biochemistry - pnas

356 BIOCHEMISTRY: HOTTA AND BASSEL PROC. N. A. S.

21 Goldberg, B., and H. Green, J. Cell Biol., 22, 227 (1964).22 Love, R., G. P. Studzinski, A. M. Clark, and E. R. Tressan, Paper #51 read at XI Inter-

national Congress of Cell Biology, Providence, Rhode Island, August 20-September 5, 1964.23 Eagle, H., C. Washington, M. Levy, and G. Barton, "End product control of amino acid

synthesis in cultured human cells," in preparation.24 Todaro, G. J., H. Green, and B. Goldberg, these PROCEEDINGS, 51, 66 (1964).

MOLECULAR SIZE AND CIRCULARITY OF DNA IN CELLS OFMAMMALS AND HIGHER PLANTS*

BY YAsuo HOTTA AND ALIX BASSEL

BOTANY DEPARTMENT, UNIVERSITY OF ILLINOIS, URBANA

Communicated by N. J. Leonard, December 21, 1964

The idea that DNA might be organized as series of rings in chromosomes of higherorganisms was first elaborated by Stahl on genetic grounds.' The credibility ofthis viewpoint is enhanced by physical demonstrations of circular DNA in somebacteria and viruses,2 9 and by the self-regulation of selective strand transcriptionin DNA circles as revealed by the studies of Spiegelman and co-workers.3 Thisreport presents two pieces of evidence relevant to theories of chromosome organiza-tion: (1) that circles can be found in DNA from mammalian cells, and (2) that thenative molecules of DNA in higher plants and animals vary markedly in size.The studies here reported were carried out on isolated wheat nuclei and boar

sperm. The first material was chosen because nuclei could be readily isolated fromdormant wheat embryos in nonaqueous media4 since such embryos are naturally de-hydrated. The second material was chosen after experience with wheat nuclei in-dicated that the abundance of nonhistone protein in such nuclei required consider-able mechanical manipulation in order to effect extraction of the DNA.

Methods.-Isolated wheat embryos were disintegrated at temperatures not exceeding 250C byblending without the addition of solvent in an "Omnimixer" (Servall). In the course of blending,the dry powder was periodically sifted through a 35-.s nylon mesh, the retained portion being re-turned to the Omnimixer. Nuclei were then isolated by use of cyclohexane-carbon tetrachloridemixtures. Such preparations were stored in 95% ethanol after a series of washes with ethyl ether,ethanol : ether (1: 1), ether, ethanol: ether (2: 1), and ethanol. A stock solution of the protease,Pronase (Calbiochem), was prepared at a concentration of 2 mg/ml. The pH was adjusted to 5with HC1, heated to 80'C for 10 min, cooled, the pH readjusted to 7.0 with NaOH, and solidsodium chloride added to a concentration of 1.0 M. The solution thus prepared was DNase-freeand could be stored for at least several months at - 20'C without loss in activity.

Defatted wheat nuclei were suspended in a solution adjusted to pH 8 and containing the fol-lowing components: 1% cetyltrimethylammonium bromide ("cetavlon"), 1.0 M NaCl, 0.01 Methylenediaminetetracetic acid ("versene"), and 0.01 M Tris buffer. This solution, less the ceta-vlon, will henceforth be referred to as "buffered saline." The suspension of nuclei was maintainedat 0C for 20 min, diluted with an equal volume of 0.01 M versene-Tris (pH, 8), the fibers werecollected with a glass rod and transferred to buffered saline solution. After gentle overnightagitation at 0C, an equal volume of stock Pronase was added and the mixture incubated at 60'Cfor 4 hr. The suspension was cooled to 37°C, crystalline RNase added to a concentration of 0.4mg/ml, and incubated for 30 min.5 The Pronase treatment was then repeated. All digestionswere carried out in a dialysis bag suspended in buffered saline solution. After the final incubationthe suspension was clarified by centrifugation, the supernatant fluid containing the DNA. Toremove cetavlon from the DNA, one alcohol precipitation step was introduced into the procedure

