Further studies of DNA-nucleoprotein gels and residual protein of isolated cell nuclei

0 1968 by Academic Press Inc. Experimental

533

Cell Research 49, 533-557 (1966)

FURTHER STUDIES OF DNA-NUCLEOPROTEIN GELS AND RESIDUAL PROTEIN OF ISOLATED CELL NUCLEI M. MACKAY, University Department

C. A. HILGARTNER

and A. L. DOUNCE’

of Rochester

School of Medicine and Dentistry, of Biochemistry, Rochester, N.Y. 14620, USA Received

May 22, 1967

this paper were carried out in an attempt to define as well as possible the variables that affect the gel-forming properties and to obtain further data on the of cell nuclei and DNA-nucleoprotein, nature of residual protein. \Ve believe that study of these gels is of importance because of the postulated relationship between gel structure and chromosomal structure. h model for chromosomal structure has previously been proposed [S], in which tracts of DNA are held together by tracts of residual protein consisting of an unspecified number of -S-S- cross-linked peptide chains. This structure should lend itself readily to the formation of gels like those which can be obtained under certain conditions from isolated nuclei or chromosomes. Scission of the DNA or the residual protein components would destroy the linear continuity of the DNA-residual protein array and thereby cause degelation. The residual protein component could be cleaved by the rupture of either peptide or -S-S- bonds. It has previously been emphasized in a quantitative study of DNA-nucleoprotein gels [S] that it is not the presence of histone but rather an intact DNA-residual protein structure which is necessary for gel formation. The observations reported in this paper extend previous work from this laboratory on nucleoprotein gels and residual protein [7-11, 16, 191.

THE

experiments

reported

in

EXPERIMENTAL

The materials used in these experiments were obtained either from rat liver or calf thymus. * The authors gratefully acknowledge support of the National Cancer Institute, United Public Health Service, Grants CA 00994-17 and 17S1, which made this work possible. Experimental

States

Cell Research 49

534

M. Mdmy,

C. A. Hilyartner,

and A. L. Ilorrnce

Isolation of cell nuclei Rat liver.--The procedure used for isolating

rat liver nuclei in 0.44 M sucrose and citric acid has been described elsewhere [15]. It was found, however, that the number of sucrose washes needed before the Chauveau specific gravity floatation step in order to produce a clean preparation of nuclei could be reduced to a total of two in those preparations where HCl was used to adjust the pH. These two preliminary centrifugations before the Chauveau step were carried out in an International refrigerated centrifuge at 2000 rpm (1155 g) and 1400 rpm. The quality of each preparation of nuclei was noted with a phase microscope. Calf thymus.---As described by Dounce et al. [8], calf thymus was homogenized for 5 min in a Waring blendor run at 38 volts, using 0.44 M sucrose containing sufficient 1 M citric acid to maintain a pH of 3.7-3.8. The homogenate was filtered through 8 layers of coarse cheesecloth (Curity no. 60). and after the pH had been adjusted, it was filtered once through 8 layers of no. 90 and twice through 8 layers of no. 120 cheesecloth. It was then centrifuged in the International refrigerated centrifuge at 2000 rpm. The pellet was resuspended once in 0.44 iVi sucrose, the pH was checked, and centrifugation was repeated at 1350 rpm. It was found that three subsequent. water washes (with the pH adjusted to 3.8 with dilute citric acid) were sufficient to purify the nuclei, and it was not necessary to include the Chauveau specific gravity floatation step. Extraction

procedure used with cell nuclei

The components to be extracted from or to be prepared from isolated nuclei include lipids, globulins, histones, DNA and residual protein. Lipid extraction.-In some experiments nuclei were used without being subjected to any procedures to remove lipids (Bla). In other experiments, nuclei were lyophilized, and then were extracted either at room temperature or in the cold, first with Bloor’s 75 per cent ethanol: 25 per cent ether, and then four times with chloroformmethanol 1:l (v/v) to remove lipids @lb). After air evaporation of the organic solvents, lipid-extracted nuclei were usually stored in a dessicator jar at -20°C. Extraction of globulin~~ and histones.-Globulins1 and histones in some experiments were extracted separately by a slight nlodification of the method of Dounce et al. [15] (B2a) using 0.9 per cent NaCl at pH 5.8 to remove globulins followed by 0.1 N HCl in the case of liver nuclei, or 0.2 N HCl in the case of thymus, to remove histones. (b) In other experiments, globulins and histones were removed together by a modification of the method described by Dounce and Hilgartner [8] (B2b), using 0.1 N HCl for rat liver and 0.2 Iii HCI for calf thymus nuclei. As shown in Fig. 1, globulins can be removed as efficiently from rat liver nuclei isolated at pH 3.8 by making a series of rapid extractions with 10 ml aliquots of 0.9 per cent NaCl at pH 5.8 as they can by making prolonged extractions. Thus it proves not to be necessary to expose the nuclei to the saline solution for extended periods of time (e.g. 15-30 min) as was our previous practice. Likewise, histones (Fig. 2) or globulins plus histones together (Fig. 3) can also be removed as effectively 1 The term globulin is used to indicate material soluble in 0.9 % SaCl. Ribosomesare undouhtedly also included.

DNA-nucleoprotein

gels and residunl protein

of cell nuclei

,535

by immediate extractions with 10 ml aliquots of HCl as by extractions over time periods of 15-30 min. However, although 0.1 K HCl suffices to remove the histones quantitatively from rat liver nuclei, it is necessary to extract calf thymus nuclei with 0.2 S HCl following the extractions with 0.1 S HCl in order to effect quantitative removal of the histones, as is shown in Fig. 4.

Fig. 1.

Fig. 2.

Fig. 3.

I:ig. l.--Extraction of globulin fraction in 0.9 % NaCl from liver cell nuclei with and without standing. 0, Immediate extraction; n, 30 min extraction; p , 15 min extraction. Fig. 2.-Extraction of histone in 0.1 1v HCl from rat liver nuclei after globulin extraction, with and without standing. O, Immediate extraction; n, 4 hr extraction; v , 15 min extraction; 0, Log-Log Plot of circles. Fig. Y.--Extraction of rat liver nuclei with 0.1 S HCl to remove globulins and hislonc together. O, Immediate extraction; C , 4 h extraction; o, 15 min extraction. ITigs I-8. Abscissu: Extraction number; ordinate: optical density, 2i5 my.

Fig. 5 shows that very little additional protein is removed by 0.5 S HCl after histones have been extracted with 0.2 N HCl. In the work with calf thymus nuclei reported here, two rapid extractions with 0.1 LV HCl followed by two rapid extractions with 0.2 N HCl was the procedure finally decided upon. (c) Extractions with acid urea: It was found that, after extractions of globulins and histones, additional protein could be extracted by the use of 8 M urea in 0.1 X HCl (B2c). Here, too, prolonged extraction periods do not appear to increase the amounts of protein extracted. \Vith both rat liver and calf thymus nuclei, almost all of the extractable protein can be removed with 4 rapid extractions (see Fig. 6 for the results in the use of rat liver). In the case of the rat liver nuclei, ten ml aliquots of acid urea were used in each extraction performed on the yield of nuclei from 4 rats. The pH of extraction, as measured by the glass electrode, was about 2.4. Rxtraclions of DXA.-Hydrolyzed DNA suitable for estimation by the SchneiderDische test [20] was obtained by hydrolysis of the DNA-nucleoprotein sample (or of whole nuclei) in 5 per cent trichloracetic acid at 95°C for 15 min, followed by centrifugation to remove the denatured protein (B3). \\‘hen it was desired to obtain the ratio of residual protein to DNA, if the DNA-nucleoprotein sample had not previously been subjected to lipid extraction it was delipidized prior to hydrolysis Experimental

