Characterization of the most Rapidly Renaturing Sequences ...

J. Mol. Biol. (1973) 81, 299-325

Characterization of the most Rapidly Renaturing Sequences in Mouse Main-band DNA

THOMM R. CECH, ALITA ROSENFELD AND JOHN E. HEARST

Departmertt of Chemist y University of California

Berkeley, Calif 94720, U.S.A.

(Received 27 April 1973, and in revised form 5 September 1973)

The most rapidly renaturing sequences in the main-band DNA of Mwr rnusc&.~, isolated on hydroxyapatite, are found to consist of two discrete families: a presumed “foldback” DNA fraction and a fraction renaturing bimolecularly. The latter family, which we call “main-band hydroxyapatite-isolated rapidly renaturing DNA”, has a kinetic complexity about an order of magnitude greater than that of mouse satellite DNA. It shows about twice as much mismatching as renatured mouse satellite, as judged by its thermal denaturation curve. In situ hybridization localizes the sequences to all chromosomes in the mouse karyotype, and to at least several regions of each chromosome. The in situ result and solution hybridization studies eliminate the possibility that the main-band rapidly renaturing DNA is composed of mouse satellite sequences attached to sequences of higher buoyant density. Nuclease S1 digestion experiments disclose that even at low molecular weight there are unrenatured “tails” attached to the rapidly renaturing sequences. When the main-band DNA fragment size is increased the amount of rapidly renaturing sequences remains constant, but the amount of attached tails of unrenatured DNA increases as judged by S1 nuclease digestibility, hyperchromicity snd buoyant density. It is concluded that at least 5% of the mouse genome is composed of segments of the rapidly renaturing sequences averaging about 1500 base pairs, alternating with segments of more complex DNA averaging about 2200 base pairs. This interspersion of sequences is compared to that found in several other organisms. The properties of the foldback DNA are similarly investigated as a function of DNA fragment size.

1. Introduction

The genomes of higher organisms contain large amounts of repeated DNA sequences. Some ideas about the function and evolution of these repeated sequences have been derived from investigations of their renaturation rates (Britten t Kohne, 1966) and sequences (Southern, 1970), as well as tests of homology between repetitious sequences of different species (Flamm et al., 1969a; Hennig et al., 1970; Rice, 1972). In order to determine what role the rapidly renaturing sequences might have in gene regulation and “chromosomal housekeeping” (Walker et al., 1969) in higher organisms, it has become important to look at the arrangement of these sequences in relation to those of higher complexity. At the molecular level, high molecular weight DNA has been examined for the presence on the same fragment of sequences with different renaturation rates (Britten & Smith, 1970; Kram et al., 1972). At the chromosome level, in situ hybridization (John et al., 1969; Pardue & Gall, 1970) has proved to be a valuable technique for determining the distribution of rapidly renaturing DNA.

299

300 T. R. CECH, A. ROSENFELD AND J. E. HEARST

One type of sequence arrangement is shown by mouse satellite, the very highly reiterated “simple sequence” DNA of Mus nzuscu1u.s. It occurs in stretches that are long relative to DNA normally isolated in the laboratory (Bond et al., 1967; Flamm et al., 1969a). The sequences occur in the heterochromatin of both interphase cells (Yasmineh & Yunis, 1970) and metaphase chromosomes (Jones, 1970; Pardue $ Gall, 1970).

In Drosophila melanogmter there are several families of rapidly renaturing DNA that localize to the chromocenter of polytene salivary chromosomes. Some of the repeated sequences (Gall et al., 1971; Peacock et al., 1973) seem to occur in long stretches like mouse satellite. Another family (Botchan et al., 1971) shows a different sequence arrangement : segments of rapidly renaturing DNA alternate with segments of more complex DNA (Kram et al., 1972). In vivo RNA contains sequences complementary to the more complex DNA, but not to the segments of highly reiterated DNA (Botchan, 1972). This suggests the possibility of transcriptional control of the more complex DNA (gene) by the simple sequence DNA “spacers”. The heterochromatic arm of the X chromosome in D. hydei also contains both rapidly renaturing and more complex sequences (Hetig, 1972).

We thought it worthwhile to examine the most highly reiterated sequences of the main-band DNA of Mw mu.scuEus for the occurrence of rapidly renaturing DNA acting as a spacer between segments of more complex DNA. Such a study would help to determine the generality of this type of sequence arrangement, and, if it were present, the known techniques of mouse tissue culture (e.g. cell cycle synchroniza- tion) could be applied to the question of the biological function of such an arrangement of DNA. Preliminary observations in this laboratory (Kram et al., 1972) sug- gested that even at fairly small fragment size some mouse satellite DNA might be attached to sequences of main-band buoyant density, as judged from the cesium chloride density gradient profile of renatured total mouse DNA isolated with hydroxyapatite. We now present evidence that this heavy-density peak of hydroxyapatite-

isolated rapidly renaturing DNA (h.a.r.r.DNA) is not of the mouse satellite sequence, but is from mouse main-band.

2. Materials and Methods (a) DNA preparation

DNA was extracted from SVT2 mouse tissue culture cells, an aneuploid cell line. The cells were grown in Dulbecoo’s modified Eagle’s medium supplemented with 2% calf serum (both obtained from Grand Island Biological). For labeled DNA, 1 to 5 @i [3H]thymidine, O-15 PCi [14C]thymidine, or 20 &i [3aP]phosphoric acid (Schwarz-Mann) was added per ml during log phase, at least one cell cycle before DNA extraction, Mono- layers were washed several times with isotonic buffer, after which the cells were removed from the plates by trypsin and swollen in hypotonic buffer. Nuclei were freed by homo- genizing in O-025 M-KCl, 0.05 M-Tris (pH 7*4), 0-006 aa-MgCls, 0.25 M-sucrose, 0.1% Triton X100. After 2 or 3 homogenization steps, each followed by centrifugation at 500 g for 5 to 10 min in an IEC PR6 centrifuge, the suspension of purified nuclei was heated to 65°C and lysed by the addition of recrystallized Sarkosyl (NL97, Geigy) to a final concentration of 2%. After 10 min at 65% the solution was diluted to 200 to 400 pg/ml, made 1 M in N&lo, and 1% in detergent, extracted with chloro- fonn/isoamyl alcohol (24: l), ethanol precipitated, and resuspended by rotation at 4°C in O-01 x SSC (SSC is 0.15 m6-NaCl, 0.015 M-sodium citrate, pH 7.0). After incubation with heat-treated ribonuclease (Calbiochem 5 times crystallized pancreatic RNsse; 60 pg/ml for 30 min at 37’C) snd pronase (Calbiochem B grade; 250 pg/ml for about 4 h at

MOUSE MAIN-BAND RAPIDLY RENATURING DNA 301

37°C after preincubation at 10 mg/ml for 2 h at 37”C), the DNA was extracted several times with ohloroform-isoamyl alcohol and again ethanol precipitated.

Alternatively, DNA was extracted from SVTZ whole cells scraped from the plates into CAPS/EDTA buffer (0.001 M-cyclohexylaminopropane sulfonic acid (Calbiochem), 0.10 M- EDTA, pH 10.5). The homogenized cell suspension was lysed with detergent at 66°C and the DNA purified aa described above. Native molecular weights greater than 4 x lo7 were easily obtainable by this method. The yields of h.a.r.r.DNAt and “foldback” DNA from the whole cell were indistinguishable from those obtained from the nuclear DNA.

(b) Shea&g of DNA and deterrnin&on of molecular we@hta

DNA of single-strand molecular weight 2 x lo6 to 5 x lo6 was prepared by forcing the solution through a 27 gauge needle with maximum thumb pressure; the resulting molecular weight, was an increasing function of syringe size. Sonication (Biosonic, 200 mA) of DNA in 0.05 M-phosphate buffer in an ice bath while bubbling N, through the solution produced average single-strand molecular weights of 0.7 x lo5 to 1.8 x 105. After sonication, solutions were dialyzed against 7.5 x SSC for rapid removal of oligonucleotides.

Single-stranded DNA molecular weights were determined by boundary sedimentation from the measurement of 8if13 m 0.9 M-NaCl, 0.1 M-NaOH, and native molecular weights from the measurement of a.$'? m 1.0 M-NaCl, 0.01 M-Tris, pH 8.0. Correction to szO,w and calculation of molecular weights were performed according to Studier (1965).

(c) Isolation of rapidly renaturing sequences

After dialysis against 0.05 M-phosphate buffer, pH 6.8, DNA solutions were denatured with NaOH at room temperature, warmed to 60°C, and neutralized with NaH2P04 such that, the final buffer was 0.12 M-sodium phosphate (Kram et al., 1972). After renaturation at 60°C for the desired Cot (moles DNA phosphate 1-l s), the solution was added to a slurry of hydroxyapatite (synthesized according to Levin (1962)) in O-12 M-phosphate buffer in a 70°C water bath. The slurry was eluted stepwise 4 or more times with 0.12, 0.16 and 0.45 M-phosphate buffers, each elution consisting of resuspension (Vortex Genie mixer), 1 min temperature equilibration in the 70°C bath, centrifugation in a clinical centrifuge at 70°C. and decanting of the supernatant. The first two or three 0.45 M eluanta were combined as h.a.r.r.DNA; they contained 86 to 95% of the total DNA eluted at 0.46 M-phosphate buffer. The use of radioactive DNA was found essential for accurate determination of h.a.r.r.DNA yield, defined as the total radioactivity eluted at 0.45 M- phosphate buffer divided by the total recovered at all three phosphate concentrations. Between 95 and 100% of the DNA cts/min put on the slurry were normally recovered, with no apparent dependence on the molecular weight of the material being fractionated.

