Inverted Repetitious Sequencesin the Macronuclear DNA Of
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Proc. Nat. Acad. Sci. USA Vol. 72, No. 2, pp. 678-682, February 1975 Inverted Repetitious Sequences in the Macronuclear DNA of Hypotrichous Ciliates* (electron microscopy/site-specific endonuclease/DNA structure) RONALD D. WESLEY Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colo. 80302 Communicated by David M. Prescott, November 27,1974 ABSTRACT The low-molecular-weight macronuclear renaturation kinetics of denatured macronuclear DNA are DNA of the hypotrichous ciliates Oxytricha, Euplotes, and the presence of a Paraurostyla contains inverted repetitious sequences. Up approximately second-order, indicating to 89% of the denatured macronuclear DNA molecules single DNA component with a complexity about 13 times form single-stranded circles due to intramolecular re- greater than Escherichia coli DNA (M. Lauth, J. Heumann, naturation of complementary sequences at or near the B. Spear, and D. M. Prescott, manuscript in preparation). ends of the same polynucleotide chain. Other ciliated (ini) The macronuclear DNA pieces possess a polarity, i.e., protozoans, such as Tetrahymena, with high-molecular- weight macronuclear DNA and an alternative mode of one end is different from the other, because a large region at macronuclear development, appear to lack these self- one end of each molecule melts at a slightly lower temperature complementary sequences. than the rest of the molecule, and because RNA polymerase The denatured macronuclear molecules of hypotrichs binds exclusively at only one end (7). are held in the circular conformation by a hydrogen- The experiments reported here demonstrate that single- bonded duplex region, which is probably less than 50 base pairs in length, since the duplex regions are not visible by strand nicks or gaps and inverted repetitious sequences are electron microscopy and since the circles in 0.12 M phos- other important structural features of macronuclear DNA. phate buffer are not retained during hydroxylapatite chromatography at 600. The existence of extremely small MATERIALS AND METHODS circles, with contour lengths shorter than the smallest pieces of native DNA, suggests that inverted repetitions Preparation ofOxytricha Macronuclear DNA. The hypotrich containing nicks (broken phosphodiester bonds in duplex Oxytricha was cultivated aseptically in 0.1% Cerophyl (Cero- DNA) or gaps (interruptions with missing nucleotides) are phyl Laboratories, Kansas City, Mo.), with axenically grown present at internal positions as well as at the ends of na- Tetrahymena as the food organism (4). Oxytricha DNA was tive molecules. Estimates from length measurements of native and denatured Oxytricha macronuclear DNA indi- labeled by feeding Tetrahymena that had been cultured in cate an average of 1.7 nicks per duplex molecule. Thus, in defined medium (8) containing [8H]thymidine (New England order to account for the high frequency of circle formation, Nuclear, 25 MCi/ml, 20 Ci/mmol). The resultant specific a restriction-type enzyme(s) must exist which inserts activity of the purified DNA was 2500 cpm/,4g. single-strand, site-specific nicks or gaps at internal posi- For DNA isolation, the hypotrichs were collected on a 10 tions in the macronuclear DNA of Oxytricha. usm nylon mesh; macronuclei were prepared by the technique Most ciliated protozoa have a macro- and a micronucleus. of Prescott et al. (5) and stored at -20°. After conjugation, a new macronucleus is derived from a Thawed macronuclear preparations were lysed at 600 for single, diploid micronucleus by a temporally well-defined 1-2 hr in a solution of 0.5% Sarkosyl, 0.15 M NaCl, 0.1 M developmental program. In hypotrichous ciliates macronuclear EDTA (pH 8.0) containing 0.5 mg/ml of Pronase CB (Calbio- development is characterized by a biphasic cycle of extensive chem, predigested for 90 min at 370 to eliminate DNase DNA synthesis (1) which involves the formation of polytene activity). To separate the DNA from protein and RNA, chromosomes that are cut into band-length pieces (2). The 4.86 ml of the nuclear lysate was added to 6.23 g of solid transection of the chromosomes is followed by degradation of CsCl (Schwarz/Mann) and centrifuged to equilibrium at most of the DNA in each band (3). Bostock and Prescott (4) 33,000 rpm for 62 hr in the Spinco no. 65 fixed-angle rotor at have shown that only some micronuclear DNA sequences are 20°. Following centrifugation, four-drop fractions were col- retained during the formation of the macronucleus. lected from the bottom of the tube, 0.5 ml of SSC (0.15 M The following information is available about the DNA that NaCl, 0.015 M sodium citrate, pH 7.5) was added to each remains in the mature macronucleus of hypotrichs. (i) The fraction, and the DNA band was located by its A260. Pooled DNA is in small pieces having the physical size