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VOL. 53, 1965 BIOCHEMISTRY: HOTTA AND BASSEL 357

either after the collection of fibers or after Pronase digestion. Fresh boar sperm was washed threetimes with physiological saline solution at room temperature. Sperm do not lose motility by thisprocedure. Following this, several methods were tested for rapid cooling and inactivation ofnucleases, the simplest of which was to add 95% ethanol cooled with solid CO2. The alcoholicsuspension was centrifuged and the residue washed successively with ethanol:ethyl ether (1: 1),ether, petroleum ether, carbon tetrachloride: cyclohexane (1:4), and ethyl ether. Further washeswere the same as those for isolated nuclei. In preparing DNA, defatted sperm were removed fromthe ethanol by centrifugation, washed with cold concentrated saline solution, and suspended inthe same solution which was made 1.0 M with respect to mercaptoethanol and the pH adjustedto 9.0. The procedure is a modification of that devised by Bodenfreund, Fitt, and Bendich.6After maintaining the suspension for 30 min at 00C, it was lightly centrifuged and the residueresuspended in concentrated saline containing 0.25 M mercaptoethanol, adjusted to pH 8. Pro-nase was added to a final concentration of 0.5 mg/ml, and the suspension incubated at 600C for4 hr. At the end of this interval more Pronase was added (0.3 mg/ml) and the suspension incu-

0.2B. 0.8

A B1a2 1/ /

(.1.~~~~~~~ ¶~~~0.74FIG. 1.-Tracings of sedimentation patterns 0.1 /

taken at approximately '/a of cell distancebelow meniscus. Arrows indicate points atwhich S20 values were calculated. Rotorspeed was 24,630 rpm. (A) Pig DNA (S20 at / °arrows: 88.5, 55.3, 44.8, 36.2, 19.1, 16.2); l0.6(B1) wheat DNA prepared under optimal 0 L 20, ,_conditions (S20: 102, 56.8, 44.8, 36.2, 20.1, TUBE NO.14.5); (B2) wheat DNA prepared as in B.but stirred during isolation at about 1200 FIG. 2.-Elution profile of wheatrpm (S20: 64.8, 34.8, 32.7, 29.0); (B3) wheat DNA from a methylated albumin-DNA prepared with sodium lauryl sulfate kieselguhr column. Solid line is that(S2o: 45.0, 32.8, 31.9, 31.2). From the stand- of unstirred preparation (same as B1point of molecular size, such preparations are in Fig. 1); dotted line is that ofinferior to those obtained by cetavlon treat- stirred preparation (same as B2 inment. Fig. 2).

bated for an additional 8 hr. The suspension was then cooled, RNase added to a concentration of0.4 mg/ml, and digestion carried out for 30 min at 370C. Pronase was again added (0.3 mg/ml),and after incubation at 600C for 8-12 hr, the suspension was clarified by centrifugation, the super-natant fluid containing the DNA.For sedimentation analyses with the analytical centrifuge (Spinco, model E), DNA solutions

were adjusted to the desired concentration and dialyzed against 1 M NaCl; 0.01 M Tris (pH,7.3). A 3-cm Kel-F cell was used in these studies, and the DNA solution was added by means of ahypodermic syringe (#20 needle) at a rate of 0.2 ml/min. Samples were run at 5, 10-15, and15-20 /Ag/ml, and pictures were taken every 4-5 min with 4-10-sec exposures. Sedimentationvalues were calculated from microdensitometer tracings of the negatives.

In studies with the preparative ultracentrifuge (model L, Spinco), a swinging bucket rotor(SW 25) was used. A 5-20% sucrose gradient was formed over a base of 4 ml 60% (w/v) sucrosein siliconized tubes and a 1.0-1.5-ml sample of DNA solution was placed on top. All solutionswere buffered with Tris (0.01 M) at pH 7.4. As markers 30S ribosomes labeled with H3-uridineor C14-uracil and prepared from E. coli were used. After centrifugation at 23,000 rpm for 8-10

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hr at 20C, 1-ml fractions were collected through a 20-gauge needle puncturing the bottom of thetube. For electron microscopy, grids were prepared according to the method of Kleinschmidt andZahn.7 DNA was diluted from stock preparations with 2.0 M ammonium acetate 0.01% cyto-chrome c to a concentration of 2 jug/ml. Samples were rotary-shadowed with uranium oxide atan angle of 6-10° and a distance of 11 cm. Grids were examined with a Siemens-Elmiskop I at60 kv.