Two problems occur with the use of hydroxyapatite at high molecular weights. (a) AS discussed by Flamm et al. (1969a), high molecular weight DNA is sheared by hydroxyapatite fractionation at high temperature. We have measured a decrease in single-strand molecular weight from 11-l x 10” to 7.0~ lo6 after alkali denaturation and h.a.r.r.DNA isolation. (b) A slightly greater percentage of the material is eluted at 0.16 M-phosphate buffer than with low molecular weight DNA. The 0.16 M-phosphate elution is used to wash away poorly formed duplexes. In a typical experiment 1.8% of the starting material was &ted in the 0.16 M-phosphate buffer washes with sonicated DNA, 2.4% with needla- sheared DNA, and 2.9% at high molecular weight.

(d) Equilibrium density-gradient centtifugation

Analytical CsCl centrifugation was performed at 35,000 revs/min in titanium double- sector centerpieces (Hearst & Gray, 1968) in the Spinco model E. Harshaw optical-grade CsCl was used. Buoyant densities were measured relative to M~crococcw, lysodeikticus marker DNA, which had a measured density of 1.733 g/ml relative to Eacherichti coli DNA, p0 = 1.710 g/ml. Density differences between bands were calculated from the density gradient (+/dr)buoyancy = 9.35 x IO-I* w2 ? according to Schmid & Hearst (1971).

Preparative isolation of mouse satellite and main-band fractions was performed in

t Abbreviation used: h.a.r.r.DNA, hydroxyapatite-isolated rapidly renaturing DNA.


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FIQ. 1. SVT2 mouse nuclear DNA banded in neutral CsCl density gradients. M. lyeodekkticua DNA, p. = 1.733 g/ml, was added as a density marker. (a) Unfractionated DNA (1.6 K); (b) mouse satellite fractions from a preparative Ag+ /Cs,S04, gradient, Ag+ removed by dialysis; (c) main band (2.2 H) isolated as in (b); (d) 4 tunes expansion of soan (c) to show residual mouse satellite in the main-band preparation.

Ag+/Cs,S04 gradients according to Cornea et al. (1968) for 64 h at 36,000 revs/min in the Spinco fixed-angle 60 Ti rotor. Purity of fractions was determined by analytical C&l gradient centrifugation (see Fig. 1) after removal of Ag+ by extensive dialysis against 1-O M-N&~, 0.01 M-EDTA, 0.06 rd-phosphate buffer, pH 68.

Renatured satellite DNA was separated from main-band h.a.r.r.DNA by CsCl density- gradient centrifugation in the Spinco fixed-angle 66 rotor for 84 h at 36,000 revs/mm The use of polyallomer tubes and addition of 10 d per tube of 20% recrystallized Sarkosyl or sodium dodecyl sulfate were found to decrease the absorption of renatured DNA to the tube during cent&fug&ion.

Radioactivity was measured by dispersing a portion in a mixture of 1 ml water plus 10 ml scintillation solution (1 part Beckman Triton Xl00 to 2 parts Amersham-Searle PPO-POPOP fluor in toluene) and counting in a Beckman 200 liquid-scintillation oounter.


C&l solution (4 to 70 ~1) from a preparative density gradient or 160 d of O-12 M-phosphate buffer from a hydroxyapatitc eluant were routinely counted in this m&nner.

(f) Bwe composition 3aP-labeled SVT2 DNA was hydrolyzed by pancreatic DNase and venom phos-

phodiesterase according to Schildkraut & Maio (1908). Mononucleotides were separated by DEAE-paper chromatography according to Furlong (1967). After autoradiography the paper was cut into l-in squares and counted in Beckman TLA fluor in toluene. Some radioactivity (1 to 3%) remained at the origin.

(g) Nudeme SI digestion

The single-strand specific nuclease Si was obtained from Aa~erg~us otisae crude a-amylase (S&n&) according to the procedure of A. Skoultchi t D. Housman (personal communication). The procedure involved dissolving the crude a-amylase in 0.02 M-

potassium phosphate buffer (pH 6*9), 5% glycerol, removing insoluble material by centrifugation, chromatographing on DEAE-cellulose with a linear O-0 to O-4 M-N&~ gradient, and asmying the fractions for single-stranded and double-stranded DNA nuclease activity. A DNA sample to be treated with S1 was dialyzed against 0.10 M-N&~, then made 0.0001 M in ZnS04 and 0.025 Y in potassium acetate buffer giving a final pH of 4.5. Normally the radioactive sample being assayed gave a single-stranded DNA concentration of less than 6 pg/ml, in which case 20 rg of heat-denatured calf thymus DNA was added/ml to prevent the anomalous reaction kinetics observed by Sutton (1971) at low substrate concentration. The 0.2 to 1.0 ml solution wa8 equilibrated at 45°C and 76 units (as defhied by Sutton) of Si enzyme in 60% glycerol were added per 20 pg of single-stranded DNA. Samples (usu&y 100 r-1) were collected at times from zero to 30 or 60 min after nucleate S1 addition snd precipitated for 10 min in ice with the addition of an equal volume of 20% trichloroacetic acid and 1 pg cti thymus DNA as a carrier. The precipitates were collected on Whatman GF/C filters by suction filtration. These were dried and counted in 10 ml Beckman TLA fluor in toluene.

(h) In situ hyb&o%urttin 3H-labeled complementary RNA was transcribed in w&o with E. coli polymerage as

described previously (Kram et al., 1972), except that the 0.25 ml reaction mixture was 1 x lo-* M in [3H]ATP (22.7 Ci/mmol) and 4x lo-* M in GTP, CTP, and UTP. The calculated specific activity was 4.7 x IO7 disintslmin per pg. In & hybridization was performed according to Gall & Pardue (1971) with few modi&&ions. Nuclei and mets- phase chromosomes fixed on glass slides were treated with pancreatic RNsae, washed in 2 x SSC, and deproteinized in 0.2 M-KC1 for 30 min at room temperature. Alkali de- natumtion was for 2 min in 0.05 M-Ns,OH at room temperature. In preparing acid- denatured chromosomes, the slides were deproteinized with HCl and treated with RN-e, followed by a second 0.2 M-HCl treatment for 30 min at room temperature. In this procedure it may be the flame drying of the slides tmd/or the 60°C incubation during hybridization that denatures the DNA rather than the HCl treatment (Comings et al., 1973).

3. Results

(a) Detection of non-satellite rapidly renabring DNA in mou.se

The rapidly renaturing sequences of ilfus musedus DNA, isolated on hydroxyapatite at a C,t of 6 x 10-s, form two buoyant density classes when centrifuged to equilibrium in a CsCl gradient. The lighter peak shown in Figure 2(a), containing 8 to 10% of the total cell DNA, is renatured mouse satellite; its buoyant density and high degree of aggregation correspond to that seen when isolated satellite of this molecular weight is denatured and renatured (Bond et al., 1967; see also Fig. 2(b)).


FIQ. 2. Repidly rermturing mouse DNA banded in neutral C&l density gradients. (a) Total mouse DNA and (b) isolated mouse satellite DNA were sonicated to a single-strand moleculrtr weight of 1 to 2 x 106. The total DNA was then alkali-denatured and renatured 8t 00°C in 0.12 M- phosphate buffer to C&=6x 10-e, and the reassocieted DNA ~8s eluted from hydroxyapetite. The mouse satellite (b) was heat-denatured and renatured as above but without the use of hydroxyepetite. M. lyaodeikticus was used 8s the marker DNA.

The broad peak at p. = I.707 g/ml is always present in h.a.r.r.DNA isolated from sonicated mouse liver DNA or cell line 3T3 or SVT2 nuclear or whole cell DNA. The renaturation of sonicated, isolated satellite DNA does not produce such a peak in a CsCl gradient, as shown in Figure 2(b). Furthermore, when mouse main-band DNA, separated from satellite either by Ag+ /Cs,SO, or repeated CsCl gradients, is sonicated, denatured, renatured to a Cot of 6 x 10m2, and fractionated on hydroxyapatite, 5 to 6% of the main band elutes as renatured DNA in the 0.45 M-phosphate buffer fractions. This material bands in a shallow peak at a buoyant density of 1.707 g/ml, identical to the density of the heavy peak of h.a.r.r.DNA from total sonicated DNA of mouse shown in Figure 2(a).

The Cot of 6 x 10d2 was chosen to allow renaturation of sequences with the complexity of mouse satellite and to exclude “intermediate” sequences repeated less than lo4 times. Cot curves of mouse main-band DNA show an early transition ending at a Cot of about lo-‘, but the rapidly renaturing and intermediate sequences in this genome are not sharply separated in the Cot curve.

(b) Two cla.sse~ of rapidly renaturing DNA in mouse main-hand

(i) Foldback DNA

The DNA of many prokaryotic and eukaryotic organisms contains a small fraction which, when denatured and then placed in renaturation conditions, re-forms a double-


stranded structure extremely rapidly (Alberts & Doty, 1968; Walker & McLaren, 1969; Britten & Smith, 1970). B&ten & Smith cite the “folding-back” of a single strand of DNA containing a sequence inversion as a possible origin of this fraction in calf DNA. We will use the term “foldback DNA” to designate DNA that binds very rapidly to hydroxyapatite by processes other than bimolecular renaturation. (The molecular mechanism denoted by the term foldback is only one of several possibilities, although it does seem the most consistent with the data presented in this study.)