Cell Research 49

536

M. Mackay,

C. A. Hilgartner,

and A. I,. Dounce

of the DNA by the method previously described [15]. The residue left after hydrolysis was dried by washing first with ethanol and then with ether, and was then weighed. Isolation of DNA (a) Undegraded DNA was prepared by the method described elsewhere [7], in which the DNA-nucleoprotein is made 0.5 per cent in sodium dodecyl sulfate, and

Fig. 5. Fig. 4.-Extraction first 0, 0.1 N HCl; Fig. B.-Extraction NaCl at pH 5.8. 0, Mass of nuclei only Figs 4-5. Abscissa:

of calf thymus

nuclei with

0.2 N HCl after 0.1 N HCl. Globulins

removed

A, 0.2 N HCl.

of histones from calf thymus nuclei after removal of globulins with 0.9 % 0.2 N HCl: A, 0.5 N HCl; 0, 0.5 N HCl after 4 extractions with 0.2 N HCI. appr. equal for o and A.. Extraction number; ordinate: optical density, 277 mp.

is then subjected to exhaustive digestion with the proteolytic enzyme mixture obtained from Streptomyces griseus (Pronase, CalBiochem). The resulting viscous solution is made 1 M in NaCl by the addition of solid NaCl and after centrifugation to remove the salted-out proteins, fibrous DNA is recovered by the addition of half a volume of ethanol. Thereafter, non-fibrous DNA can be recovered by the addition of up to a total of one or two volumes of ethanol. Preparation of residual protein (1) DNA-nucleoprotein material from which the globulins and histones and usually the lipids had been removed was suspended in 0.01 M magnesium acetate and was subjected to exhaustive hydrolysis with crystalline DNAase (Worthington), at either a pH of 5.8 (acetate buffer-Dla) or 7.6 (tris buffer-Dlb). The pH was generally monitored, and was periodically adjusted by addition of dilute NaOH. The residual protein, which formed a flocculent precipitate during the digestion, was washed several times with water, and as a rule was lyophilized thereafter. (2) The same procedure as the above was also used on DNA-nucleoprotein which had been extracted with acid urea (D2a or D2b). Preparation of gels and stability tests on gels DNA-nucleoprotein gels were prepared from whole nuclei, from DNA-nucleoprotein from which globulins and histones had been extracted, and from DNAresidual protein materials which had been subjected to acid urea extraction. Experimental

Cell Research 49

DNA-nucleoprotein

gels and residual protein

of cell nuclei

537

(a) Gels were prepared from whole nuclei or from globulin-and-histone-free nucleoprotein, by suspending the material in distilled water, and raising the pH to a value of 8 or 9 with dilute ammonium hydroxide. (b) Rat liver or calf thymus DNA-nucleoprotein which had been extracted with acid urea would not gel when suspended in distilled water at pH 8 or 9. However, gels could be formed from these materials either by suspending the material in 0.5

Fig. 7.

Fig. 6.-0.1

N HCl-8 M urea extraction of rat liver nuclei after removal of globulins in 0.9 y0

KaCI and histones in 0.1 A\r HCI. 0, Immediate extraction; 0, Log-Log plot of circles.

Fig. 7.-Extraction

extraction;

A, 30 min extraction;

Y, 15 min

of rat liver nuclei directly with 0.1 ATHCILS M urea (immediate extraction).

Figs 6-7. Abscissa: Extraction

number;

ordinate:

optical

density,

270 m,u.

per cent sodium dodecyl sulfate and raising the pH to 8 or 9, or by adding 2-3 mg of trypsin to the DNA-residual protein suspension in distilled water at pH 8 after which the material gradually became a gel. A description of the qualitative tests for gelation is given by Dounce and Hilgartner [S]. Q uantitative studies of the gels [9] were not carried out on this work. (c) The stability of the gels was tested by observing their behavior in 1 M NaCl or in 8 M urea, according to one or another of the following procedures: (i) by adding enough crystalline NaCl to bring its concentration in the gel to 1 M; (ii) by diluting the gel with a very strong NaCl solution so that the final concentration of NaCl was 1 M; or (iii) by dissolving sufficient urea in the gel to make the latter 8 M in urea when brought to a predetermined volume, usually twice the original volume. In (ii) and (iii), the control gels were similarly diluted with distilled water only. Formaldehyde treatment of acid urea extracted DNA-nucleoprotein of rat liver nuclei and recovery of DNA The residue left after extraction with 0.1 N HCl-8 M urea was suspended in 1.3 per cent formaldehyde -0.01 M NaCl for various time periods and sodium dodecyl sulfate (dupanol) was added to a concentration of 0.5 per cent, before raising the Experimental

Cell Research 49

538

M. Mackay,

C. A. Hilgartner,

and A. 1,. Dorznce

pH to 7-8. The residue was then digested by adding pronase to the dupanol solutions, and NaCl was added to a concentration of 1 M to precipitate undigested or partly digested protein that had been freed from its attachment to DNA. After centrifugation, DNA was recovered from the supernatant solution by adding 4 vol of ethanol, followed by the addition of f vol more of ethanol if the half volume had failed to precipitate any DNA. If fibrous DNA was obtained it was removed and ethanol was added up to a total of one volume to precipitate the non-fibrous DNA. Polarographyl (a) Sulfhydryl sulfur: A weighed sample of lyophilized residual protein was treated with 6.0 ml of pH 7.0 buffer (containing 0.1 M HCl, 0,i M Tris, and 8 M urea), plus 1.0 ml 0.25 per cent gelatin, plus 1.0 ml 2 x 1O-s M methylmercuric iodide, at ambient temperature, for a period of two days or more. A blank was prepared similarly except that it contained no sample. Blank and sample were then centrifuged, and the diffusion currents i, and i, were determined on the supernatant fluid by means of a dropping mercury electrode. - SH-blocked residual protein was subjected (b) Disulfide sulfur: The sedimented to exhaustive dialysis against water, re-lyophilized, and reweighed. It was then treated with 6.0 ml of pH 9.2 buffer (containing 0.5 M KCl, 0.2 M (NH,),SO,, and 8 M urea), plus 1.0 ml 0.25 per cent gelatin, plus 1.0 ml 2 x 1O-3 M methylmercuric iodide, at ambient temperature, for a period of two days or more. As before, a blank was prepared similarly, except that it contained no sample. Blank and sample were then centrifuged, and the diffusion currents determined on the supernatants. (c) Total sulfur: The residual protein sample was treated with pH 9.2 sulfite-urea buffer without previously being blocked with methylmercuric iodide, thus determining the total sulfur content. (d) A later modification of the method of determining disulfide sulfur gave more residual protein was reproducible results. In this modification, the ~ SH-blocked dissolved in 7.6 per cent NaOH prior to treatment with sulfite-urea, methylmercuric iodide, and gelatin. RESULTS

I. Experiments Nuclei

With DNA-Residual Protein Gels. Effect of Extraction With Various Media on Gel-Forming Capability

of

In previous work we removed globulins from cell nuclei with 0.9 per cent KaCl at pH 6 (a pH of low proteolytic activity [17, 241) and then removed histones in 0.1 or 0.2 9 HCl [lS] at a temperature as close to 0°C as possible in each case. \Ve routinely observe that the removal of globulins does not destroy the capacity of liver or thymus nuclei to form gels at pH 8 to 9; and gels formed from nuclei extracted in this way are stable in 1 M NaCl and in 8 M urea. 1 These polarographic determinations were carried out in the laboratory of Dr William Forbes by Dr John Kirman, Mrs K. Oka and Dr C. R. Hamlin. The latest and most accurate determinations were done by a procedure worked out by Dr Hamlin. Experimental

