20 X 1061 daltons. Larger circular molecules fell into size classes with 0.3 means of 40 and 60 X 106 daltons, and a few molecules of about 80 X 106 daltons were found. The average unit mass for all molecules was 19.17 X 106 daltons (Figs. 5 and 6). Clearly, therefore, the unit circle size in Dytiscu is about E c twice that of Colymbetes. Partially denatured molecules were prepared by spreading w 2 the DNA in formamide concentrations above 90%. Such a z high formamide concentration was necessary because of the co generally high (G + C) content of the rDNA. 0 cIn In Dytiscus each of the 20 X 106-dalton circles contained one major region of denaturation extending for less than 1/4 of the circumference of the circle. The 40, 60, and 80 X 106-dalton molecules had 2,3 and 4 such regions, respectively 0.1 (Fig. 7). A denaturation map was constructed by aligning all molecules by eye such that apparently homologous regions were in register. The percent of molecules showing denatura- tion in a given region was plotted for each region of the repeat 20 30 40 unit (Fig. 8). Some denatured molecules of rDNA FRACTION NUMBER partially Colymbes were observed, but insufficient material was available to FIG. 2. DNA from ovariole tips of the beetle Colymbetes prepare a denaturation map for this species. fuscus, centrifuged to equilibrium in neutral CsCl. The heavier peak (p = 1.721 g/cm3) consists of amplified rDNA from oocytes, DISCUSSION while the main peak (p = 1.693 g/cm3) is derived primarily from follicle cells and nurse cells. Breaks indicate fractions of rDNA The first suggestion that amplified rDNA might occur as pooled for study. circular molecules came from light microscopic observations Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Ribosomal RNA Genes 1821 en 10 r Z02 Fd i n A [1 _ nf¶I fnrd n n ni 10 20 30 40 50 MOLECULAR WEIGHT x 10-6 FIG. 4. Frequency distribution of molecular weights of 51 circular rDNA molecules from the beetle Colymbetes fuscus. Molecular weights have been multiplied by 10-. 10 7 (I, Lii X -J 50 : t 0 o L El on a 20 40 60 80 MOLECULAR WEIGHT x 10-6 FIG. 5. Frequency distribution of molecular weights of 46 circular rDNA molecules from the beetle Dytiscus marginalis.

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