In order to determine how much of the 5 to 6% rapidly renaturing DNA found in the main band of mouse might be foldback DNA, hydroxyapatite isolations were performed at 70°C immediately after neutralization of alkali-denatured main band DNA. The 0.45 M-phosphate eluant yielded the foldback fraction, defined as those molecules that formed double-stranded regions in the first I.25 to 2 minutes of a 20 to 60-minute renaturation experiment. Two minutes corresponded to a C,t of 2 x low3 to 6 x 10e3t. The supernatant was immediately returned t,o the 60°C bath to allow renaturation of main-band h.a.r.r.DNA, and after a C,t of 6~ low2 had been reached this material was fractionated on a second hydroxyapatite slurry in the usual manner. In three such hydroxyapatite isolations performed on different samples of 3H-labeled, sonicated main-band DNA, 2.91,tO.4% (maximum deviation) of t,he DNA was collected as foldback, and 2*5fO*l% as main-band h.a.r.r.DNA. Hereafter main-band h.a.r.r.DNA will refer to material from which foldback DNA has been removed.

The CsCl density profiles of these two kinetic fractions are shown in Figures 3(a) and (d). When allowed to renature for two to three days in high salt (equivalent C,t M 50)f, the main-band h.a.r.r.DNA fraction forms high molecular weight aggregates at densities of 1.691 and 1.703, g/ml (Fig. 3(e)), the lighter of which is presumed to be residual mouse satellite (see Figs l(d) and Z(b)). Such aggregates, presumably the result of complementary “tails ” of simple sequence remaining after the primary bimolecular renaturation, have been observed with several simple sequence DNAs (Waring t B&ten, 1966; Flamm et al., 19693; Botchan et al., 1971). In contrast, the foldback fraction still bands very broadly in CsCl after the high C,t incubation (Fig. 3(b)). The failure of the foldback DNA to aggregate is consistent with it originat ing from inverted DNA sequences, but is not consistent with a very simple sequence renaturing bimolecularly at COt1,2 < lo- 4. Its broad density distribution reflects the low molecular weight of the sonicated DNA from which it is isolated. (Super- imposed on the broad foldba.ck band is a highly aggregated peak at renatured satelhte density; sometimes a highly aggregated main-band h.a.r.r.DNA peak is also seen. A kinetic method of isolating foldback DNA cannot eliminate all products of bimolecular renaturation, though their concentration can be reduced t,o a small fraction of t,hat of the foldback DNA.)

When the foldback DNA is again denatured and chromatographed as before on hydroxyapatite (C,t m 10m4 with C,, based on foldback concentration), a variable but significant amount of the DNA is recovered as single-stranded DNA in t.he 0.12

t Co is based on the total main-band DNA ooncentretion. A class of DNA comprising 6% of the total would therefore renature to a Cot of 6 x 10m5 when the total main-band DNA haa renatured to a C,t of 1 x 10m3.

2 Equivalent C,t is defined w C,t corrected for salt concentration (if not 0.12 M-phosphate buffer) and for single-strand molecular weight (if not sonicated). Corrections are according to Wetmur t Davidson (1968), with the assumption of negligible increase in renaturation rate above 3.2 M monovalent cation concentration.

21


and 0.16 M eluants. In three experiments, the amount of foldback recovered as double-stranded DNA after rechromatography has varied from 40 to 90% of the original foldback material. As expected from the low C,t, the rechromatography almost completely removes the mouse satellite and any main-band h.a.r.r.DNA that contaminate the foldback (Fig. 3(c)). However, the removal of these two second- order renaturing families can account for less than one-fourth of the foldback that

,(b)

FIG 3. An8lytical ultracentrifugation in neutral C&l of rapidly renaturing DNA from mouse main-band. Ag+/Cs,S04-isolated main-b8nd DNA was sonioated to l-8 x lo5 mol. wt, single- stranded. Foldback fraction: (8) isolated 8t Cot < 3x 10e3; (b) same sample as shown in (8) aggregated (incubated at 60°C in high selt to an equivalent Cot of about 60); and (a) denatured again and collected on hydroxyapatite after renaturation to 8 Cst of 2 X 10V4, and then re8ggre- gated. Main-band h.e.r.r.DNA: (d) isolated at Cot = 6x 10-s; (e) aggregated 8s in (b); 8nd (f) banded preparatively in CsCl to remove satellite peak, rechromatographed on hydroxyapatite at C,t = 2 x 10-c, and reaggregated.

elutes as single-stranded DNA during rechromatography. This loss of the foldback characteristic is in agreement with a previous observation (B&ten & Smith, 1970) on the instability of foldback DNA.

That the foldbaok DNA is not an artifact of the hydroxyapatite isolation, but is actually changing from a single-stranded to a double-stranded form when the solution is neutralized, is shown by the optical renaturation study in Figure 4. A large fraction of the hyperchromicity is recovered rapidly (Cot < 2 x lo-*), as fast as the temperature jump is accomplished. Because the sample is in 0.06 M-phosphate buffer, a salt concentration that slows bimolecular renaturation by a factor of ten


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FIQ. 4. Optical renaturation kinetios of isolated main-band rapidly renaturing DNA frections. Foldbeok DNA (--a--@--) isolated et C,t = 4 x 10-B and main-bend h.a.r.r.DNA (-B-B-) isolated et C,,r = 6X 10-s. both from sonioated Ag+/Cs,SOl-prepared main-band DNA. Each symbol ( n ) represents the average value of two successive determinations on the same sample of main-bend h.a.r.r.DNA.

The experiments1 procedure hes been described (Botchnn et al., 1971). The h.a.r.r.DNA was dialyzed 8gninst 0.12 M-phosphate buffer, pH 6.8, the foldb8ok DNA against 0.06 na-phosphate. Samples were denatured for 3 to 6 min at 92 to lOO”C, the observed hyperohromism (corrected for thermal expansion) being 27*2% for the foldbsok and 26& 2% for the h.e.r.r.DNA. At t = 0 the temperature ~8s ohanged to 60°C for the h.e.r.r.DNA or 66°C for the foldback DNA, 30 Y being required for npproximete thermal equilibrium. (The first 3 time-points on the foldbnck curve end the first point on the h.a.r.r.DNA curve are therefore expeoted to be nrtifioi8lly high.) The nbsorbenoe at 60°C before the sample was melted WM 0,200 o.D.~&~ for the foldback, O-161 O.D.Fgm for the h.a.r.r.DNA. A0 is the absorbance at 100°C at time zero, oorreoted according to Fig. 6(a) for hypoohromism due to the bese sta&ing of denatured DNA upon reducing the temperature to 60°C. A0 ~8s 0.243 O.D.yLm for the foldback, 0.181 O.D.Tti= for the h.a.r.r.DNA. Am is the native absorbance of the DNA, oaloulatad aa &/1.36.

The solid line ( ) through the m&n-band h.a.r.r.DNA is the theoretioal ourve for two oomponenta, one (62.6% of the fraction) with a second-order rate constant ka of 296 1 mole-l 880-l (C&s = 6.8~ lo-s), plus a 37.6% component not renaturing during the course of the experiment. A oomparnble fit to the data oan be obtained by assuming a 60% component with a kp of 436 (C,,t,/, = 4.6~ 10e9) plus a 60% component with 8 ka of 26 (Cotllz = 8.0~ 10-a). Further increasing the estimate of the proportion of the second component fits the data less well. The dashed line (-----) through the foldbaak DNA date is not theoretical, but simply connects the points. The horizontal lines (( ) h.e.r.r.DNA; (-----) foldback DNA) give the expeoted degrees of completion for the reactions, onlouleted 8s (A’--A m)/(Ao-A 61)‘, where A’, is the absorbanoe of the renatured sample at 6O“C before the experiment.

(Wetmur & Davidson, 1968), the renaturation occurs before an equivalent Cot of 2x 10w6. (The renaturation curve in 0.12 M-phosphate was found to be very similar to that in 0.05 M-phosphate buffer.) Of the absorbance increase observed upon melting the foldback DNA, 40% was not recovered during the renaturation, and hydroxyapatite chromatography of the sample after the kinetics experiment resulted in 42% not being recovered in the double-stranded fraction. These results are consistent with partial loss of the foldback characteristic due to single-strand nicking of the DNA.

(ii) Main-ban43 h.a.r.r.DNA

Removal of the foldback DNA by the procedure described above and removal of

308 ‘I?. R. CECH, A. ROSENFELD AND J. E. HEARST

aggregated mouse satellite? by preparative CsCl gradient centrifugation resulted in the recovery of a homogeneous family of DNA sequences shown in Figure 3(f). The DNA was then denatured, renatured, and rechromatographed on hydroxyapatite, which served as a final purification step and more importantly disrupted the high molecular weight aggregates that had facilitated the removal of mouse satellite DNA in the preparative C&l gradient. These aggregates would have affected the results of the melting experiments and the S, nuclease digestion (to be discussed). This purified main-band h.a.r.r.DNA (1.8 x lo5 mol. wt single-stranded) is a family of sequences with the following properties.

(1) It forms a single peak in CsCl density gradients with a reproducible density of 1.705 g/ml, decreasing to 1.703 g/ml after additional renaturation to high cot.

(2) The renaturation kinetics (Fig. 4) can be interpreted as a single, rapidly renaturing kinetic class following second-order renaturation with Cot1/2 = 6.8 x 10e3. A simple interpretation of the renaturation results would be to postulate a sequence of 2800 base pairs repeated about 30,000 times, but the basic sequence from which this sequence was derived could be much shorter (Southern, 1971).