Cell Research

49

If the histones are removed in 0.1 N HC1 from liver nuclei or in 0.2 A’ HCl from calf thymus nuclei following removal of the globulin fraction with 0.9 per cent NaCl at pH 6.0, the nuclear residue from rat liver may or may not form gels on raising the pH to 8-9; in the case of thymus nuclei, gels always formed. The cause for variability of results with liver nuclei is not get certain, but autolytic reactions during the period of globulin extraction (proteolysis and/or nucleolysis) may well be involved. It has been found possible to remove the globulin and histone fractions together by extracting the nuclei directly in 0.1 X HCI in the case of liver nuclei [23], and we have found that the same is true in the case of thynus nuclei extracted with 0.2 :V HCl (see Fig. 3 for the use of rat liver). Rapid extractions were found to remove as much protein as prolonged extractions. \\‘hen the globulin plus histone fraction is removed from liver nuclei by exhaustive extraction in 0.1 9 HCl, the extracted nuclei routinely form gels when the pH is raised to a point between pH 8 and pH 9. Thymus nuclei also routinely form gels in the same pH range after exhaustive extraction \vith 0.2 S HCI. Gels formed from either liver or thymus nuclei are stable in 1 X NaCl and 8 M urea. \Vhen liver or thymus nuclei are exhaustively extracted with acid as just described to remove histones and globulins together, the addition of 1 M NaCl before raising the pH to a value between 8 and 9 prevents the immediate formation of gels \vhen the pH is subsequently raised. The chief reason for this seems to be that the interaction of the sodium chloride with the protein component of the DXA-residual protein complex in acid solution causes this complex to remain insoluble and inc.apable of hydration after the pH is raised. However, gels can be subsequently formed by the addition of sodium doderyl sulfate or of trypsin, both of which would affect the protein component rather than DKA. In the case of thynus nucleoproteiil, gels thus formed by treatment with trypsin are stable for long periods of time: we have indeed thus far been unable to destroy gels made from calf thpmus nuclei by the use of trypsin. On the other hand, gels made from rat liver nuclei and also from chicken erythrocgte nuclei [7] are slowly degelled by trypsin. Pronase treatment of whole nuclei from rat liver or calf thymus prevents gel formation; when added to the gels made from whole rat liver nuclei, pronasc slowly destroys the gels. Gels made from calf thymus nuclei can also be destroyed with pronase, but this is somewhat variable, perhaps because of a mixing problem [9:. Following the extraction of the globulin plus histone together in 0.1 A’ HCl Experimental

Cell Research 49

540

M. Mackay,

C. A. Hilgnrfner,

and A. L, Dounce

with rat liver nuclei or in 0.2 Al’ HCI with thymus nuclei, we have extracted the nuclear residue further using 0.1 N HCI-8 M nrea solution. (See Fig. 6 for the extraction curve obtained with rat liver nuclei.) Such extraction fractionates the so-called residual protein into two portions, In the case of rat liver nuclei isolated at pH 3.8, approx. 3/5 becomes soluble and Z/S remains together with the DNA in the nuclear residue. The ratio of residual protein t.o DNA is thereby changed from about 25 to a value between 0.7 and i-0. When the globulin and histone fractions are removed separately from rat liver nuclei, the fraction of residual protein that is soluble in 0.1 LV HC1-8 ,V urea is only in the neighborhood of $ to 4 of the total residual protein. This finding may mean that the acid-urea-soluble fraction of the residual protein consists in part at least of acid-denatured globulin. After four extractions with 0.1 il: HCl--8 Al urea, neutralization of the residue in the presence of 0.5 per cent sodium dodecyl sulfate to a pH between 8 and 9 causes the formation of gels as usual, but such gels usually are not stable in molar NaCl owing to insolubility; they may, however, be partially stable in 8 M urea. Also such gels dilute out more rapidly than gels made from whole nuclei or from the usual DNA-residual protein residue which remains after extraction of the globulin-histone fraction in 0.1 nr HCl. This indicates that the DNA-protein structure of acid-urea-extracted gels has been damaged in some way, probable by some kind of denaturation or conformational change involving the protein component. It is possible, nevertheless, to obtain entirely fibrous DNPl from the HCIurea treated residue by digesting exhaustively with pronase and then isolating DNA by the use of 0.5 per cent sodium dodecyl sulfate and ethanol it;]. The ~ustonlar~ fibrous nature of this DNA indicates that it has not been grossly denatured, although slight depurinization may have occurred. Therefore we surmise that the chief damage to the DNA-residual protein structure caused by the 0.1 N HCl--8 M urea is concerned with the protein component of the structure. Such damage might indicate cleavage of sensitive peptide hands involving aspartic acid, for example, or conformational change, or both. On the other hand, we have exhaustively extracted a sample of whole liver nuclei isolated at pH 3.8 with 8 M urea in 0.1 N HCX, until each subsequent extract removed very small amounts of protein. The curve of extraction is shown in Fig. ‘7. In this case the residual material did not form a gel on neutralization to a pH value between 8 and 9, but when sodium dodecyl sulfate was added to a concentration of 0.5 per cent before neutralization, the usual gel formed. This gel remained stable in the deterrent solution for Experimental

Cell Research 49

I),VA-nucleoprotein

gels and residucrl protein

of cell nuclei

511

several weeks. At the end of this time it was found to be stable in molar h’aC1 and 8 M urea. Either the direct extraction of the nuclei with 0.1 LV HCl-8 M urea failed to produce the damage caused by the extraction with 0.1 X HCl8 M urea subsequent to extraction of the globulins and histones in 0.1 I\~ HCl, or whatever damage had been done was reversed on prolonged standing at room temperature. The most likely type of damage that could be reversed would seem to be some type of conformational change in the protein component. Although gels made in 0.5 per cent sodium dodecyl sulfate from 8 M urea0.1 S HCl-extracted residue of rat liver and calf thymus nuclei that had previously been extracted with HCl to remove histone and globulin \vere usually partially stable in 8 M urea, they were not stable in 1 3f IVaCl, as has already been stated. It was, however, possible to form gels \\-ithout adding sodium dodecyl sulfate by adding a small amount of trypsin (3-4 mg) to the residue suspended in water and then raising the pH to 8-9, or by adding the trypsin to the viscous solution formed by the residue in water at pH 8-9. These gels were at least partially stable in 1 M NaCl. It is likely that the salting out of the DK\‘A-residual protein complex when NaCl is added to the gel made LIP in sodium dodecyl sulfate is the cause of instability of these gels in 1 ,I2 KaCl. \Ye cannot state at present why the gel made from whole rat liver nuclei that had been extracted directly with 0.1 S HCl in 8 ~11urea \I-as soluble and stable in 1 111NaCl. I:se of formaldehyde to investiynte damage to .D,YA caused by extraction DLVA-nucleoprotein with 0.1 S HCl-8 M urea

of

It was reasoned that if rupture of hydrogen bonds occurred when n-hole nuclei or DN;A-nucleoprotein was extracted with acid urea, the addition of formaldehyde before reneutralization would prevent recombination of the strands and result in the recovery of non-fibrous DNA from the acid ureaextracted material. Nuclear residues which had been extracted with 0.1 ~1~ HCl to remove the globulins and histones together and then with acid urea were therefore treated with 1.3 per cent formaldehyde in 0.01 M NaCl, and following neutralization the DNA was isolated by the use of pronase followed by dodecyl sulfate, etc. [7]. In this case mainly fibrous DNA was obtained, showing that extensive denaturation had not occurred. However, when the globulins were extracted separately at pH 5.8 with 0.9 per cent NaCl and the nuclei were washed with water before proceeding to the histone extraction, only non-fibrous DNA was obtained after subsequent acid-urea extraction and formaldehyde treatment, indicating that here extensive denaturation 35