Results.-The physical characteristics of the DNA preparations are summarizedin Figures 1, 2, and 3. These characteristics are similar in a number of respects to

those obtained for E. coli under optimalconditions of extraction.8 From the

. WHEAT standpoint of sedimentation values,the population of DNA molecules isheterogeneous. Preparations whichhave been least subject to mechanicalAMA k shearing show a spread of molecular

0.05 _ 12/ y \ / *. \ / sizes, the largest of which is of the orderof 108 avograms (Fig. 1). The applica-tion of even mild shearing forces renders

J . *.the preparations more homogeneousZo P 5 10 15 20 P.i 30 and reduces the high molecular weights 0.1 - PIGnc component (Figs. 1 and 2). Stirring for

4-6 hr at 400 rpm with a 1.5 X 1-cmTeflon bar in a 50-ml Erlenmeyer flaskproduced no change in sedimentation

0.05 -t JA I ,, A I profile; references to mild stirring in

the preparative procedures pertain tospeeds no greater than 400. Speedshigher than 1000 rpm invariably altered

0 1 20 . the sedimentation profiles in the direc-% 10 15 20 25 30 tion just described.SUCROSE TUBE NO. MENISCUS To test whether the more rapidly

FIG. 3.-Sedimentation profiles of DNA in sedimenting components were productsa 5-20% sucrose gradient. 1 ml of DNA of molecular aggregation, several con-solution was layered at the top. Wheat:(A) 45 ug; (B) 100 Ag; (C) 200 Mg. Pig: centrations of DNA were run in a pre-(A) 40 Mg; (B) 80 Mg; (C) 160 /g. Dotted parative ultracentrifuge, and the sedi-line represents the labeled 30S ribosomemarker which was added to the C tubes. mentation profile was determined byOne-ml fractions were collected by puncture reading the absorbance of successiveof tubes with #20 hypodermic needle. Du-plicate runs of identical samples indicated fractions obtained upon puncturing thethat profiles might be shifted no more than bottom of the tubes. The resultsone tube.

(Fig. 3) show that the profiles in theregion of rapidly sedimenting components do not alter with change in concentra-tion. This was also confirmed by labeling the DNA of pollen cells (Trilliumerectum) and following sedimentation patterns at concentrations of 40, 13, and 4,ug/ml. The results were identical with those shown in the figure. Thus, thehigher molecular weights of DNA obtained by the gentler preparative proceduresmost probably reflect the native condition of the DNA and do not represent arte-factual aggregates.Taken together, the three sets of analyses permit the following conclusions:

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DNA molecules from higher organisms attain sizes at least as great as 108 avograms.The fact that such sizes have been obtained from tissues which require more ex-tensive mechanical manipulations (with accompanying molecular shearing) thando bacteria makes it probable that actual molecular weights are much greater thanthose measured. Heterogeneity of molecular size certainly exists in the prepara-tions, but the experiments do not discriminate between artefactual and real hetero-geneity. If the results are interpreted entirely in terms of experiments with E.coli, the conclusion would be drawn that the native DNA molecules in higher organ-isms are probably of extremely large but undetermined size.A different approach to the problem is possible by means of electron micros-

copy. Given a criterion by which the intactness of a molecule may be recognized,size may be measured directly. This advantage is, however, offset by the limitednumber of molecules which can thus be measured. Short of a formidable organiza-tion for scanning electron micrographs (which we do not have), nothing evenapproaching a statistical survey is possible. Despite this important limitation,DNA preparations were examined under the electron microscope. The number ofmolecules picked up on any single grid usually varied from 40 to 120, although incertain reconstitution experiments as few as eight were found. No attempt willtherefore be made to express the observations in terms of percentages and henceto match them with the data obtained by physical analyses. The observationshere reported pertain to characteristics of individual molecules and to the con-ditions under which they were obtained.