(3) Although the main-band h.a.r.r.DNA shows some hyperchromicity at lower temperatures, most of the melt occurs as a fairly sharp transition over a range of about 15 deg. C with a T, of 77°C (Fig. 5). If the native T, of this DNA were 86°C (the same as that of native total mouse DNA), the decrease in T, upon renaturation (AT,) would be 9 deg. C. This corresponds to about 6% mismatching (Ullman & McCarthy, 1973), or perhaps as much as 13% (Laird et al., 1969). There is about twice as much mismatching as in renatured mouse satellite DNA, which has a AT, of 4 to 5 deg. C (Fig. 5(a) ; see also Flamm et al., 1967). The fact that the T, is dependent upon base composition and is anomalously high for native mouse satellite DNA (Bond et al., 1967) adds to the uncertainty of the above estimate of mismatching.

(4) The base composition of main-band h.a.r.r.DNA, 40*6&0*2% G + C (average of three measurements f standard deviation), is very similar to that of the main-band DNA from which it is isolated: 41.1 f0*3% G + C (10 measurements).

(c) Attachment of more complex DNA as a function of fragment size

As shown in Figure 6, the main-band h.a.r.r.DNA and foldback DNA could be distributed in the genome in several possible patterns : (a) in short, isolated segments; (b) in long, uninterrupted segments, as seems to be the case for mouse satellite

t The peak with po= 1691 is assumed to be renatured mouse ‘satellite not only beoause of its buoyant density, but also because its emount veries between different preparations of main-bend DNA. For instance, 1.2% of the main-band DNA of Fig. l(d) banded at the density of renatured satellite efter h.a.r.r.DNA isolation, about that expeoted from the small amount of satellite remaining in the native DNA. The p0=1.691 peak of Fig. 3(d) end (e) beoomes undetectable in main-band h.a.r.r.DNA isolated from mouse main-band whioh has been recycled on a preperative CsCl gradient following the Ag+/CsaSO, gradient. The CsCl purificetion is undesirable, however, because it involves seleotive removal of light-side main bend along with the eetellite, reducing the yield of m&-band h.a.r.r.DNA.

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Opt

ical

m

eltin

g cu

rves

of

var

ious

fra

ctio

ns

of m

ouse

D

NA.

(a

) To

tal

unsh

eare

d na

tive

SVT2

m

ouse

D

NA

(-A-A

-);

rena

ture

d m

ouse

sa

tel-

lite

DN

A,

2 x

10s

mol

. w

t. si

ngle

-stra

nded

(-m

-m-);

de

natu

red

mou

se

DN

A w

ith

the

porti

on

rena

turin

g be

fore

C

,t =

1 re

mov

ed

(-O-e

--).

(b)

Mai

n-

band

h.

s.r.r

.DN

A (w

ith

fold

back

an

d m

ouse

sa

tellit

e re

mov

ed)

isol

ated

at

eq

uiva

lent

C

ot =

6

x 10

m2

et

diffe

rent

si

ngle

-stra

nd

mol

ecul

ar

wei

ghts

: 1.

8 x

lo5

(--m

--m--)

; l-7

x 1

0s (

-a-@

-);

and

1.0~

10

’ (--

A--A

--).

(c)

Purif

ied

fold

back

fra

ctio

ns

from

m

ain-

band

D

NA;

sy

mbo

ls

oorre

spon

d to

m

olec

ular

w

eigh

ts

in

(b).

Sam

ples

ha

d a

conc

entra

tion

of

0.08

to

0.68

0.D

.T’~

“” an

d w

ere

dial

yzed

ag

ains

t 0.

12 M

-pho

spha

te

buffe

r, pH

6.

8. T

empe

ratu

re

incr

ease

w

as

et t

he r

ata

of 1

6 de

g. C

/h,

abso

rban

ce

and

tem

pera

ture

be

ing

mon

itore

d by

a B

eckm

an

DU

sp

ectro

phot

omet

er

equi

pped

w

ith

a G

ilfor

d au

tom

atic

ab

sorb

ance

re

cord

er.

Cur

ves

are

norm

aliz

ed

to h

igh-

tem

pera

ture

ab

sorb

rsno

e an

d co

rreot

ed

for

ther

mal

ex

pans

ion.

310 T. R. CECH, A. ROSENFELD A-ND J. E. HEARST

(0)

/

--__-_----_- _ - - __ - - _ - -- _

\

FIQ. 6. Models for the arrangement of mouse main-band h.a.r.r.DNA. In model (a) each stretch of h.e.r.r.DNA ( ) is isolated from others by long stretches of more complex DNA (-----). In (b) the h.a.r.r.DNA occurs in large blocks, perhaps only one block on each ohromatid. In (c) the h.a.r.r.DNA is clustered, with stretches of more complex DNA interamlating the rapidly renaturing sequences. The wavy lines indicate regions of the genome edjsoent to those containing h.a.r.r.DNA.

(Waring & B&ten, 1966; Pyeritz et al., 1971) or (c) in a clustered arrangement, alternating with DNA of higher kinetic complexity, as is the case for a h.a.r.r.DNA in D. rnelanogaster (Kram et al., 1972). In order to investigate the distribution of these DNA classes, hydroxyapatite isolations were made at several molecular weights: 1.8 x 105, l-7 x 106, and 1.0~ lo7 single-stranded. Because of the dependence of renaturation rate on the square root of L, the single-strand molecular weight (Wetmur & Davidson, 1968), the DNA was renatured to C,t = 6x 10m2 x (Lsonio./L)1’2, where L,,,,,, = 1.8 x lo5 mol. wt. The applicability of the L-1’2 dependence of renaturation rate has not been demonstrated for partially non-renaturing molecules; it is expected to be an overcorrection if renaturation is monitored by hypochromism and to be accurate if hydroxyapatite is the assay.

(i) Main-band h.a.r.r.DNA As shown in Table 1, the main-band h.a.r.r.DNA yield increases slowly as a function

of molecular weight: a 60-fold increase in the fragment size results in a twofold increased yield. The increase in yield is most easily explained by the main-band h.a.r.r.DNA being covalently linked to a more complex DNA that is unrenatured at the equivalent Cot of 6 x 10w2. The increased yield would then reflect on increased amount of these single-stranded “tails “, with the amount of double-stranded h.a.r.r.DNA remaining constant. Of the possible sequence distributions shown in Figure 6, only the type of arrangement of model (c) explains the observed yield of mouse main-band h.a.r.r.DNA as a function of molecular weight. The mouse satellite- type distribution of Figure 6(b) would not cause any appreciable dependence of yield on molecular weight, while the model in Figure 6(a) would predict a linear increase in yield (as soon as the fragment size exceeded that of the h.a.r.r.DNA region).

In order further to demonstrate the applicability of model (c) in Figure 6 to the distribution of main-band h.a.r.r.DNA, and to assign limits to the average lengths of the two types of segments, 3H-labeled main-band h.a.r.r.DNA of different single- strand molecular weights was treated with S, nuclease (Fig. 7(b)), which digests

TABL

E 1

Pro

perti

es

of m

ouse

mai

n-ba

nd

rapi

dly

rena

turin

g D

NA

ae

a ju

nctio

n of

DN

A

fragm

ent

size

1.

2.

3.

4.

6.

6.

7.

8.

Dou

ble-

%

%

n-

.-L*-

Si

llgb

Yiel

d %

D

oubl

e-

stra

nded

D

oubl

e-

Hyp

er-

-L---

:-:&-

. st

rand

ed

\%

‘,‘,

of m

fbin

- cw

l PO

L

mol

. w

t be

nd

stra

nded

ness

yi

eld

DN

A)

6%

(O/o

of m

ain-

ww

dige

stio

n)

bend

D

NA)

PO

t-A

ll ny

pw-

chro

mia

ity

atre

nded

mas

uu

r”uw

Jl~y

stre

nded

ness

from

ab

ove

70°C

fro

m

,A,\

L-.-

&in-

bend

h.

e.r.r

.DN

A So

nioe

tad

1.8

x 10

6 26

67

1.

8 1*

70&

w

71

19.0

l-6

71

Nee

dle-

ahea

red

1.7

x 10

6 3.

6 41

1.

4 1.

7076

64

8.

2 29

U

nshe

ered

10

.3 x

106

6.

0 26

1.

3 1.

7130

23

6.

0 18

Fold

baak

So

niae

ted

1.8

x 10

5 2.

6 92

2.

3 29

-l W

I N

Stdk

+she

Sred

1.

7 x

106

4.3*

67

2.

9 18

.3

68

Um

hear

ed

10.3

x 1

06

9.b*

69

6.

6 14

.6

46

Kine

tic

fract

ions

w

ere

isol

ated

fro

m

Ag+/

Css

S04

purif

ied

m&-

bend

D

NA

as d

escr

ibed

in

th

e te

xt

end

the

lege

nds

to

Figs

3

end

7. T

he

yiel

d (a

olum

n 2)

is

oor

reot

ed

for

mat

erie

l be

ndin

g et

th

e de

nsity

of

re

nstu

red

mou

se

sate

llite.

So

me

fold

back

D

NA

yiel

ds

(*)

may

be

ove

rest

imet

ed

by

abou

t 10

%

due

to

incr

ease

d m

ain-

band

h.

a.r.r

.DN

A in

th

e fo

ldba

ok

fract

ion

at

high

m

olec

ular

w

eigh

ts.