- G81805

Experimental

Cell Research 49

542

M. Mackay,

C. A. Hilgartner,

and A. L. Dounce

had occurred. Since mainly fibrous DNA had been obtained after formaldehyde treatment when all extraction steps remained the same as above except for the omission of the water wash, it appears likely that removal of NaCl before the HCl extractions and subsequent acid-urea treatment caused the failure to recover fibrous DNA. The stabilizing effect of NaCl on Dr\‘A is well known. Two nucleoprotein samples that had not been treated with 0.1 S HCl8 ,‘I# urea were also treated with 1.3 per cent formaldehyde in 0.01 i1f NaCl. Here the globulins and histones had been removed separately. In one case there was a water wash between removal of the globulins and removal of histones while in the other case this wash was omitted. After the addition of the formaldehyde, the nucleoprotein was neutralized and DNA was isolated by the use of pronase, followed by sodium dodecyl sulfate and alcohol [S]. In both cases fibrous DNA was obtained. This experiment indicates that the residual protein protects the DNA to a considerable extent at least against the denaturing action of the 0.1 N HCl used in extracting histones. In the case of material extracted subsequently with acid-urea, it appears likely that there may be a reversible denaturation which can be prevented if the NaCl concentration is not allowed to fall too low. As a control, pure calf thymus DNA was subjected to various extracting media used in the above studies to see what the ef’fects \vould be on DNA alone. In each case the DNA was recovered by bringing the solution to 0.5 per cent sodium dodecyl sulfate, raising the p&I to 7-5, adding 1 M NaCl and precipitating the DNA4 with ethanol. DNA dissolved in a small aliquot of mater dispersed rapidly in a solution of 8 111urea-O.1 N HCl. About one-third of the DNA was recovered in 2 vol ethanol and additional non-fibrous DNA was precifibrous form with 1. pitated by adding one or two volumes of ethanol, indicating rather severe denaturation. Attempts to dissolve DNA in 0.1 N HCl alone were unsuccessful, since a rubbery mass was formed when the DNA was added in the dry state and a particulate but sticky precipitate was formed when the DN4 was dissolved in a small amount of water before being added to the 0.1 N HCl. Since these precipitates were not soluble in 0.5 per cent sodium dodecyl sulfate at pH 8, the DNA could not be recovered in its original form.

Damage to DIVA-residual

protein

by freezing

Failure to obtain gels from DNA-nucleoprotein residues which had been frozen raised the question of possible damage being done to DNA when the Experimentaf

Cell Research 49

DATA-nucleoprotein

gels and residual

protein

of cell nuclei

543

latter is frozen in an aqueous solution. Since it was found that pure DNA4 dissolved in water and frozen could be subsequently reprecipitated in fibrous form, it appears that freezing damages the protein component of DNAresidual protein rather than the DNA, probably by causing some kind of conformational change leading to insolubility or failure of the protein component to become hydrated.

Extraction of SflCl

of rut

liver

nuclei

isolcrted

nt pH

3.8 with

various

concentrations

Globulins were extracted from the nuclei using 0.9 per cent KaCl at pH 5.8. This extraction was followed by extractions with 1 M, 3 M, or 5 JI NaCl at this PH. Only trace amounts of DNA were detected in the 1, 3 and 5 M NaCl extracts, whereas trichloracetic acid-precipitable proteins hvere found in all the extracts as would be expected, since the NaCl must have extracted the globulin fraction, as well as at least some histone. The stability of rat liver nuclei and DNA-nucleoprotein in strong NaCl solutions was tested by making gels from these materials in various concentrations of NaCl, with DNA concentrations being approximately equal in all cases. Thick gels xvere made by adding enough alkali to bring the pH between 8 and 9, and the appropriate amount of a concentrated NaCl solution was added to bring the final concentrations in the test gels to 1, 2, 3 or 4 ~11 NaCl. The gels made from whole nuclei were stable in all concentrations of NaCl. However, on being diluted to 1 : 5 with more 4 M NaCl, the gel made up in 4 M NaCl was not stable. The undiluted DNA-nucleoprotein gels were stable in 1 M, 2 &I and 3 M NaCl but were not stable in 4 M NaCl. The DNA-nucleoprotein gels in 1 M, 2 M, and 3 M SaCl lost their gel properties on being diluted 1 to 5 in these same solvents respectively. Fibrous DNA was however recovered from all of the degelled samples of gels made from both whole nuclei and nucleoprotein as described above, by using pronase treatment followed by the use of 0.5 per cent dodecyl sulfate and alcohol to isolate the DNA [7], indicating that any gel instability was probably not due to damage to the 1)N.I components. This would be anticipated from the known stabilizing action of XaCl on DNA.

Effect

of disulfide

bridge-cleaving

reagents

on 1)X&nucleoprotein

gels

Bailey’s reagent [28] which cleaves disulfide bridges at pH 7.6 by means of sulfite in the presence of tetrathionate in 8 M urea, results in the sulfonaExperimental Cell Research 49

544

M. Mwkay,

C. A. Hilgartner,

and A. L. llounce

of both sulfurs of a cleaved disulfide bridge. In several experiments, Bailey’s reagent was found to Iyse gels made from whole rat liver nuclei and from rat liver D~A-nucleop~otein (from whic.h the globulins and histones had been removed by means of 0.1 N HCI). Bailey’s reagent was also found to render soluble the residual protein of cell nuclei (prepared by exhaustive DNA-ase digestion of DNA-nucleoprotein), which remains very insoluble if its disulfide bridges are intact. Bailey’s reagent has subsequently been used routinely in our laboratories to destroy gels made from rat liver and from calf thymus nucleoprotein, and to solubilize residual protein. These experiments have been repeated a number of times. Two closely related new reagents for the disruption of disulfide bridges, described by Cleland [5], are dithioerythritol and dithiothreitol. At neutral or alkaline pH, these reagents ~~ndergo successive interchanges with disulfide bridges, resulting in the conversion of a disulfide bridge into two free sulfhydryl groups, with the accompanying formation of a very stable disulfidecontaining six-membered ring from the dithio-sugar. Several experiments were done to examine the effects of dithioerythritol on DNA-nucleoprotein a suspension of rat liver gels and residual protein. In one experiment, residual protein in 8 1%urea, at pH 9, was made 0.023 M in dithioerythritol. After centrifugation, it was found that about half the bulk of the residual protein had dissolved. The sediment was resuspended in 8 M urea at pH 8.2, and made 0.017 M in dithioerythritol; apparently under these conditions, no further solution occurred. A gel was prepared by suspending the yield of thymus nuclei obtained at pH 3.8 from 60 g calf thymus by the method of Dounce et al. [9] in 100 ml water, and adding enough dilute NH,OH to produce a pH of approximately 9.0. Two ml of this gel was treated by stepwise additions of 0.2 ml of 0.1 ill dithioerythritol (a total of 9 additions). The material took on an amber hue partial degelation. A control sample treated by stepwise as it underwent addition of 0.2 ml distilled water underwent no color change or degelation. Using a gel made by suspending the DNA-nucleoprotein obtained from the livers of six rats in a total volume of 10 ml and raising the pH to 9.0 with dilute NH,OH, it was found that addition of solid dithioerythritol produced only a slight decrease in pH, When dithioerythritol was added to a final concentration of 0.2 M, this very concentrated gel underwent partial degelation. When the reagent was added to a concentration of 0.5 M, the gel retained only very slight and slow recoil which might have been due to the DNA component alone. At this point the concentration of DNA-residual protein was about 2 mg/ml. tion

Experimenfnl

Cell Research 49

I)i~l~~-nucleoproteiIr

gels and residucrl protein

of cell nuclei

545

RESULTS

II. Experiments

with Residual Protein Itself. Effect of Extraction of DNA-Nucleoprotein With 8 M Urea-O.1 N HCZ on the Residual