In all grids of wheat DNA only filaments of variable length were found. InFigure 4, a typical wheat preparation is shown in which one of the moleculesmeasures 31.4 IA or the equivalent of 61.54 X 106 avograms. What appears to besignificant is the uniform morphology of the filament. To the extent that this uni-formity represents a uniformity of molecular organization, and to the extent thatthe ineffectiveness of either proteases or ribonuclease in disrupting the filament re-flect an absence of protein or ribonucleic acid segments, the conclusion may bedrawn that the indications from physical analyses with respect to molecular sizeare fundamentally correct. The question of heterogeneity cannot be answered,however, from measurements of filament length. As might be expected, factors ofmechanical shearing affect results-so much so that attempts to use the drops col-lected through a #20 hypodermic needle from the tubes used in sedimentation studieswere unsuccessful. Grids prepared from drops representing the heaviest fractionsshowed few large-sized molecules such as were found by direct sampling.The answer to the question of molecular heterogeneity lay in the observation that

samples of pig sperm DNA contained circles. These are shown in Figures 4-7.The circles were of various dimensions. The smaller ones were easily identifiedand traced; the larger ones were more difficult to follow, and only in a limitednumber of cases could the conclusion be drawn that the circles in question were un-interrupted by breaks. The circumferences of 19 of the smaller circles were traced,and the lengths of these ranged from 0.5 to 9.7 Am, that is, from 1 X 106 to 19 X 106avograms. The circumference of the largest circle in which no evidence could befound for physical interruptions was 16.8 ,u, or nearly 33 X 106 avograms.The validity of these observations and measurements depends upon the assump-

tion that the circles were not artefacts of preparation. To test the assumption we

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360 BIOCHEMISTRY: HOTTA AND BASSEL PHoc. N. A. S.

.

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FIG. 4.-Filament of wheat DNA. Total length-31.4 ,s. A possible break in the filament is indi-cated by arrow. Lengths on either side of the arrow are 17.0 and 14.4 JU. Bar = 1 1A.

had recourse to published experiments with X phage.9 These have unambiguouslyestablished the circularity of the DNA and have also shown that circles could bereversibly broken and reformed. Moreover, the sites of closure appear to bespecific, the specificity manifesting itself either in end-to-end aggregation betweenmolecules or within them. Based on the studies of X phage, a series of experimentswere conducted in which the same DNA preparation was treated under conditionswhich were identified as either favorable or unfavorable to the formation of circles

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6

.. .. ........ ...... ..........

in~~ ~ ~ ~ ~ ~ ~ ~ .

.... ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Y..

FIG. 5.--Circles of pig DNA remaining after exposure of preparation to pH 12. Outer circlewhich may be readily traced is 12.2 M in circumference. Inner circle is difficult to trace on theprint because of the "puddles" of denatured DNA. It measures 22.3 JA in circumference.

FIG. 6.-Preparation of pig DNA which had been heated and fast-cooled, a condition un-favorable for circle formation. One circle measuring 5.48 MA may be traced on the print. In oneregion of the circle (indicated by arrow) the shadowing is poor and the double strands appear tohave separated. The linear filament begins at the wedge of the arrow and may be traced to theleft margin of the photograph.