The

perc

enta

ge

doub

le-s

trand

edne

ss

(ool

umn

3) i

a th

e pr

opor

tion

undi

gest

ed

by

S, n

uale

ese

afte

r 30

min

. C

ohnn

n 4

give

s th

e pr

oduc

t of

the

va

lues

of

ool

umn~

2

end

3. T

he

buoy

ant

dens

ities

(c

olum

n 6)

are

fo

r h.

s.r.r

.DN

A is

olat

ed

at e

quiv

alen

t C

ot =

6

x 10

m2

and

are

conv

erte

d to

es

timat

es

of d

oubl

e-st

rand

edne

as

(aol

umn

6) b

y as

sum

ing

thst

th

e bu

oyan

t de

nsity

of

son

iaet

ed

h.s.

r.r.D

NA

aorre

spon

da

to

the

perc

enta

ge

doub

le-s

trand

edne

sa

give

n by

th

e Sr

res

ults

on

the

sa

me

DN

A.

Hyp

erah

rom

iaity

(o

olum

n 7)

is

give

n aa

a p

er-

cent

age

of

the

sbso

rban

ce

at

70°C

end

is

oor

rect

ad

for

sing

le-s

trand

un

stac

king

, th

e am

ount

of

sin

gle-

stra

nd

DN

A ta

ken

from

th

e S

1 da

te

(aol

umn

3).

The

estim

atea

of

co

lum

n 8

are

norm

aliz

ed

to

the

pem

enta

ge

doub

le-s

trmnd

edne

ffl

for

soni

cate

d sa

mpl

es

give

n by

th

e S

1 da

ta.

The

brao

kete

d nu

mbe

rs

are

set

equs

l to

th

e ao

rresp

ondi

ng

num

bers

in

col

umn

3 fo

r th

is

norm

aliz

atio

n.


30 0 15 30

Timetmin)

(a) (b) Cc)

FIQ. 7. S, single-strand nuclease digestion of renatured mouse DNA fractions. Each symbol represents the ratio of trichloroacetio acid precipitable cts/min before and after incubation with Si nuolease. Acid-preoipitability at 60 min averaged 6% below the 30.min values. (a) -A-A-, denatured mouse DNA with h.a.r.r.DNA removed; -O-O--, high molecular weight renatured mouse satellite. (b) Main-band h.a.r.r.DNA fraotions : 1.8 x 10s (W), 1.7 x 10s (.), 1.0x 10’ (A) single-stranded molecular weights. The filled symbols represent material isolated from Ag+/CssSO, main-band DNA. The sonicated sample ( n ) shown in Fig. 3(f) was recycled through hydroxyapatite before the S1 digestion. The higher molecular weight samples ( 0, A) were isolated at an equivalent C,t of 6 x 10-s after removal of foldback DNA, then aggregated to high C,t and purified from residual mouse satellite by centrifugation in preparative CsCl gradients. In order to restore the DNA to its renatured but non-aggregated state the latter samples were dialyzed against 0.06 M-phosphate buffer, alkali-denatured, and renatured to an equivalent C,t of 4x10-1. The open symbols correspond to h.a.r.r.DNA of similar molecular weights, but isolated from satellite-free main-band DNA (isolated by successive CsCl gradients), with no recycling through hydroxyapatite. (0) Foldback DNA; the symbols refer to same moleoular weights as in (b). Filled symbols: foldback isolated at low C,t from Ag+/CssSO, main-band DNA was again denatured and renatured to an equivalent Cot of 2 x 10m4 to 1 x 10m3, and reisolated on hydroxyapatite. Open symbols: foldbaok isolated from CsCl-prepared satellite-free main band DNA, with no reoycling through hydroxyapatite.

single-stranded regions of renatured DNA (Sutton, 1971). As shown in Figure 7(a), denatured mouse DNA (with rapidly renaturing portions removed) is 98% degraded by the enzyme within ten minutes, and renatured high molecular weight mouse satellite is 14% degraded (reflecting the mismatching which is also apparent from the increased pc of this DNA). High molecular weight native mouse DNA is totally resistant to the S, nuclease.

The amount of h.a.r.r.DNA not digestible by S, nuclease, multiplied by the yield of h.a.r.r.DNA at that molecular weight, should be a constant fraction of the genome. As shown under “double-strand yield” (Table 1, column 4) this is the case for main- band h.a.r.r.DNA. When a sample of main-band DNA purified from satellite by successive CsCl gradients was sheared to three molecular weights, and main-band h.a.r.r.DNA was isolated (the open symbols of Pig. 7(b)), the product of the hydroxyapatite yield and the S-resistant fraction was again found to be nearly constant. A slight tendency for decreasing double-strand yield seen in both molecular weight series could be due to a greater fraction of the main-band h.a.r.r.DNA being collected in the foldback fraction, or to increased mismatching at high molecular weights (Waring & Britten, 1960; Bond et al., 1967; our unpublished observations on mouse satellite DNA) with the mismatched regions being susceptible to S, digestion.


The average length of the main-band h.a.r.r.DNA segments shown in Figure 6(c) can be estimated from the degree of S, digestibility of the h.a.r.r.DNA at sonicated molecular weight. The fraction (F) of h.a.r.r.DNA that is double-stranded and therefore S1 nuclease-resistant is given by the equation: F = [A(1 - m)]/(A + L), where A is the average length of the rapidly renaturing segments in the native DNA, L is the average fragment size of the sonicated DNA, and m is the fraction of mismatched bases in the renatured A segment (see Appendix for derivation). If mismatching is neglected and the one-third S, digestibility of main-band h.a.r.r.DNA at sonicated molecular weight is assumed to be entirely due to the more complex DNA tails, then A = 2L = 3.6 x IO5 single-stranded. An upper limit for the amount of mismatching, determined from the T, of h.a.r.r.DNA from sonicated main-band DNA, is m = O-14. If it is assumed that all of these mismatched bases are susceptible to S, digestion, then the h.a.r.r.DNA is estimated to occur in segments with an average single-strand molecular weight no greater than 6.3 x 105.

The average size of the intercalating, more complex stretches of DNA can be determined from the h.a.r.r.DNA yield at high molecular weights, where at least two such regions are included in most fragments. The difference between the hydroxyapatite yield (column 2, Table 1) and the E&-undigestible double-stranded (“pure “) h.a.r.r.- DNA (column 4) shows that the interspersed complex sequences comprise at least 2.1% of the genome. This is 1.2 to 1.5 times the amount of “pure” h.a.r.r.DNA, giving an average molecular weight of 4 x lo5 to 11 x lo5 single-stranded for the intercalating regions. Variations in the length of the complex DNA segments could explain the yield increase between the 1.7 x log and 1 x IO7 molecular weight fragment sizes.

Buoyant density profiles of h.a.r.r.DNA at different molecular weights detect the presence of unrenatured DNA tails and have been used to determine the lengths of the two types of segments when h.a.r.r.DNA occurs intercalated with more complex DNA (Kram et al., 1972). The mean CsCl buoyant densities of the mouse main-band h.a.r.r.DNA increase with molecular weight, and the resulting estimates of double- strandedness are compared with the S, digestion data in Table 1. The predicted shapes of the density distributions (Fig. 6 in Hearst et al., 1973a), based on the average lengths of rapidly renaturing and more complex regions quoted above, are consistent with those actually observed.

Another method of estimating the degree of double-strandedness is from the hyperchromicity (Fig. 5(b)), Assuming that the hyperchromicity above 70°C represents melting of fairly well-matched hybrids plus unstacking of any single-stranded tails, the percentage double-strandedness can be estimated for the higher molecular weight main-band h.a.r.r.DNAs relative to the sonicated sample (see Table 1). This method gives a higher estimate of the amount of single-stranded material than does the S1 digestion, although the amount of single-strandedness clearly increases with molecular weight.

(ii) Ir’oldback DNA The amount of foldback DNA as a function of the molecular weight of isolation is

given in Table 1. The large increase in yield with molecular weight has also been reported for the foldback DNA fractions of calf (B&ten & Smith, 1970) and toad (Davidson et al., 1973). It suggests that the mouse foldback DNA is not as tightly clustered in the genome as is main-band h.a.r.r.DNA. Also unlike the latter, the


double-stranded yield (column 4) is not constant but increases with molecular weight. (This was confirmed in the case of the molecular weight series shown as the open symbols of Fig. 7(c).) A possible explanation could be that the molecular weight has increased enough to include two non-adjacent complementary sequences on the same strand.

When the fragment size is greatly increased, the degree of double-strandedness in the foldback DNA decreases somewhat as judged by S, digestibility (Fig. 7), hyperchromicity (Fig. 5), or buoyant density (unpublished results). The buoyant density profiles suggest that there may be more than one class of foldback DNA, depending on the molecular weight of isolation. The thermal denaturation at all molecular weights shows a sharp hyperchromic transition over a range of 10 to 15 deg. C, with a T, only about 2 deg. C less than native mouse DNA. Walker & McLaren (1969) have also observed the steep melt of mouse foldback DNA.

Further evidence against the foldback DNA resulting from cross-linked or incom- pletely denatured DNA is the loss of the foldback characteristic after treatment with S, nuclease, presumably because of digestion of single-stranded hairpin regions. After S, digestion and denaturation, a five-minute renaturation and rechromlttography on hydroxyapatite yielded only 14% of the original material as double stranded.