Protein

Component

Rat liver DNA-nucleoprotein was estracted \vith 8 41 urea which was made 0.1 N HCl in the hope of removing any extraneous proteins which might be present, including nuclear membrane proteins. In later experiments, acid urea was found to extract more protein from DNA-nucleoprotein than did 8 M urea at pH 6 or at pH 3.3. In the case of the liver cell nuclei isolated at pH 3.8, as previously stated, about l/2 to 3/S of the residual protein remaining after extraction of globulins and histones with 0.1 N HCl is estracted by acid urea, lowering the ratio of residual protein to DNA from about 2.3 to 0.7-1.0. ,I careful study \vas done to establish the nature of this cft’ect. The main questions to be answered were (1) does acid urea treatment produce a valid chemical fractionation of the residual protein? and (2) does acid urea produce detectable degradation of the residual protein? These questions were tested by performing amino acid analysis on whole residual protein, the soluble extract, and the insoluble residue, by performing gel filtration studies on these materials, by examining the gelability of the insoluble residues, and by estimation of the deoxyribonucleotidcs in the soluble extracts. Amino acid nnrtlyses Amino acid analyses were performed on both rat liver and calf thymus DNA-nucleoprotcin material. l The DNA in the whole residual protein and in the insoluble residue after extraction with acid urea was in each instance removed by hydrolysis with 5 per cent TCA at 95°C. The acid urea was removed from the soluble extract by exhaustive dialysis, and this acid-urea soluble fraction was recovered by lyophilization. Tables 1 and ‘2 give the results of the amino acid analyses of these materials. The similarity of the analyses indicates that the portion of residual protein extracted in the HClurea is not appreciably different from the unextracted portion in respect to amino acid composition. 1 We are indebted to Dr H. Takei who perform4 of Dr William Forbes.

these amino acid analyses in the laboratory

Experimental

Cell Research 49

546

M. Mackay,

Column

C. A. Hilgartner,

and A. L. Dounce

chromatography

All chromatographic analyses were carried out by means of ascending column chromatography using Sephadex G-200 and monitoring the absorbancies of the column effluents automatically by means of a Gilford spectrophotometer recording at 280 and 260 rnp (to be described in detail elseTABLE

1. Amino

acid

analysis

extract

(RPS),

Amino acid Lysine Histidine Ammonia Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half cystine Valine Methioninc Isoleucine Leucine Tyrosine Phenylalanine Cysteic acid

of’ rat

liuer

and insoluble

residual residue

protein

(RLRP),

soluble

(RPR).

RLRP & CY”)

RPS (6 “0)

RPR (g %I

7.31 2.73

7.82 2.94

7.88 2.98

7.35 9.58 4.29 5.16 313.04 4.86 4.49 5.01 1.48 5.46 1.04 3.92 8.30 4.15 4.94 6.84

7.55 10.25 4.50 5.07 13.03 5.12 5.99 5.34 0.35 6.07 2.35 5.10 8.90 4.10 5.39 0.13

8.16 10.17 4.58 6.33 14.21 4.32 4.33 4.55 5.11 1.67 4.85 9.66 4.76 6.42

where). Effluent volumes were measured by means of a drop-counting fraction collector. The samples of whole residual protein, and of insoluble residue, were dissolved in Bailey’s solution and then diluted with an equal volume of column eluant. Except for the acid-urea soluble extract from rat liver nuclei all runs were made using a pH 9.0 buffer composed of 0.1 IV NH,OH-0.2 N NH, Cl as eluant; the rat liver soluble extract was run using an eluant composed of 2 M urea-O.01 N HCl. Whole residual protein.-IThole residual protein (not delipidized) obtained by method B2b and Dl (digestion with DNA-ase at pH 7.6) from both rat Experimenfal

Cell Research 49

DNA-nucleoprotein

gels und residual protein

of cell nuclei

547

liver and calf thymus was entirely excluded, having elution volumes 11, equivalent to those of the samples of Blue Dextran (Pharmacia) which were used to check and calibrate the columns. Soluble e&wt.-The protein of the acid-urea-soluble extract of rat liver IDEA-n~~eleoprotein was entirely excluded and therefore, by this test, showed TAClI,1: 2. dmino

mid analysis of calf thymus residual protein insoluble residue (RPR). CTRP (g ?A)

Amino acid Lysine Histidine Ammonia Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glgcine Alaninc Half qstine Valine ~Iethionine Isoleucine Leucine Tyrosine Phenylalanine Cysteic acid

RPS (not done)

(CTRP)

rend

RPR (g 76)

7.87 2.97

9.89 3.29

7.92 9.97 5.23 5.82 15.25 4.89 4.62 5.52 1.29 4.70 1.09 4.10 9.38 4.52 4.85 -

7.17 9.01 4.43 5.59 13.28 4.59 4.00 4.86 1.27 4.05 1.00 3.13 7.85 4.33 4.61 7.65

no evidence of containing small fragments. (However, since it had been subjected to exhaustive dialysis, degraded fragments of small size might have been lost.) On the other hand, the soluble extract of calf thymus DNAnucleoprotein showed only a very slight excluded peak, and otherwise apparently consisted of degraded protein materials of polydisperse nature. Insoluble residues.-The insoluble residue of rat liver lIEA-nucleoprotein contained an excluded component, a large amount of polydisperse material of intermediate sizes and moderate amounts of small fragments which were retarded as much as the salt. Thus this material may have undergone con-

548

Al. ~~acka~, C. A. Hilgartner,

and A. L. Dounce

siderable degradation in the course of extraction with acid urea. The insoluble residue of calf-thymus DNA-nucleoprotein on the other hand was almost entirely excluded, but in several instances did contain small amounts of somewhat retarded (and therefore presumably degraded) materials. These results are summarized in Table 3. TABLE 3. Results from gel filtration of whole residual protein e.hmt (RFS), und insoluble residue (RPR). Rat liver Sample

K,

HP RPS RPR

0 0 0

(RP), soluble

Calf thymus

1

2

3

KLI

-i--t++ i--t++ + + -t +

0 0 + i

0 0 i-

0 0 0

-t -t -t

1

2

3

-f -f -t + + t-t-t-b

0 -t -t + + +

0 -I- -t 0

t i-

Ve- vo KD=--. Vi 1, 2, and 3 stand for relative positions in the et&ion curves (Le., “excluded”, “intermediate” and “retarde , Rough quantities of UV-absorbing materials are indicated by a scale going from 0 to + 1 + +. RP, residual protein; RPS, portion of residual protein soluble in 0.1 N HCl-8 M urea; RPR, portion of residual protein insoluble in 0.1 N HCl-8 M urea. See text.

Estimution

of nucleic acid fragments

in the soluble acid-urea estrrrcts

As a further means of establishing whether or not detectable degradation of DNA-nctleoprotein is brought about by extraction with acid urea, several experiments were done to estimate the quantities and types of nucleic acid fragments present in the soluble extracts. The acid-urea extract was first dialyzed against 5 changes of distilled water for a total of 12 h, and then lyophilized. In one experiment, one milligram of lgophilizecl extract was heated in 4 ml 3 per cent perchloric acid at 95°C for 15 min. The spectrum of the hydrolgsate, taken on a PE 20% recording spectrophotometer, indicated the presence of nucieotides. A yuantitative assay for RNA nucleotides was not done, but a Dische test showed that 9 per cent of the extract by dry weight consisted of (leoxyribonLIcleoti(les. In another experiment with the lyophilized extract, base ratios of the nwleic acid component were measured spectrophotometricallp by eluting the spots obtained by means of descending paper chromatography following KOH and perchloric acid digestion of 10 mg of extract. Some RNA was found in the KOH hydrolysate, but the concentrations were too low for base

