CORE Metadata, citation and similar papers at core.ac.uk Provided by Caltech Authors Proc. NaMl. Acad. Sci. USA Vol. 74, No. 10, pp. 4382-4386 October 1977 Biochemistry Sequence relationship between long and short repetitive DNA of the rat: A preliminary report (long repetitive DNA/sequence homology/rat genome) JUNG-RUNG WU, WILLIAM R. PEARSON, JAMES W. POSAKONY, AND JAMES BONNER* Division of Biology, California Institute of Technology, Pasadena, California 91125 Contributed by James Bonner, August 8, 1977 ABSTRACT Long and short repetitive sequences of rat DNA fragments. Long repeated DNA has been used to drive whole can be isolated and characterized. Long [>1.5 kilobases (kb)] DNA tracers of various lengths to determine the interspersion sequences can be separated from short (0.2-0.4 kb) sequences by exclusion chromatography after renaturation of 4-kb DNA period of these sequences in long whole DNA. Our data are fragments to a repetitive Cot and digestion with the single- consistent with the model that short repeated sequences are strand-specific SI nuclease. (Cot is the initial concentration of present in the long repeated sequence elements in the rat ge- DNA in mol of nucleotides/liter multiplied by time in sec.) Long nome. repetitive DNA can be driven by an excess of whole rat DNA to measure its repetitive frequency. Excess long repetitive DNA can also be used to drive tracer quantities of either long (self- MATERIALS AND METHODS renaturation) or short repetitive DNA. Both the extent and the Preparation of DNA. Unlabeled DNA was extracted from rate of the renaturations are found to be similar, suggesting that rat ascites cells and labeled DNA was extracted from Novikoff long and short DNA fragments share sequences. When long DNA 3-4 kb long were prepared repetitive DNA is used to drive whole DNA tracers of various hepatoma cells.t fragments lengths, a 3.2-kb interspersion period is found. These data are by shearing DNA for 45 min at 7500 rpm in a Virtis 60 ho- consistent with the concept that short repetitive sequences are mogenizer (7) in 30 mM NaOAc (pH 6.8). DNA was sheared present within long repetitive DNA sequences in the rat ge- to 0.35 kb at 50,000 rpm in the Virtis 60 and in 66% glycerol (7). nome. The DNA was then passed over Chelex 100 (Bio-Rad), filtered, and precipitated with EtOH. Recent studies on repetitive DNA sequence organization Sizing DNA Fragments. Single-stranded DNA fragment (1-4, t) have revealed that there are two size classes of repeated lengths were determined by sedimentation through alkaline DNA sequences. In many organisms, interspersed sequences sucrose gradients. Isokinetic sucrose gradients (8) were formed 0.2-0.4 kb long comprise 50-70% of the repeated DNA while in SW41 tubes in 0.1 M NaOH using a Vmix of 10.4 ml, Cflask the remainder of the repeated sequences are substantially = 16.0% (wt/vol), and Cr, = 43% (wt/vol). Gradients were longer, more than 1.5 kb (1 kb = 1000 base pairs or nucleotides) centrifuged from 16 to 24 hr at 40,000 rpm. All tubes contained in length. The existence of two size classes of repeated sequences two markers of known molecular weight and samples were run poses a number of interesting questions. First, are long and short at least two times. Molecular weights were calculated from sequences kinetically different? This question is answered by sedimentation rates by the Studier (9) equations. measuring the repetition frequency and complexity of purified Preparation of Long Repeated DNA Fragments. DNA long and short repeated sequences. A second question is perhaps sheared to an average length of 4 kb was denatured and incu- more interesting: Do sequences that appear in short DNA se- bated at 65° in 0.3 M NaCI/10 mM piperazine-N,N'-bis(2- quences also appear in long sequences? This question is relevant ethanesulfonic acid) (Pipes) at pH 6.8 to an equivalent Cot of to the "integrator gene" hypothesis (5). Long "integrator" se- 5 using a factor of 2.31 for the reaction rate increase due to the quences also present throughout the genome as short inter- Na+ concentration. (Cot is the initial concentration of DNA in spersed sequences suggest batteries of control elements as mol of nucleotide/liter multiplied by time in sec.) After incu- proposed by Britten and Davidson (5, 6). bation, samples were diluted with an equal volume of 50 mM In this paper we present data concerning the kinetic pa- NaOAc/0.2 mM ZnSO4 at pH 4.2, and dithiothreitol was added rameters of long and short repeated DNA sequences and we to a final concentration of 5 mM. The final reaction mixture was examine the sequence relationships between