FIG. 7.-A small circle of pig DNA measuring 2.85 ,.A. Such circles are readily identifiedand traced.

in X phage.10 In each of the experiments solutions of DNA were dialyzed to theequilibrium point against 0.6 M NaCl :0.01 M Tris buffer (pH 7.4). Favorableconditions were supplied by heating preparations to 60'C for 30 min and theneither slow-cooling for 18 hr, or slow cooling to 450C, maintaining at that tempera-ture for 3 hr, and slow cooling to room temperature over an additional 8 hr. Un-favorable conditions were supplied either by heating the preparations to 750C for10 mmn and cooling quickly, or by adjusting the pH of the DNA solution to 12.0, al-lowing it to remain as such for 30 min at 0WC, and then titrating the solution backto a pH of 7.4.Examination of the grids prepared from the various samples yielded the following

observations. "Favorable" conditions led to no evident increase in the frequencyof circles observed. As stated earlier, expression of such frequency in percentageterms would yield only fictitious values because of the small number of moleculesscanned. The result is significant inasmuch as many of the filaments found on agrid were no longer than those of X phage. If circles could form from such shortfilaments, one would have expected a noticeable increase in at least the small circlesand a decrease in the number of short filaments. This however, was not observed,

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An average of 2-3 circles per grid was found, similar to the number found in gridsprepared directly from the original solution. The same result was obtained under"unfavorable" conditions. In this set, the alkali-treated preparations provided,as might be expected, a somewhat different picture. "Puddles" were observed inthe micrographs (Fig. 5) and these presumably represent denatured DNA. Somefilaments, however, were also observed; whether these were originally circles whichhad been fragmented in the course of grid preparation or whether they were fila-ments which had remained undenatured or had become renatured cannot be de-cided. The fact that the rings did persist, however, strongly favors the interpreta-tion that the component loops remain attached at some point during denaturationand hence return to their original configuration.

Discussion.-The mechanical difficulties in preserving the intactness of verylarge DNA molecules are so well known that they require no elaboration. Thesignificant finding is that despite these difficulties two reasonably clear conclusionsmay be drawn which have an important bearing on speculations regarding thestructural organization of chromosomes. The first-and perhaps expected-con-clusion is that chromosomes do contain DNA molecules of large size and thatpresent data can only set a lower limit but not an upper limit to native values.Speculations on small DNA molecules of constant size as building blocks of longerfilaments are not supported in these observations. The second conclusion is thatthe DNA molecules are heterogeneous in size and that at least some of the mole-cules have a circular configuration. One could speculate along with Stahl thatcircularity is a general property of the DNA molecules, and explain the relativelysmall numbers of circles found on a grid as due to shearing or nuclease effects en-countered in the course of preparation. Indeed, one might also point to the pre-diction made by Hayashi et al.3 that circularity would not be confined to the bac-teria and might constitute an elementary unit of transcription. The correctness ofthis view is, however, beyond the compass of these experiments. If circularity is ageneral attribute, then some physical axis must be present, the duplication of whichwould represent the decisive event in chromosome reproduction. Such an axiscould also be constructed out of DNA.Summary.-Examination of DNA extracted from wheat embryo and boar sperm

by sedimentation analysis and electron microscopy indicates the following: (1)molecular size is variable, the upper limit being undetermined but exceeding 2 X10 avograms; (2) at least part of the DNA from boar sperm is in the form of circles.

We wish to express our thanks to Dr. P. J. Dziuk for providing us with boar sperm.

* This work was supported by the National Science Foundation (GB-1381).1 Stahl, F. W., in Proc. Soc. de Chim. Phys., 11th Annual Reunion (Pergamon Press, 1962), p.

194.2 Cairns, J., J. Mol. Biol., 6, 208 (1963).3 Hayashi, M., M. Hayashi, and S. Spiegelman, these PROCEEDINGS, 51, 351 (1964).4Stern, H., and A. E. Mirsky, J. Gen. Physiol., 36, 181 (1952).6 Marmur, J., J. Mol. Biol., 3, 208 (1961).6Bodenfreund, E., E. Fitt, and A. Bendich, Nature, 191, 1375 (1961).7Kleinschmidt, A., and R. K. Zahn, Z. Naturforsch., 14G, 770 (1959).8 Hanawalt, P. C., and D. S. Ray, these PROCEEDINGS, 52, 125 (1964).9 Hershey, A. D., E. Burgi, and L. Ingraham, these PROCEEDINGS, 49, 748 (1963).10MacHattie, L. A., and C. A. Thomas, Jr., Science, 144, 1142 (1964).

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biochemistry - pnas

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