.lI,VA-nucleoprotein

gels nnd residual

protein

549

of cell nuclei

analysis. The base ratios listed in Table 4 were found for the hydrolyzed DNA in the perchloric acid digest. These analyses indicate that the acitl-urea soluble extracts contain at least measurable quantities of degraded DNA. The base-ratios suggest that this DNA%might be single stranded. It should be noted however that the acid-urea TAISI,I- 4. Base ratios

h/T G/C Pu/Py A+T G+C

of DAVA extracted from the acid-urerr JIN.4 residual protein.

soluble

0.718 1.65 1.08

(Lower than for rat liver DSA) (Higher than for rat liver DNA) (WIthin the range of normal rat liver

1.02

(Lower

than for rat liver

fruction

of

DSA)

DSA)

extraction may produce some depurinization, and that any purines liberated in this way would have been lost in the dialysis of the acid-urea extract, thus altering the base ratios of the remaining material. Polurographic

determincction

of -S-S- and -SEI groups

in residrml

otein

Polarographic analysis for -S-S- and -SH groups were carried out on samples of residual protein isolated by several methods. The methods of extraction are indicated in the tables and footnotes to it. The results which arc shown in Table 5, are reported as micromoles of -SH, -S-S-, or total -SH plus -S-S- per g protein. Proper electrophoresis

of residual

protein

components

In a previous paper [8], it was pointed out that finding t\vo components on electrophoresis of whole rat liver residual protein does not necessarily prove that there are two non-identical types of protein chains in this protein, since even if the residual protein consisted of two identical peptide chains, reduction of the disulfide bridges might cause the addition of more thioglycollate molecules to one chain than to the other, resulting in an artifactual difference in the electrophoretic mobility of the two chains. Electrophorcsis of residual protein solubilized by means of Bailey’s reagent would be espetted to answer this theoretical objection, since both sulfurs of a disulfide bridge arc sulfonated by the reagent, resulting in equivalent modification of the two chains. Lipid-containing rat liver residual protein prepared by method B2b and 111 (pH 5.8) from pH 3.8 nuclei, and solubilized by Bailey’s reagent \vas Experimental

Cell Research

49

41. ~~ac~a~, C. A. ~ilgart~ler,

550

and A. I,. Dounce

subjected to electrophoresis on cellulose acetate paper in glycine buffer, 0.04 M at pH 9.0; and as before, this resulted in a ~olnponel~t staining at the origin, plus two other components, which migrated approximately equal distances in opposite directions. This finding supports the notion that there are at least two kinds of peptide chains in residual protein. TABLE

5. Polarographic

determination r~s~d~ffl

Source

Sample

RL RL

RP’ RPb (RPb) RPRC RPS’

RL

RPRb

RL RL RL

RL CT CT

RPS’ RPRb RPS’

of

-SH and -S-S groups in

p~o~~~n.

Extraction

CSH (P~iut)

G3s (D-k)

ct

Bl, B2a, Dla Bl, B2b, B2c, Dla Bl, B2b, B2c, Dla

lost 58.3

11.2

69.5

B2b, B2c, B3, D2b B2b, B2c, dialysis

41.6 35.4

19.9 16.9

61.5 52.3

B2b, B2c, D2b See footnotes and B2b, B2c, dialysis See footnotes and B2b, Bk, D2b See footnotes and B2b, B2c, dialysis See footnotes and

21.4

77.1

98.5

3.6

6.8

10.4

51.7

24.3

76.0

19.3

35-1

51.7

73.7

11.2 11.4

84.9

text test text text

RL, Rat liver; CT, calf thymus; RPR, portion of residual protein insoluble in 0.1 N HCl-8 M urea; RPS, portion of residual protein that is soluble in 0.1 N HCl-8 M urea (see text). a Dialyzed against water and recovered by IyophiIizing. b DNA was removed by digestion with DXAase I at pH 7.6 and then the residual protein was delipidized. ’ Dried with EtOH (2 x ) and ether (2 x ).

Gel filtration

to estimate the molecular

weight of residual protein

It is known that gel filtration media such as the Sephadex cross-linked dextrans and the Bio-Gel polyacrylamide media can give information from which one can estimate the Stokes radius of the molecules, and that in at least certain cases, molecular weights can be obtained from these data which agree fairly well with molecular weights estimated by ultracentrifugatio~ [l, 211. An attempt was made to estimate the Stokes radii and the molecular weights of the components of solubilized residual protein. The lipid-containing residual protein, which was prepared by method B2b and Dl (pH 5.8) from pH 3.8 rat liver nuclei, was made soluble by means of Bailey’s reagent. Chromatography was performed in 0.04 M glycine buffer, pH 9.0, using in Experimental

Cell Research 49

DNA-nucleoprotein

gels trnd residual protein

of cell nuclei

551

turn Sephadex G-25, G-50, G-75, G-100, G-200, and Rio-Gel P-300 in downlvard-flow columns, collecting fractions of 3 ml by drop-counting. The positions of the protein peaks were established by spectrophotometric estimation of the fractions at 280 and 260 m,u. In every case, the residual protein was excluded from the gel-filtration medium, permitting the tentative conclusion that the components of residual protein are very high in their molecular weights (over 400,000) and/or hare very high axial ratios. The experiments described above were done with residual protein materials from \vhich the nuclear lipids and RNA had not been removed. 4 later esperiment was done in order to rule out the possibilities that nuclear lipids or RNA (or both) might have influenced these previous results. ,I sample of residual protein from which the lipids had been extracted was rendered soluble by treatment with 0.1 X NaOH, the pH was promptly lowered to 9.5 by adding solid NH,Cl, and the sample was then put through a column of Sephadex G-200 arranged for upward flow; monitoring of the column effluent n-as done automatically by means of a Gilford spectrophotometer fitted with flow cells and set up so as to record the absorbancies at 280 and 260 mp. This experiment resulted in an excluded peak of considerable magnitude which according to spectrophotometric analysis appeared to correspond to high molecular weight protein free of nuclei acid. The material of this peak gave positive quantitative ninhydrin [27] and Ellman biuret tests. There \vas also a polydisperse retarded component lvhich showed spectral characteristics of nucleic acid. Material corresponding to the beginning of the peak showed contamination with peptide material; the amount of such contaminating peptide diminished rapidly for material further under the peak, as judged by the quantitative ninhydrin test, the biuret test, and especially the spectral curves. It is thus evident that there is an inherently high molecular weight protein component present in the residual protein fraction \vhich is not merely an aggregate of protein of lower molecular weight with lipid or RNA. It should be noted that this residual protein sample \vas not treated with Railey’s reagent, but the treatment with 0.1 S NaOH presumably brought about cleavage of the disulfide bonds. DISCUSSION

The experiments reported in this paper fall into two somewhat complementary categories, i.e. experiments on DNA-residual protein gels, and work on the residual protein itself. Both categories confirm and extend previous observations on DNA-residual protein structure and the relationship of this Experimental