(d) Chromommal localization of main-band h.a.r.r.DNA In order to determine whether the clusters of main-band h.a.r.r.DNA are localized

on specific chromosomes or certain regions of chromosomes, 3H-labeled complementary RNA transcribed in vitro from main-band h.a.r.r.DNA using E. wli RNA polymerase was hybridized in situ to denatured metaphase chromosomes of mouse (Balb/c) embryonic primary cells and SVT2 cells. The DNA used as a template was sonic&ted main-band h.a.r.r.DNA prepared identically to that shown in Figure 3(f) -free of foldback DNA and contaminating mouse satellite DNA. It was then digested with S1 nuclease (20% digestion due to its highly aggregated state), which was removed by phenol extraction. The complementary RNA was therefore complementary to the rapidly renaturing main-band h.a.r.r.DNA sequences, and perhaps to some attached sequences that renatured during the high Cot aggregation (equivalent Cot M-50).

Autoradiogrems developed after 18 days showed localization of grains to all the chromosomes (Plate I). Karyotyping according to chromosome length (Plate I(b)) revealed no obvious preference of hybridization for a particuler size-class of chromosomes. Three base-denatured diploid spreads with 96 to 189 grains per spread (an average of 150) were analyzed in detail. It was estimated that only about four chromosomal grains per spread were background grains, based on the distribution of background grains in clear areas near the chromosome spreads. (This background estimate agreed well with the number of grains seen in the hybridization of mouse satellite complementary RNA to human chromosomes, discussed below.) On the average, the one-third of s, chromosome that included the centromere contained 30*9&3.6% (standard deviation) of the grains, the middle one-third contained 37*7*4*00,/,, and the telomeric one-third contained 31*4&2*5%. G iven the coarse resolving power of autoradiography (about 1 pm), it is not possible to say whether the centromeric labeling was due to gmins on the chromosome arms near the centromere or grains in the centromere itself. It is possible that a minute amount of satellite DNA in the template could have been responsible for some of the centromeric labeling.

PLATE I. In situ hybridization with RNA complementary to main-band h.a.r.r.DK.4. (a) The chromosomes were from colcemid-arrested mouse embryonic primary cells (second transfer), and were alkali-denatured. Hybridization was for 16 h with 510,000 cts/min (0.03 pg) complementary RNA in 200 ~1 of 2 x SSC, after which the slide was washed and treated with RNsse. Suto- radiographic exposure time was 18 days. Heavy Giemsa staining of the centromeric heter~~- chromatin should not be confused with exposed silver grains, though centromerio grains often occur; a few examples are marked with arrows. The Y chromosome, unlabeled in this spread, was not consistently under-labeled. (b) ThP chromoscmc+ of spread (a) ft~ arranged according to ClcrrPening chromatid length.

, fWOl<, ,‘. J, I

wm ,

PLATE II. I?L si~u hybridization to acid-denatured mouse chromosomes. The chromosomes and RNA complementary t,o mouse main-band h.a.r.r.DNA are the same as were used in the Plate I study. Hybridization was for 16 h at 62°C with 660,000 cts/min (0.03 rg) complementary RNA in 100 pl of 2 x MC. Autoradiographic exposure time was 43 days.


In situ hybridization to acid-denatured chromosomes gave similar results, with about the same average number of grains per chromosome per day of autoradiography as was seen with the base-denatured chromosomes. An acid-denatured diploid spread is shown in Plate II. Hybridization was performed as in the Plate I study, but autoradiographic exposure was 2.4 times longer. Four diploid spreads had an average of 365 grains (the range was 268 to 417), with 25.4% 1.6% on the centromeric, 40*7f2*7% on the middle, and 33*8-&2.Oo/o on the telomeric third of an average chromosome. Background on chromosomes was estimated as six grains per spread. The interphase nucleus below the spread in Plate II shows some localization of grains to regions darkly stained by Giemsa, i.e. to heterochromatin. Normally about 20% of the area of an interphase nucleus appeared darkly stained, while 20 to 55% of the grains in the nucleus were in this darkly stained area.

Confidence in the specificity of the in situ hybridization results was gained from several control experiments processed simultaneously with the study shown in Plate I. RNA transcribed in vitro from mouse satellite DNA localized almost exclusively to the centromeres of mouse primary cell and SVT2 cell metaphase chromosomes, though not to the Y chromosome. These observations agree with those of Jones (1970) and Pardue & Gall (1970). The same mouse satellite complementary RNA showed no hybridization to human diploid chromosomes after 18 days of exposure, the five to ten grains per spread equaling the background level on the slide. The mouse main- band h.a.r.r.DNA, however, did hybridize to acid and base-denatured human chromosomes (Hearst, et al., 1973b). The number of grains was five times below the level seen on mouse chromosomes, but five times above the background level. Further- more, there was a decided preference for centromeres and telomeres, and some preference for certain chromosomes such as those in group D (Denver classification).

(e) Cross-hybridization of mou.se satellite and main-band h.a.r.r.DNA The observation of Flamm et al. (1969a) that mouse main-band DNA seems to

contain a few per cent of sequences complementary to mouse satellite led to our investigating the possible complementarity between mouse main-band h.a.r.r.DNA and mouse satellite DNA. The predicted native buoyant density of the rapidly renaturing portion of main-band h.a.r.r.DNA (I.697 to 1.698 g/ml), its base composition, its kinetic complexity, and the broad chromosomal localization seen in the in situ hybridization make it unlikely that it has a sequence complementary to the satellite DNA. Nevertheless, more direct tests of sequence homology were made.

In the first test (Fig. 8), a small amount of l*C-labeled pure mouse satellite DNA was added to 3H-labeled total mouse DNA and portions of the mixture were sheared to single-strand molecular weights of 1 x IO5 to 2 x lo5 and 4 x 106. Each mixture was then denatured and renatured, and reassociated DNA was collected on hydroxyapatite. Any pure satellite sequences in the main-band rapidly renaturing DNA would have competed for the 14C-labeled satellite according to their concentration relative to that of all the satellite DNA; however, no such hybrids were found upon banding in CsCl. Because the heavy peaks of Figure 8(a) and (b) also contain foldback DNA and complex DNA attached to the rapidly renaturing main-band DNA, the upper limit on the amount of “pure” rapidly renaturing main-band h.a.r.r.DNA that could be exactly complementary to satellite DNA is 4%. This result is not neces- sarily contradictory to that of Flanun et al. (1969a) because the shearing of the main band in the above experiments could have freed satellite sequences in the main band

FIG

. 8.

Cro

ss-h

ybrid

izat

ion

betw

een

mai

n-ba

nd

h.a.

r.r.D

NA

and

mou

se

sate

llite.

(a

) A

mix

ture

of

200

clg

3H

-lebe

led

tota

l m

ouse

D

NA

(11,

800

cm/m

m/&

an

d 3

pg 1

4C-la

bele

d m

ouse

sa

tellit

e D

NA

(660

0 ot

s/m

in/&

w

as

soni

oate

d to

a m

olec

ular

w

eigh

t of

1

x lo

5 to

2

x lo

6 si

ngle

-stra

nded

, de

natu

red,

re

natu

red

to

a C

,r of

2.

4x

10ea

, an

d fra

ctio

nate

d on

hy

drox

yapa

tite.

Th

e pr

opor

tion

of

r*C

-labe

led

sate

llite

“trac

er”

to

the

3H-la

bele

d sa

tellit

e in

th

e to

tal

mou

ac

DN

A w

as

such

th

at

no

mor

e th

an

0.2%

of

th

e “C

ot

s/m

in

in

the

h.a.

r.r.D

NA

coul

d re

pres

ent

r4C

:‘*C

hy

brid

s.

The

O-4

6 aa

-pho

spha

te

elua

nts

cont

aine

d 11

.0%

of

th

e 3H

rad

ioac

tivity

an

d 87

%

of

the

“C.

The

first

tw

o 0.

46 a

r-pho

spha

te

buffe

r fra

ctio

ns

wer

e m

ade

0.00

2 M

in

EDTA

an

d 0.

3%

in r

ecry

stah

ixed

Sa

rkos

yl,

and

solid

C

sCl

we8

ad

ded

to

a fin

al

dens

ity

of

I.700

g/

ml.

Cen

trifu

gatio

n w

as

for

68 h

at

36,0

00

revs

/mm

in

th

e Sp

inco

an

gula

r 66

rot

or,

afte

r w

hich

th

e tu

be

ws9

pun

ctur

ed

with

a

23-g

auge

ne

edle

an

d &d

rop

fract

ions

co

llect

ed.

Porti

ons

of e

ach

fract

ion

(70

~1)

wer

e co

unte

d fo

r 10

mm

aa

des

crib

ed

in

Mat

eria

ls

and

Met

hods

. Th

e ha

lf-t&

&m

ohel

co

unts

w

ere

corre

cted

fo

r sp

illove

r fro

m

the

r4C

cha

nnel

, bu

t no

cor

rect

ion

was

ne

eded

in

th

e op

posi

te

dire

ctio

n.

(b)

Sam

e as

(a)

ex

cept

th

at

the

DN

A m

ixtu

re

was

she

ared

to

abo

ut

4 x

IO’J

mol

. w

t. si

ngle

-stra

nded

an

d re

natu

red

to

an e

quiv

alen

t C

ot o

f 6

x lo

- a.

R

enat

ured

m

ater

ial

elut

ed

at

0.45

x-p

hosp

hate

in

clud

ed

17.7

%

of t

he

*H

and

92%

of

the

“C

ct

a/m

in.

Con

trol

expe

rimen

ts

in w

hich

3H

-labe

led

D.

