Extrachromosomal Copies of Transposon Tcl in the Nematode
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Proc. Natl. Acad. Sci. USA Vol. 81, pp. 4018-4022, July 1984 Biochemistry Extrachromosomal copies of transposon Tcl in the nematode Caenorhabditis elegans (transposable element/repetitive DNA/extrachromosomal DNA/transposon excision/target site duplication) KE-SAN RUAN* AND SCOTT W. EMMONS Departments of Molecular Biology and Genetics, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, NY 10461 Communicated by Frank Lilly, March 14, 1984 ABSTRACT Extrachromosomal copies of the 1.6-kilobase tory of D. Hirsh. Culture conditions, including the prepara- transposable element Tcl are present at the level of between tion of synchronous populations, have been described (5, 6). 0.1 and 1.0 copy per cell in Caenorhabditis elegans strain Ber- DNA Isolation. The method of DNA isolation from nema- gerac. Extrachromosomal elements were detected and studied todes was as described by Emmons and Yesner (5). using Southern hybridizations employing a Tcl-specific probe. Restriction Endonuclease Digestion, Gel Electrophoresis, The amount of extrachromosomal Tcl DNA was roughly con- and Southern Hybridization. Enzymes were obtained from stant during development in Bergerac, which has approxi- New England Biolabs and were used following the supplier's mately 300 integrated chromosomal copies of Tcl in its haploid specifications. Agarose (type II, medium electroendosmo- genome. Extrachromosomal Tcl DNA was not detected in sis) was from Sigma. Electrophoresis was carried out in Tris strain Bristol, which has 30 chromosomal copies of TcW. Three borate buffer (89 mM Tris base/89 mM boric acid/2.5 mM forms ofextrachromosomal DNA were detected. The predomi- Na2EDTA, pH 8.3). Transfer to nitrocellulose (Schleicher & nant form was a 1.6-kilobase linear molecule with ends corre- Schuell) was according to Southern (7). The immobilized sponding to the ends of an integrated Tcl element. The other DNA was hybridized to a 32P-labeled DNA probe in a solu- two forms were, respectively, relaxed and supercoiled circular tion containing 50% formamide and dextran sulfate (8). De- copies of the element. Structural assignments were based on tails of the hybridization procedure were as follows. The ni- electrophoretic mobility, the results of sedimentation velocity trocellulose filter was prehybridized (10 ml/100 cm2) in 50% and equilibrium density gradient experiments, and on the sizes (vol/vol) formamide (MCB, Cincinnati, OH)/3x SET buffer of the products produced by treatment of purified extrachro- (20x SET = 3 M NaCl/0.02 M EDTA/1 M Tris buffer, pH mosomal DNA with restriction endonucleases. The suggestion 7.9)/0.1 M sodium phosphate buffer (pH 7.0)/5x BFP solu- is made that these extrachromosomal transposable elements tion (lx BFP = 0.02% bovine serum albumin/0.02% Fi- are the products of excision events known to be occurring at coll/0.02% polyvinylpyrrolidone) containing denatured soni- high frequency in somatic cells in Bergerac. cated Escherichia coli DNA at 100 ,g/ml. Prehybridization was at 37°C for 2-4 hr. Hybridization was carried out in a The genome of the soil nematode Caenorhabditis elegans similar solution, except that BFP solution was lx and sodi- contains multiple conserved copies of a 1.6-kilobase (kb) um dodecyl sulfate (to 0.3%) and sodium dextran sulfate 500 DNA sequence with the properties of a transposable element [to 10% (wt/vol)] were added. Hybridization was at 37°C (1, 2). This sequence, denoted TcJ, is present at dispersed overnight. The hybridized filter was washed at 37°C with genomic sites that vary in number and location in various two or three changes of 2x SET buffer/0.1 M phosphate strains. Structural and sequence analysis has shown that Tcl buffer, pH 7.0/0.2% sodium dodecyl sulfate, with 30 min be- elements have 54-base-pair (bp) perfect terminal inverted re- tween changes. Finally, the filter was rinsed in 0.2x SET peats and an internal open reading frame of 819 nucleotides buffer, dried, and exposed for 24-48 hr to x-ray film (Kodak (Fig. 1) (3). Sequence studies of two insertion sites leave un- XR5) at -70°C with an intensifying screen. The probe used resolved the question of whether Tcl creates a target site to detect Tcl sequences was plasmid pCe2002. This consists duplication on insertion (Fig. 1) (4). of a 1.7-kb Sst I fragment carrying a Tcl element subcloned Tcl elements studied in one strain, Bergerac, undergo fre- from pCe2001 (1) into pNO1523 (9). The probe was labeled quent, spontaneous excision (1). Excision has been shown to by nick-translation to approximately 108 cpm/,mg with [a- occur in the somatic tissues, while in the germline Tcl ele- 32P]dCTP (New England