Cell Research 49

562

31. Mmkay,

C. A. Hilgartner,

and A. L. Dormce

to chromosomal structure (see especially [S, 91). The various experiments will be discussed in the order in which they are listed in the section on results. The fact that nuclear gels can be obtained after extraction of rat liver ~1eoxyribo~L~cleoprotein with 0.1 ,V HCI-8 M urea, and that such gels are at least partially stable in 8 M urea, indicates to us that the DNA is still firmly attached to the residual protein component which remained insoluble in the HCl-urea. The observation that the DNA is protected against acid denaturation by the insoluble part of the residual protein indicates the same thing. Effective protection against the results of acid denaturation could hardly be afforded by a protein like histone, which is dissociated from the DNA by acid, but it might be given by a residual protein component that is attached to DNA at one or both ends of the double-stranded helix in such a \vay as to keep the two strands in register after hydrogen bond rupture, so that renatLIrati~~n could easily occur. In this connection it should be noted however, that the residual protein affords DNA very little or no protection against heat denaturation [cij. Possibly the residual protein could favor renaturation after acid denaturation, merely by preventing one or both of the DNA strands from curling up after rupture of the hydrogen bonds by acid. In this paper we also report the efrects of various reagents on the gelability of DNA-nucleoprotein as well as the effects of the same reagents on the stability of the gels in question in 8 ,V urea and in 1 ;V NaCl. We do not at the present time completely understand the apparently anomalous lack of stability of certain of the DN=i-nucleoprotein gels in 8 ill urea or strong iYaC1 or on simple dilution which was observed, but it appears certain that one factor of importance is damage to or change in the residual protein component of the gels, such as could be caused by conformational change or denaturation. This is well illustrated by the phenomenon of gel formation caused by the addition of trypsin which can occur under certain conditions. The trypsin evidently removes a non-essential part of the residual protein which can impede gel formation presumable after conformational change in the residual protein. In the case of DNL4-nucleoprotein gels from liver, further addition of trypsin or prolonged standing without adding more enzyme results in degelation, apparently because of eventual tryptic cleavage of vital portions of the residual protein that link DNA strands together. In the case of gels made from thymus DNA-nucleoprotein, such cleavage for some reason does not take place. The fact that it does not take place offers a very good control against the possibility that the eventual destruction of gels of rat liver DNA-nucleoprotein by trypsin could be the result of the action of contaminating DNA-ase. This situation has also been discussed elsewhere [7 1. Experimental

Cell liesearch 49

I)i~T~il-ntrcleoprofeir7 gels and residual profein

of cell nuclei

553

\Vhy gel formation should be interfered with by very strong NaCl is not clear, but the phenomenon is probably concerned in some way with conformational change in or dehydration of the protein component of the DNAresidual protein. There is no evidence iIl~licating that strong NaCf solutions cleave the residual protein from the DNA, and statements made in the past by other authors about the dissociation of DNA-nucleoprotein that is brought about by strong salt solutions should be taken to refer only to the dissociation of histone from the DXi4, except in cases where the residual protein has already been cleaved by enzymatic reactions or other means. Our present and previous observations on the deslruction of DNA-nucleoprotein gels by disulfide-cleaving reagents give strong presumptive evidence for the crucial role of disnlfide linkages in gel structure. In spite of a quantitative variability which xve do not yet understand, the polarograpllic results presented in this paper seem to leave no doubt of the occurrence of both -SH and -S-S- bonds in whole residual protein as well as in both the soluble and insoluble residual protein fractions obtained by extracting the DNAresidual protein complex with acid urea. %Thg the acid urea soluble fractions of rat liver residual protein show less -SI-I and -S-S- by analysis than do the acid urea insoluble residue cannot yet be stated, except for the possibiIity that much of the acid-urea-soluble material may not be residual protein at all, as suggested subsequently. We have found previously [‘i] that disruption of DNA-nucleoprotein gel structure by -SH ~onipoL~nds is not necessarily caused by disulfide bond rupture, since in certain instances, especially at low concentrations of the -SH compound used, proteases activated by -SE-I compounds are directly responsible for gel destruction [7]. The use of Bailey’s reagent however of protease activation, since in this more or less rules out the possibility case the 8 A# urea which is present should inactivate the proteases and other enzymes by dcnaturation. A41sothe presence of the tetrathionate in Bailey’s reagent may furnish a second mechanism for protease inactivation by bringing about the addition of sulfite groups to available -SH groups of the proteases. The possible role of high pH in destroying DNA-nucleoprotein gels in the presence of -SH col~ll)ounds can also be ruled out by the use of Bailey’s reagent, since this reagent operates at a pH value close to neutrality. Cleland’s reagent also rules out the effect of pH in complicating the interpretation of the degelling action of -SH reagents. It is true that this reagent was used in the vicinity of pH 9, but even at a pH this high, DNh-nucleoprotein gels are stable inde~nitel~ in the absence of the reagent. The action of Bailey’s reagent on isolated residual protein is of interest Experimental

Cell Research 49

551

M. Mackay,

C. A. Hilgarfner,

and A. L. Dounce

in connection with the relationship of -S-S- bonds to DN,4-nucleoprotein gel structure as well as to the structure of residual protein itself. This reagent undoubtedly causes solubilization of the isolated residual protein by -S-Sbond cleavage, not merely by the action of the 8 M urea contained therein, since 8 M urea alone does not dissolve isolated residual protein at the pH of the reagent. The above observations on disulfide bond behavior appear to leave little doubt that intact disulfide bridges play a crucial role in maintaining the DNL4-nucleoprotein gels and by inference, structure of residual protein, chromosomes. The experiments reported in this paper on the effect of 0.1 LV HCl-8 111 urea in dissolving approximately three-fifths of the residual protein of rat liver nuclei isolated at pH 3.8 were undertaken before the work of \J’ang appeared on the fractionations of residual protein by another procedure [25, 261. At the present time it is perhaps too early to try to compare the two results, since we have not yet repeated \vang’s work. His method of fractionation is no doubt milder than the use of 0.1 N HCl-8 114urea, but it is quite possible that proteolysis may have occurred at some point in his procedure, since apparently no specific steps were taken to avoid this possibility. Our method of fractionation is evidently severe enough to have produced some degradation of the residual protein, as we have stated in the section on results, but it is uncertain to what extent the soluble part of the residual protein represents protein cleaved from DNA by the acid urea. 4 small amount of deoxyribonucleotide material appears in the soluble extract, but whether this material is firmly bound to fragments of residual protein in the soluble extract is not yet known. The bulk of the DNA4 remains in the insoluble residue fraction. It has been found in preliminary experiments that the ratio of acid ureasoluble residual protein to total residual protein is lower in rat liver nuclei isolated at pH 5.8 than in nuclei isolated at pH 3.8. This taken together with the very recent observation that the fraction of acid-urea-soluble residual protein is lower in pH 3.8 nuclei when the globulins and histones are measured separately than when they are removed together in 0.1 ,I; HCl suggests that acid denaturation may under certain conditions throw part of the globulin fraction in with the residual protein fraction, and that a fair portion of the material that passes into solution when nuclear residues containing DNA-residual protein are extracted with acid urea represents denatured globulin. Nuclear membrane protein may or may not be soluble in acid urea. The amino acid analyses indicate that the acid-urea-soluble portion of the Experimental

Cell Research 49

IISA-nucleoprotein

gels and residunl

protein

of cell nuclei

555

residual protein is not markedly different in amino acid analysis from the acid-urea-insoluble portion. This could be true even though the acid-ureasoluble fraction of residual protein contains a considerable amount of denatured globulin. Roth portions contain -SH and -S-S- groups and the analysis indicates that the soluble portion cannot be a typical histone. The fact that a considerable portion of the residual protein of rat liver and calf thpmus nuclei can be extracted in 0.1 N HCl-8 31 urea make it necessary to use caution in interpreting the extraction of “histone” from cell nuclei with strong acid, such as for example the extraction of calf thymus nuclei with 0.5 iV H,SO, or HCl. The finding of small quantities of cysteine-containing components [B, 18, 221 in histone samples should also be interpreted with caution, for the same reason. It is of interest that whole residual protein from rat liver and calf thymus differ in amino acid analyses. This difference may be important in trying to ascertain why rat liver DN,4-residual protein gels are attacked by trypsin, whereas calf thymus DNA-nucleoprotein gels are resistant to this enzyme. The electrophoretic data presented in this paper confirm the previously reported results [8] that there are at least two types of peptide chain in residual protein. However, the total number of peptide chains in residual protein still remains unknown. Little is known of the structure of residual protein itself. The data from Sephadex gel filtration show that the components of whole residual protein have molecular weights that are too high to be measured by the methods employed by us. In this paper and previously 181 the presence of two types of peptide chains that have different isoelectric points and are -S-S- crosslinked is reported, but the total number of such chains is unknown. The observations recorded in this paper appear to support the model for basic chromosomal structure at the molecular level which has been previously proposed [8]. The model can easily be expanded to include the work of Bendich [2, 31 on spacers in purified DNA, since one could visualize the intercalation of many short spacers of the Bendich type in DNA4 with more occasional residual protein spacers, linked in the same way as previously proposed by us [8]. \\Te -\vould prefer however to link only one strand of each DNA double helix to the spacer material in all cases [9]. The two approaches would thus be fused in a completely consistant manner. Our work on the role of -S-S- bonds in DNA-nucleoprotein structure is also consistent with the work of Bendich et al. [4] on the isolation of DNA from mammalian spermatazoa. It is hoped that the findings reported in this paper will encourage further Experimental

Cell Research 49

556

M. Mackay,

C. A. Hilgartner,

and ,4. I,. .Llounce

studies of the properties of DNA-nucleoprotein gels, since we believe that such studies, largely neglected in the past, can lead to new information about basic chromosomal structure.