&m

- g&

r D

NA

was

su

bstit

uted

fo

r th

e eH

-labe

led

mou

se

DN

A w

ere

perfo

rmed

at

bo

th

mol

ecul

ar

wei

ghts

. Th

e lo

w

leve

ls

(10

to

20 a

ts/m

in

abov

e ba

ckgr

ound

) of

14

C fo

und

unde

r th

e m

ain-

band

h.

a.r.r

.DN

A pe

aks

in

(a)

and

(b)

do

not

diffe

r ei

gniiic

antly

fro

m

the

leve

ls

at

corre

spon

ding

de

nsiti

es

in t

he

cont

rol

grad

ient

s,

and

ther

efor

e pr

obab

ly

repr

esen

t tra

iling

mat

eria

l fro

m

the

rV:3

H

sate

llite

peak

ra

ther

th

an

hybr

idiz

atio

n w

ith

the

mai

n-ba

nd

h.a.

r.r.D

NA.

(c

) H

ybrid

izat

ion

of

the

light

st

rand

of

m

ouse

sa

tellit

e D

NA

with

m

ain-

baud

H

.a.r.

r.DN

A.

The

“C

light

st

rand

w

as

isol

ated

fro

m

high

m

olec

ular

w

eigh

t sa

tellit

e D

NA

on a

pre

para

tive

alka

line

C&l

gr

adie

nt

acco

rdin

g to

Fla

mm

et

al.

(196

7).

The

self-

reas

soci

atin

g fra

ctio

n of

the

lig

ht

stra

nd

(7%

) w

as

rem

oved

by

hyd

roxy

apat

ite

fract

iona

tion

(Fla

mm

el

al.,

19

69o)

, af

ter

whi

ch

the

sing

le-s

trand

m

olec

ular

w

eigh

t w

as a

ssum

ed

to b

e re

duce

d to

abo

ut

7 x

10e.

H.a

.r.r.D

NA

wac

is

olat

ed

from

aH

-labe

led

mai

n-ba

nd

DN

A (6

x 1

0e s

ingl

e-st

rand

ed;

purif

ied

by

Ag+/

Cae

SO*

and

CsC

l gr

adie

nts)

at

an

equ

ival

ent

Cot

of

6 x

lOW

a, w

ith

a 19

.6%

yi

eld

in t

he

0.46

&f-p

hosp

hate

fra

ctio

ns

(incl

udin

g fo

ldba

ck

DN

A).

A m

ixtu

re

of

22 p

g of

the

3H

-labe

led

h.a.

r.r.D

NA

(936

0 ot

s/m

in/&

pl

us

3 H

of

th

e W

-labe

led

sate

llite

light

st

rand

(4

330

cm/m

m/H

ca

lcul

ated

fro

m

base

co

mpo

sitio

n)

wae

di

alyz

ed

agai

nst

0.12

M

-pho

spha

te

buffe

r-099

1 M

-ED

TA,

ther

mal

ly

dena

ture

d,

and

rena

ture

d at

60

°C f

or

27 h

at

Cc

= 0.

1 O

.D.;“

“A-

(equ

iv.

C,t

= 7)

. an

d ba

nded

in

C&l

&s

des

crib

ed

for

(a).

A co

ntro

l gr

adie

nt

was

tre

ated

in

the

ab

ove

man

ner,

with

no

n-ra

pidl

y re

natu

ring

mai

n-ba

nd

DN

A su

bstit

uted

fo

r th

e h.

a.r.r

.DN

A.

The

arro

ws

show

th

e pe

aks

of t

he

light

st

rand

sa

tellit

e (p

,, =

1.69

7)

and

mai

n-ba

nd

DN

A (p

c =

l-717

) fro

m

the

cont

rol

grad

ient

. --o

--&-,

3H;

--n---

n-.,

14C

.


from attachment to more G + C-rich sequences, allowing the satellite sequences to band at satellite density.

The above experiments require any mouse satellite sequences in the 3H-labeled main-band DNA to compete with pure 3H-labeled satellite sequences for the r*C- labeled satellite tracer. This competition was eliminated by incubating only the light strand of mouse satellite DNA with main-band h.a.r.r.DNA at about the same molecular weight. As seen in Figure 8(c), most of the satellite strand remains at its denatured density, with only a little evidence of the formation of 3H : 14C hybrids at intermediate densities.

The above evidence against main-band h.a.r.r.DNA containing satellite sequences in no way eliminates a possible evolutionary relation between the two.

4. Discussion (a) Muin-band h.a.r.r.DNA

We have presented evidence that a rapidly renaturing family of Mua rwuaculus DNA, an order of magnitude more complex than the mouse satellite as judged by renaturation kinetics, is clustered in the genome-segments of this redundant DNA averaging 1500f500 base pairs are interspersed with stretches of more complex DNA averaging 2200&1100 base pairs long. Fragments of DNA 30,000 base pairs long should contain at least five h.a.r.r.DNA segments. There is uncertainty in these numbers for several reasons: they depend on the degree of mismatching of the h.a.r.r.DNA, which can only be estimated; the estimated lengths based on nuclease S, digestibility, hyperchromicity, and buoyant density are somewhat different; and the h.a.r.r.DNA yield continues to increase at high molecular weight, suggesting heterogeneity in the length of the more complex DNA segments. The cross-hybridization experiments demonstrate that the rapidly renaturing sequences are not homologous to the mouse satellite sequence, but do not rule out an evolutionary relation.

A clustered distribution similar to that postulated for the mouse main-band h.a.r.r.DNA has been demonstrated for the Xenopus Levis ribosomal RNA cistrons (Brown 6 Weber, 1968; Birnstiel et al., 1968) and 5 S RNA cistrons (Brown et al., 1971). The isolated ribosomal DNA renatures rapidly (C&l, = 2 x 10e2; Birnstiel et al., 1969) due to reiteration of both gene and spacer. Its low concentration in mammalian genomes (about O-l%), however, would exclude it from the 6 x 10V2 C,t fraction of mouse h.a.r.r.DNA, a conclusion valid also for 5 S RNA and transfer RNA genes (Hatlen & Attardi, 1971).

Another case of a “spacer” distribution occurs with a D. melanogaster h.a.r.r.DNA (Kram et al., 1972) that has been referred to several times in this paper. The kinetic complexity of the D. melanogaster h.a.r.r.DNA is the same order of magnitude as that of the main-band h.a.r.r.DNA in mouse. The estimated average size of the rapidly renaturing segments is four times longer in D. melanogaster than in Mus mu-sculw, while the more complex DNA segments separating the sections of rapidly renaturing DNA are twice as long as in mouse. The Drosophila h.a.r.r.DNA is localized in the centric heterochromatin of D. melanogaster by in situ hybridization, while the centromeres of mouse chromosomes are not enriched in mouse main-band h.a.r.r.DNA.

Less highly reiterated (intermediate) sequences occur interspersed with unique DNA in the genomes of calf (Britten & Smith, 1970), D. melunoga.ster (Wu et al.,


1972), and sea urchin (Britten, 1972). In calf and sea urchin, a high proportion of DNA fragments 4000 nucleotides long contain both repeated and non-repeated sequences, suggesting a lack of clustering of at least some of the repetitive DNA segments. A detailed study of Xenopus DNA by Davidson et al. (1973) has shown that the major intermediate class (with & complexity of 500,000 base pairs) is widely dispersed. They found that about half of the repeated sequences alternate with segments of unique DNA. They formed no conclusions about a 15% more rapidly renaturing fraction, except that it could occur in a more highly clustered arrangement. Rice (1971) and Britten (1972) have shown that mouse intermediate sequences may be arranged similarly to those in Xenopus. Their studies do not relate to more rapidly renaturing families of mouse DNA like the one described in this paper; these were either excluded from the kinetic fractions they studied or mixed with DNA sequences of higher complexity.

Examining the distribution of mouse main-band h.a.r.r.DNA on a broader level, the in situ hybridization shows a very widespread chromosomal distribution. This type of distribution has been observed in several eukaryotes when RNA complementary to total DNA is hybridized in situ (see discussion by Walker, 1971) and is therefore characteristic of an intermediate repetitious DNA. Well-defined DNA fractions giving this type of distribution are the D. neohydei heavy satellite, which localizes to many bands of the polytene chromosomes as well as to telomeres and kinetochore regions (Hennig et al., 1970), and the homogeneous main band of human DNA (Corneo et al., 1970), a fraction of which (satellite III) localizes to several distinguishable regions of each chromosome (Hearst et al., 1973b). Another rapidly renaturing human DNA family, satellite II, localizes close to the centromeres of three chromosomes pairs but is also found in lower concentration in many other sites on all the chromosomes (Jones & Corneo, 1971). These examples of widespread distribution are clearly distinct from the centromeric localization of many satellite DNAs and the D. meh-wgatder h.a.r.r.DNA discussed above.

The mouse main-band h.a.r.r.DNA shows some localization to heavily Giemsa- stained areas of interphase nuclei (Plate II), though it is not restricted to these regions. This is consistent with the observation by Rae & Franke (1972) that only part of mouse interphase heterochromatin contains high concentrations of mouse satellite DNA.

The mouse satellite complementary RNA does not give discernible hybridization to the human chromosomes, in agreement with the low cross-hybridization of satellite DNA to the DNA of even closely related species (Flamm et al., 1969a). Under identical conditions, the complementary RNA transcribed from mouse main-band h.a.r.r.DNA cross-hybridizes with the human chromosomes, suggesting the possibility of sequences conserved in evolution. Microtus agrestis rapidly renaturing DNA has been shown to hybridize in situ with mouse, hamster, and human nuclei by Arrighi et al. (1970), but it is not known which sequences in their C,t = 8 fraction were responsible for the result.