the two classes of 0.15 M NaCl/5 mM Pipes/25 mM NaOAc/0.1 mM ZnSO4/5 sequences. Long and short repeated DNAs have been isolated mM dithiothreitol at pH 4.4. by nuclease digestion and agarose A-50 fractionation. Long DNA samples were incubated with S1 nuclease at 370 for 45 repeated DNA has been driven by whole DNA to determine min and the reaction mixture was chilled on ice and made repetition frequency, self-renatured to determine its com- 0.12 M in phosphate buffer. Duplex DNA strands were sepa- plexity, and used to drive short repeated DNA to examine rated by hydroxyapatite chromatography, eluted with 0.5 M cross-renaturation. We do not find any significant kinetic dif- phosphate buffer, and chromatographed on Bio-Gel agarose ference between the long repeated DNA elements and all re- A-50 (Bio-Rad) as described by Britten et al. (7). peated rat DNA sequences by these criteria. In addition, there The long unlabeled DNA used in the self-reaction and is evidence for some sequence homology between long and short long/short cross-renaturation and interspersion experiments repeated DNA sequences. A more sensitive experiment has been used to look for short sequences internal to long repeated DNA Abbreviations: kb, kilobase (1000 base pairs); Cot, initial concentration of DNA in mol of nucleotide/liter multiplied by time in sec; Pipes, The costs of publication of this article were defrayed in part by the piperazine-N,N'-bis(2-ethanesulfonic acid). payment of page charges. This article must therefore be hereby marked * To whom reprint requests should be addressed. "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate t Pearson, W. R., Wu, J. R., and Bonner, J. (1977) Biochemistry, in this fact. press. 4382 Biochemistry: Wu et al. Proc. Natl. Acad. Sci. USA 74 (1977) 4383 ~=0.9 00 0 r0 250 50P (0 0 < ~~26 39 44 40 50 60 70 - 80 90 100 TEMPERATURE, 0C FIG. 2. Melting experiment of repetitive DNA duplexes of dif- ferent fragment lengths. Repetitive DNA duplexes were isolated and fractionated as described in the legend of Fig. 1. Fractions 26, 39, and 44 were melted in a spectrophotometer equipped with a thermal cu- 10 20 30 40 50 60 70 FRACTION NUMBER vette. The temperature was raised at a rate of 0.50/min to 980. +, Fraction 44; 0, fraction 39; X, fraction 26; 0, native DNA refer- FIG. 1. Profile of rat repetitive DNA duplexes on agarose A-50. ence. DNA sheared to 4 kb was denatured and renatured to Cot of 5, di- gested with S1 nuclease, and bound to hydroxyapatite. The double- strand fraction (15%) was eluted with 0.5 M phosphate buffer and RESULTS chromatographed on Bio-Gel agarose A-50. The size of the fraction indicated was determined by alkaline sucrose sedimentation. The When 4-kb DNA is renatured to Cot of 5 and digested with SI fractions marked were used for the melting experiment in Fig. 2. nuclease and the duplexes are sized on agarose A-50, repetitive sequences are fractionated into two classes.t A typical column was isolated from 10 mg of 4-kb DNA. The DNA was denatured profile is shown in Fig. 1. Long (4 kb) fragments were used to for 5 min at 100°, incubated to Cot of 5 (13 min), and digested minimize creation of short fragments by random shear and with 250,ul of an SI nuclease preparation (the gift of Francine overlap of long repeated sequences. The incubation was carried Eden). This nuclease preparation has been extensively char- out to Cot of 5 to prevent renaturation of single copy sequences. acterized (10). The concentration used (25,ul/mg) corresponds (Long fragments have a higher effective Cot because of the to a digestion estimate of 0.85 which is 1.7 times the standard fragment length.) The long repetitive DNA excluded from incubation. After hydroxyapatite chromatography, 17% of the agarose A-50 is an average of 1.5-2.0 kb long. The short in- DNA was found duplexed (30 A20 units), and this duplex DNA cluded repetitive DNA is 0.2-0.5 kb long. was passed over agarose A-50. Fifty-five percent of the DNA To make certain that long duplexes did not contain single- was excluded. strand tails, we melted duplexes fractionated on A-50. A sample The 3H-labeled DNA used as tracer in the repetition fre- melting experiment of three fractions indicated in Fig. 1 is quency and cross-renaturation experiments was prepared as shown in Fig. 2. All fractions show more than 90% of native above. In this preparation, the enzyme-to-DNA ratio was 25 hyperchromicity. The melting temperatures range from 2.50 native for the material to 150 below Ail/mg; 19% of the DNA was bound to hydroxyapatite, and 47% below long (excluded) of the repetitive duplexes were excluded from agarose.