SUMMARY

1. Data are presented which provide further tests of a previously proposed model for the fundamental structure of chromosomes at the molecular level. 2. Observations concerning the effects on the gel-forming capabilities of the residues left after extracting with various reagents the DNA-residual protein complexes isolated from rat liver or calf thymus nuclei are presented and discussed. These results are consistent with the postulate that the formation of viscoelastic gels is dependent on linear continuity between tracts of DNA and tracts of residual protein. 3. Observations are presented concerning the use of formaldehyde to investigate the possibility of damage to DNA caused by extracting DNL4nucleoprotein with 0.1 L\’ HCl in 8 M urea. DNA appears to survive acid denaturation because of the presence of the portion of the residual protein which, together with the DNA, remains insoluble in the acid urea. It is inferred that protection might be conferred on the DNA4 by the insoluble residual protein if the latter, which appears to be covalently bonded to the DNA, keeps the strands in register after hydrogen bond rupture brought about by acid, thus facilitating renaturation. 4. It was found that DNA-nucleoprotein materials which had been frozen failed to produce the gels customarily observed on raising the pH to 8-9. Pure DNA dissolved in water and frozen could be subsequently recovered in fibrous form, indicating that freezing damages the protein component of DNA-nucleoprotein rather than the DNA. 5. Data are presented which show that Bailey’s reagent and Cleland’s reagent for the disruption of disulfide bridges destroy the viscoelastic properties of DNA-nucleoprotein gels and render soluble at pH values above neutrality the residual protein of cell nuclei. These observations on disulfide bond behavior appear to leave little doubt that intact disulfide bridges play a crucial role in maintaining the structure of residual protein, DNA-nucleoprotein gels, and by inference, chromosomes. 6. The effects on the residual protein component caused by extracting with acid urea were studied by performing amino acid analysis on whole residual protein, the soluble extract, and the insoluble residue; by performing gel filtration studies on these materials; by examining the gelability of the acid Experimental

Cell Research 49

D,Vil-nucleoprotein

gels and residrml protein

of cell nuclei

urea insoluble residues, and by analysis for deoxpribonucleotides in the acid urea soluble extracts. The evidence indicates that some degradation of the residual protein component results but the bulk of the DNA appears to remain lirmly bound to the acid urea insoluble component of the residual protein. ‘7. Polarographic data showing the concentrations of sulfhydryl and disulfide sulfur in whole residual protein, the acid urea soluble extract, and the insoluble residue are presented. 8. Paper electrophoresis of solubilized residual protein performed under conditions which answer a possible objection to previous data still indicate the presence of two types of peptide chains in the residual protein. 9. Gel filtration studies performed on solubilized residual protein indicate that the components of this material are probably greater than 400,000 in molecular I\-eight. REFERENCES 1. ACKEKS, G. I<., Biochem. 3, 723 (1964). 2. UENDICH, A., BORENFREUNI), E., KOHSGOLD, G. C., KRI~~, M. and RALIS, M. E., Amino Acids or Small Peptides as Punctuation in the Genetic Code of DNA, hcidi Nucleici c loro funzione biologica, p. 213. Istituto Lombard0 di Science e Lettere, Tipografia Successori Fusi, Pavia, 1964. 3. BERKOWITZ, W. F. and BENDICH, A., Biochem. 4, 1979 (1965). 4. BORENFKEUND, E., FITT, E. and BENDICH, A., Nature 191, 1375 (1961). 5. CLELAXD, W. W., Biochem. 3, 480 (1964). 6. DE~KIN, H., ORD, M. G. and STOCKEN, L. A., Biochem. J. 89, 296 (1963). 7. DOU~VCE, A. L., Arch. Biochem. Biophys. 117, 506 (1965). 8. DOUNCE, A. I,. and HILGARTNIXR, C. A., Exptl Cell Res. 36, 228 (1964). 9. DOUNCE, A. L., LOVE, B. B., DESIXOXE, J. and MACK.~Y, M. S., in The Cell Nucleus Metabolism and Radiosensitivity, Proc. Symp. 9-12 May, Radiobiological Institute T.N.O., p. 147. Tavlor and Francis, London, 1966. 10. DOUSCE, A. L. and hfONTY, K. j., J. &. Physiol. 41, 595 (1958). 11. Douxc~, A. L. and MONTE., K. J., J. Biophys. Biochem. Cytol. 1, 155 (1955). 12. Doun-CE, A. L. and O’CONSELL, Xl. P., .I. Am. Chem. Sot. 80, 2013 (1958). 13. DOUNCE, A. L., O’COSNEI,L, M. P. and BloxTu, K. J., Biophys. Biochem. Cytol. 3, 649 (1957). 14. DOUSCE, A. L. and SARKER, N. K., in J. S. MITCHELL (ed.), The Cell Nucleus, p. 206. Butterworth London, 1960; also Academic Press, New York, 1960. 15. Douscq A. L., SEAXAN, F. and MACKAY, M., Arch. Biochem. Biophys. 117, 550 (1966). 16. DOUNCE, A. L., TISHKOFI:, Y. T., BARNETT, S. R. and FREER, R. M., J. Gen. Physiol. 33, 629 (1950). 17. DOUNCE, A. L. and Unr&~, R., Biochem. 1, 811 (1962). 18. ORD, M. Y. and STOCKEN, L. A., in The Cell Nucleus-Metabolism and Radiosensitivity, p. 135. Taylor and Francis, London 1966. 19. SARKAR, S. K. and DOUNCE, A. L., Arch. Biochem. Biophys. 92, 321 (1961). 20. SCHSEIDER, W. C., .I. Biol. Chem. 161, 293 (1945). 21. SIEGEL, L. M. and MONTY, K. J., Biochim. Biophys. Ada 112, 346 (1966). 22. STOCKES, L. A. and ORD, M. Y., in The Cell Nucleus-Metabolism and Radiosensitivity, p. 141. Taylor and Francis, London, 1966. 23. UM.~RA, k., Ph. D. Thesis, University of Rochester Biochemistry Dept. 1963. 24. UXA%A, R. and DOUNCE, A. L., Exptl Cell Res. 35, 277 (1964). 25. WANG, T. Y., J. Biol. Chem. 241, 2913 (1966). 26. -in The Cell Nucleus-Metabolism and Radiosensitivity, p. 243. Taylor and Francis, London, 1966. 27. YEXX, E. W. and COCKISG, E. C., Analysf 80, 209 (1955). 28. DE ZOETES, L. W. and DE BRUIS, 0. A., Rec. Trau. Chim. Pays-Bus 80, 908 (1961). 36 - 681805

Experimental

Cell Research 49