The relation between simple sequence DNAs and highly condensed heterochromatin suggests a possible function for the h.a.r.r.DNA sequences in the coarse control of the transcription of the intercalating, more complex DNA (Sutton, 1972; Paul, 1972; Kram et al., 1972; Hearst & Botchan, 1973; Hearst et al., 19733). Further implication of these sequences in genetic regulation requires information on the transcription of the h.a.r.r.DNA and complex DNA sequences, and on the processing and translation of any such transcripts.


(b) Foldback DNA

The foldback fraction of DNA from mouse main-band, originally removed from the h.a.r.r.DNA as a contaminant, has interesting properties in itself. The possibility of its being an artifact caused by cross-linking or incomplete denaturation of DNA has not been eliminated at this time, but seems unlikely since (a) the foldback DNA is found in equal proportions in DNA prepared by different methods ; (b) it is found in high molecular weight as well as sonicated samples ; and (c) it retains its ability to foldback after both heat (92°C in 0.05 M-phosphate buffer) and alkali (pH 12.9 at SO’C) denaturation, though this ability is decreased after nuclease S, digestion. A similar class of DNA has been found in several eukaryotic genomes (Walker & McLaren, 1969; B&ten & Smith, 1970; Davidson et al., 1973).

Walker & McLaren (1965) studied both sonicated and high molecular weight mouse DNA deliberately cross-linked with nitrous acid. At both molecular weights the cross- linked DNA showed a ‘7 deg. C lower T, and 10% loss of hyperchromicity after denaturation and renaturation. In contrast, our studies on foldback DNA show a large decrease in the amount of double-strandedness in going to higher molecular weights; if the DNA were cross-linked, it would be difficult to explain why the large fragments do not zipper completely.

The properties of the presumed foldback DNA as a function of molecular weight disclose some intriguing but not easily explainable facts. The first unexpected result is the low degree of S1 nuclease digestibility of the foldback DNA isolated from sonicated main-band, corresponding to only 8% single-strandedness. Presumably this includes single-stranded tails, internal mismatched regions, and hairpin loops. On the average, the folding back of a DNA strand containing an inverted sequence would be expected to yield an intrastrand duplex plus a single-stranded tail of equal length. (This assumes that the point of symmetry that determines the position of the hairpin loop is located randomly along the fragment.) It is possible that the DNA is folded in such a way that some single-stranded regions are inaccessible to the nuclease; however, the 29% hyperchromicity of this DNA fraction (Fig. 5(c)), almost as great as that of renatured mouse satellite DNA, minimizes the possible extent of this type of S, resistance. Other possible explanations for the almost total double-strandedness of the foldback DNA at small fragment size include: (a) multiple inversions within each fragment, producing many closely spaced foldback regions; (b) non-random shearing of the native DNA such that the point of symmetry that becomes the hairpin loop is always in the middle of the fragment; (c) failure of hydroxyapatite to bind molecules with less than 200 base pairs so that only highly base-paired foldback DNA is collected at sonicated molecular weight.

Judging from the increase in yield with molecular weight, the foldback regions are more widely distributed in the genome than the clustered main-band h.a.r.r.DNA sequences,

If the foldback regions are transcribed, it is possible that they code for double- stranded regions of heterogeneous nuclear RNA. Jelinek & Darnell(l972) have found 300 nucleotides per 10,000 of HeLa cell heterogeneous nuclear RNA to be double stranded, these regions not appearing in the messenger RNA. Double-stranded HeLa cell RNA has also been studied by Kimble & Duesberg (1971), who found RNase- resistant fragments 500 to 1600 nucleotides long.


APPENDIX

Calculating the Lengths of Regions of Rapidly Renaturing DNA

If native DNA contains discrete regions A of DNA capable of rapid renaturation, separated by long stretches of more complex DNA (as shown in Fig. Al(a)), we want to determine the average length of the A regions by studying the properties of the DNA when isolated in its renatured state. The native DNA is randomly sheared, denatured, and renatured to a Cot sufI-lcient for renaturation of A sequences only. Fragments containing A sequences are then isolated on hydroxyapatite and are called h.a.r.r.DNA. As shown below, a correlation can be derived between the fraction of h.a.r.r.DNA that is double-stranded and the average length of the h.a.r.r.DNA regions in the native DNA. The fraction of double-strandedness can be estimated from hyperchromicity, electron microscope measurements, buoyant density, or degree of degradation by a single-strand specific nuclease.

(0) -A --i . ..-_- - - - --------- ..- - - - - - _ _ - - - - ---...

I I 1 I b-L-4

lb)

x

FIG. Al. (8) Simple sequenoe DNA ( ) of length A ooours ss 8n isoleted region of the netive DNA, oontinuous with long stretohes of more oomplex DNA (= = = =). The brclces

I) show two possible fregmenta resulting from sheering the DNA into pieoes with length i-b) The gr8ph ( ) of f(z) (the fraction double-strandedmass of a h.a.r.r.DNA fre-pent) 8s a funotion of z (the position of the right-bend end of the fmgment) for the A 2 L situ8tion ShOWn in (8). The reotangle (. . * * *) has en 8ree equal to the tot81 amount of h.a.r.r.DNA.

The position of the right-hand end of a DNA shear fragment is defined by the variable x. The fraction of a fragment of h.a.r.r.DNA in double-stranded, base- paired form isf(x). A is the average length of a region of simple sequence DNA before the native DNA is sheared. L is the average (single-stranded) length of a DNA fragment after shearing, usually determined from the alkaline sedimentation coet3kient.

We assume random shearing of the DNA; i.e. all values of x are equally probable. We assume that in order to be collected as h.a.r.r.DNA a fragment of length L must contain some of the A region; i.e. only fragments with 0 < x < A + L are considered. Mismatching and unrenatured sections of simple sequence DNA are neglected at first, and all bases in the A regions are assumed to be paired in the h.a.r.r.DNA.

22


Case I. A 2 L

As shown in Figure Al, the sheared fragments are smaller than region A. The left- hand brace of Figure Al (a) shows a fragment with 0 < x < L. Such fragments have a fraction f(x) double-strandedness that increases linearly with L. When L 5 x 5 A, the h.a.r.r.DNA fragments are completely within the A region, therefore f(x) = 1.0. The right-hand brace of Figure Al(a) shows such a fragment. Cuts with A < x < A + L have a linearly decreasing degree of double-strandedness. The graph of f(x) is shown in Figure Al(b).

F is defined as the fraction of the total sample of h.a.r.r.DNA that is double- stranded. F is given by the amount of base-paired DNA in h.a.r.r.DNA divided by the total amount of DNA (single-stranded tails plus double-stranded regions) in this h.a.r.r.DNA. This is the ratio of the area of the trapezoid in Figure Al(b) to the area of the rectangle (dotted).

F = j-t’+ ‘f(x) dx _ A Jf+"dX

. A-+-L

(1)

FIG. A2. The graph of Fig. Al(b) in the ctwe where A < L.

CaseII.A<L

As graphed in Figure A2, the fragments become more double stranded as x increases from 0 to A. At x = A, the fragment includes the whole A region as well as some single-stranded DNA. The maximum fraction double-strandedness (A/L) is reached at this point. Fragments continue to include all of A until x > L, when f(x) begins decreasing. F is again given by equation (1).

Rejknements

Mismatching will prevent the DNA in A segments from being totally double- stranded in the h.a.r.r.DNA. If m is the fraction of mismatching in renatured h.a.r.r.- DNA, the scaling factor (1 - m) is introduced into equation (1).

A certain minimum stable length of double-stranded DNA is assumed necessary for a renatured molecule to be retained on hydroxyapatite. If S is the minimum length, then fragments with x < S or x > A + L - S are not included in the h.a.r.r.DNA.

The result of these refinements is that only the shaded area in Figure A3 represents double-stranded h.a.r.r.DNA, while the rectangle (dotted) represents the total amount of h.a.r.r.DNA.


Fm. A3. Two re6nements have been added to the graph of Fig. Al(b). The A region is assumed to renature into 8 mism8tehed form, where m is the frection of mismatohed A bases. H.a.r.r.DNA is not isoleted unless the fragment has a base-paired length 2 S, where S is the minimum stsble length required for retention on hydroxyapatite.

F= (2)

If S is of the order of 50 nucleotides, omission of the terms in S in equation (2) will normally decrease F by only a few per cent. Equation (2) can then be approximated by

F = 41 - n&l A+L ’ (3)

The preceding theoretical treatment is a special case of a more general theory presented by Hearst et al. (1973a). Correspondence is obtained in the following way.

Since B (more-complex DNA) regions are assumed to be very long compared to the A regions, only one A region is ever included in the same cut. This means Y = 0 and X L- Z = L (notation of Hearst et al.). Furthermore, to assure that all fragments contain some A for retention on hydroxyapatite, let B = L. If pB = 0 and pa = 1, then the average density on this scale becomes the fraction double-strandedness of the h.a.r.r.DNA.

F = p = Pa. pa i- pfl * PO +

If the above substitutions are made, equation (4) reduces to F = A/(A + L).

We thank Theodore Gurney Jr for many helpful discussions, especially about mouse tissue culture, and for supplying cell lines. The ideas and encouragement supplied by Michael Botchan are very much 8ppreciated. Peter Botchen generously donated the RNA polymeraae. James Allen supplied the slides of human chromosomes. This work wa8 supported in part by United States Public Health Service grants no. GM11180 and no. GM15661 to one of us (J. E. H.) and a Public Health Service Fellowship (5-F03-HL 2931303) to another of the authors (A. R.)

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Characterization of the most Rapidly Renaturing Sequences ...

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