Nuclear) following the procedure ments were found to be genetically stable (5). One product of of Maniatis et al. (10) except that DNase I (Sigma) was add- excision is a religated empty chromosomal site. Experiments ed to the reaction mixture (1 ng/ml). Probe, containing ap- carried out so far have not yet established whether the exci- proximately 3 x 107 cpm, was added to a 10-ml hybridization sion site is perfectly restored or has a small rearrangement volume after denaturation of the probe DNA at 100°C. (±50 bp). These findings led us to investigate the possibility Sucrose Gradients. DNA (approximately 100 jig) was lay- that the other product of excision was a free copy of the ered in 2 ml on the top of a 10-40% sucrose gradient in 1x transposable element. We show here that extrachromosomal SET buffer. The total volume was 37 ml. The gradient was copies of Tcl can indeed be detected in Bergerac, and we centrifuged in a Beckman SW27 rotor at 22,000 rpm, 15°C, present evidence regarding their structure. for 36 hr. One-milliliter fractions were collected from the top and dialyzed against 0.2x SET buffer overnight to remove sucrose, and the DNA was precipitated with ethanol and re- MATERIALS AND METHODS suspended in 100 ul of 0.2x SET buffer. Nematodes. Bergerac worms were strain EM1002, a clonal CsCl/Ethidium Bromide Density Gradients. DNA (approx- derivative of Bergerac (BO) from the Caenorhabditis Genet- imately 200 yg) was centrifuged to equilibrium in 1x SET ics Center. Bristol worms (strain N2) were from the labora- buffer containing CsCl (Kawecki-Berylco, Reading, PA) to The publication costs of this article were defrayed in part by page charge Abbreviations: kb, kilobase(s); bp, base pair(s). payment. This article must therefore be hereby marked "advertisement" *Permanent address: Institute of Biophysics, Academy of Sciences in accordance with 18 U.S.C. §1734 solely to indicate this fact. of China, Beijing, China. 4018 Downloaded by guest on September 23, 2021 Biochemistry: Ruan and Emmons Proc. NatL Acad. Sci. USA 81 (1984) 4019 S R R S R R R El (bp) 17 136 388 415 613 635 826 849 1386 1589 -IH-HF# I 54 ATG TAA 523 1342 *-- tcaaat A A atotgt-- A CAGT--- *ACTG T *--atattt T 1610 bp atgtoc *-- 1612 bp 1610 FIG. 1. Structure of Tcl. The structure of Tcl summarized here is taken from the sequencing studies of Rosenzweig et al. (3, 4). The positions of restriction sites used in the present work, of the terminal inverted repeats, of the ends of the element, and of the longest open reading frame are labeled in base pairs. The nucleotide position of the first base of a restriction site is given: EV, EcoRV; C, Cla I; S, Sal I; R, Rsa I. In the lower part of the figure the sequences at the ends of two independent chromosomal Tcl elements in Bergerac are shown. Lowercase letters indicate sequences of the insertion sites, which are present without a Tcl element in strain Bristol. Upper- and lowercase letters together indicate sequences found in Bergerac. The adenosine and thymidine at the ends of the Bergerac-specific DNA can be interpret- ed either as a two-base target site duplication or as a part of the Tcl element itself. Thus, the precise length of the element is not yet defined. a final density of 1.58 g/ml and ethidium bromide (Sigma) at 400 pg/ml. The total volume was 23 ml. The solution was centrifuged using an Oakridge tube in a Beckman 60 Ti rotor at 33,000 rpm, 15'C, for 60 hr. One-milliliter fractions were collected from the top, extracted with 2-propanol to remove ..7 ethidium bromide, and then treated as described above for Q I 1.1 sucrose gradients. 4.1 0 U C) RESULTS IQD Extrachromosomal Tcl DNA. Low molecular weight DNA /- / / / / homologous to transposon Tcl can be detected in Bergerac A/A / / B/ DNA preparations. This was accomplished by fractionating - 6.0 (II) DNA extracted from nematodes on an agarose gel and hy- - 4.36(11) bridizing it, after transfer to a nitrocellullose sheet, to a ra- - 6.0 (I) . diolabeled Tcl-specific probe (7). The probe hybridized to a. three low molecular weight species that migrated on the gel - 4.36(I) ahead of the high molecular weight chromosomal DNA (Fig. -. -2. 3 (M) 2A, lane a). We denote these species A, B, and C, in order of 2.1 (M) c .. _.a - 1.8 (M) c -> decreasing electrophoretic mobility. Comparison of their B 7- --d B -> to those known standards (Fig. 2A, lanes c, d, mobilities of A--3 .. and e) suggested that A, B, and C are, respectively, a 1.6-kb - 11 (II) supercoiled, linear, and relaxed circular molecules. Confir- I mation of this structural assignment and further evidence - 0.65(m) that these molecules are Tcl elements is presented below. The amount of extrachromosomal TcJ DNA in Bergerac is between 0.1 and 1.0 copy per cell.