equivalent to fractions were dialyzed against SSC and stored at -20°. one to a few genes (5). The low-molecular-weight nature of Macronuclear DNA preparations were shown to be free of the DNA may have some bearing upon the unusual cytological Tetrahymena DNA contamination by analytical equilibrium structures, termed replication bands, where DNA synthesis density gradient centrifugation. The buoyant density of characteristically occurs in hypotrichous ciliates (6). (ii) The Oxytricha macronuclear DNA is 1.701 g/cm3 and that of Tetrahymena. DNA is 1.690 g/cm3 (4). Abbreviations: SSC, standard saline-citrate, 0.15 M NaCl, 0.015 The macronuclear DNAs of Euplotes and Paraurostyla were M sodium citrate, pH 7.5; RF, replicative form of phage OX174 prepared similarly by isolating macronuclei, followed by DNA. nuclear lysis and CsCl density gradient centrifugation. * This paper is No. IV in the series, "DNA of Ciliated Protozoa." Preparation of Bacteriophage 6X174 DNA. oXamS, a lysis- The preceding paper is ref. 7. defective amber mutant of OX174, and Escherichia coli (HF 678 Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) DNA of Hypotrichous Ciliates 679 4704) were kindly provided by Dr. Dan S. Ray. OX replicative the 3' end of a DNA double helix (13), was used to determine form (RF) DNA was prepared by blocking viral single- if the single-stranded circles, which rapidly form after de- stranded DNA synthesis with chloramphenicol. The procedure naturation of macronuclear DNA, were sealed by a region of was as described previously (9) except that RNA digestions duplex DNA. The exonuclease III reactions were carried out and the Sephadex G-100 chromatography step were elimi- as described by Wolfson and Dressler (14). The enzyme was nated. Single-strand breaks were then introduced into the generously provided by Dr. Tom Kornberg. covalently closed molecules (form I) by photolysis ("photo- Single-stranded macronuclear DNA circles were dialyzed nicking") for 45 min in the presence of ethidium bromide as overnight to remove the formamide (four changes against described by Smith and Vinograd (10). The nicked molecules 0.07 M Tris * HCl, pH 7.6). The solution was then made 7 mM were separated from OX RF I by ethidium bromide-CsCl in MgCl2 and 10 mM in 2-mercaptoethanol. Two units of density gradient centrifugation, the dye was removed by exonuclease III (13) were added to 0.25 Ag of single-stranded dialysis against Dowex 50, and the DNA in SSC was used circular DNA and the mixture was incubated at 450 for 20 as a double-stranded DNA size standard for electron micros- min. Incubation was at 450 to increase the specificity of the copy. enzyme for the 3' end of double-stranded DNA (13). To insure The same general protocol was used to prepare OX single- completion of the reaction, an additional unit of enzyme was stranded circular DNA, but chloramphenicol was omitted. added and allowed to act for 20 min, at which time the reaction Infected cells were vigorously aerated for 2 hr while [14C]- tube was transferred to 00. The material was then processed thymidine was added in aliquots of 5 MACi at 1, 30, and 60 min for electron microscopy. post-infection. Collection and lysis of infected E. coli were as Control experiments, in which single-stranded OX DNA described above except that sucrose gradients were centrifuged circles were incubated with exonuclease III under the same for 12 hr at 24,000 rpm in the Spinco SW 27 rotor at 5°. The conditions, were performed to exclude the possibility of con- radioactivities of aliquots were measured, and fractions con- version of circular to linear molecules by an endonuclease taining mature viral DNA were pooled and dialyzed against activity. A preparation containing 87% (160/183) phage SSC. The DNA was further purified by neutral CsCl density circles remained essentially unaltered after exonuclease III gradient centrifugation for 48 hr at 40,000 rpm in the Spinco incubation (85%, 182/214), indicating the absence of any SW rotor at 200 (po = 1.723 g/cm3). This constituted the endonuclease contaminant. single-stranded DNA standard for electron microscopy. Hydroxylapatite Chromatography. Hydroxylapatite (DNA Conditions for the Formation of Single-Stranded Circles in grade) was obtained from Bio-Rad (Richmond, Calif.). Small Macronuclear DNA. Single-stranded circles were prepared as columns (1 ml bed volume) equilibrated with 0.12 M sodium follows: macronuclear DNA (0.5-1.0 Mug) was denatured in phosphate buffer (pH 6.8) were maintained at 600. [3H]- 0.5 ml of 0.1 M NaOH, 0.02 M EDTA at 22-24° for 10 min. Thymidine labeled, single-stranded rings were prepared by the Next, 50 Ml of 1.8 M Tris-HCl, 0.2 M Tris, and 0.5 ml of standard formamide method as described above and dialyzed formamide (99% pure, Matheson, Coleman and Bell) were against 0.12 M sodium phosphate buffer. The sample (0.7 ,g added so that the final annealing pH was 8.0.