Studies of the Recombination of Nucleic Acids with a Tissue Polypeptide*

GEORGET. LEWIS,PH.D., WALTERL. BLOOM,M.D.,t ANDCHARLESW.SMART,M.S.

(from the Department of Biochemistry, Emory Unirersity School of Medicine, Emory University, Georgia)

INTRODUCTION to behave like the naturally occurring nucleo Nucleic acids are known to occur in both plant proteins which were prepared and studied at that and animal tissues, where they are thought to time. Hammersten (10) in his classical study of exist in combination with protein substances thymus nucleic acid in 1924 carried out the first within the nucleus and in the cytoplasm. Certain attempt to recombine histone and nucleic acid and to study the chemical properties of the reformed of these nucleoproteins are thought to take part in nucleoprotein. Since Hammersten's work, little the processes of cellular reproduction which are so important in the uncontrolled growth of malignant interest has been exhibited in extending investiga tissue. The chromosomes themselves seem to be tions of the chemical nature of such recombina largely made up of nucleoproteins, which thus tion products, although such study might lead to become self-duplicating units, as was re-empha indirect evidence concerning the linkage between sized by Mirsky (14). However, Stedman (20) has nucleic acids and proteins as it exists in the cell. claimed that the genetic protein is not nucleo- The general impression appears to be that this protein but a third substance called "chromo nucleic acidâ€”proteinbond is not a firm one. This somin." The importance of a more complete is suggested by the ease with which it may be understanding of the chemistry of these com broken during manipulation incident to the isola pounds is apparent to all investigators in the fields tion of nucleoprotein from tissues (17). of neoplasia and cellular reproduction. Greenstein (4, 5, 6) has carried out a series of Gulland (9) in a recent review has further investigations on the effect of the addition of pro stressed the concept of an autosynthetic molecule teins and of various ions on the physical character as a biological unit necessary for the reproduction istics of polymerized desoxyribonucleic acid. His of cells. This review also contains a discussion of studies indicate that the nucleic acid interacts the relationship of nucleoproteins to this problem definitely with several types of protein and with and of the nature of the nucleic acid to protein basic amino acids. According to Greenstein, such bond. Our present knowledge of the chemistry and combinations result in marked alteration of cytology of cellular reproduction would indicate physical properties of the polymerized nucleic acid that the nucleoproteins are extremely active and isolated from animal tissues. Similar studies have necessary components in the process. also been reported by Gulland (8). However, no Many studies have been made on the chemical chemical data were obtained on the combination nature of the bond which presumably links the products of these two chemical entities (nucleic nucleic acids with the proteins in the cell. Only a acid and protein or basic amino acids). few investigations have been designed to deter The present paper is concerned with a study of mine the chemical nature of the recombination certain chemical properties formed by nucleic acids and a simple basic protein isolated from normal products formed in vitro by nucleic acids with pro tissues. We have used the term "basic polypep- teins such as those with which they might com tide" for this low molecular weight protein. The bine in vivo. At an early period in the investiga tion of nucleic acid chemistry, Milroy (13) showed product, formed by combination of the basic that nucleic acids may combine with proteins polypeptide with polymerized desoxyribonucleic in vitro. These combination products were thought acid prepared from calf thymus, is compared with * Aided by a grant from the Georgia Division of the the corresponding product formed with ribo- American Cancer Society. nucleic acid. Definite chemical differences between t From the Departments of Biochemistry and Medicine, the two nucleic acids have been shown (9), and, Emory University School of Medicine. furthermore, their marked difference in respect to 103

polymerization is recognized ; but these variations for formation of a precipitate. The physical char are not of sufficient degree to explain completely acter and approximate amount of precipitate was the presumed difference in their biological be recorded. havior. The approach described in this paper was If the precipitates were to be analyzed, the an attempt to determine whether variations in be tubes were centrifuged and the precipitates havior could be observed when the two types of washed twice with distilled water. The entire nucleic acid were combined in the test tube with precipitate from each tube was then digested with a basic tissue constituent to form a product similar 2.5 cc. of 10 N sulfuric acid for at least 2 hours. to those which may exist as a nucleoprotein in the A few drops of 30 per cent hydrogen peroxide were living cell. then added, and digestion continued for a mini EXPERIMENTAL mum of 30 minutes. Each digest was diluted to MATERIALS 25 cc., and aliquots were take for phosphorus and A. Basic polypeptide (BPP).â€”This seems to be nitrogen analyses. Usually 1-ml. aliquots were used a basic low molecular weight protein which is pre for phosphorus determination, which was carried pared from fresh calf thymus by the method of out according to Fiske and SubbaRow (3). Ali quots of usually 10 ml. were employed for nitrogen Bloom et al. (1). The preparation used contained analysis by Wagner's boric acid modification of 13.49 per cent nitrogen. It was utilized in 0.5 per the micro-Kjeldahl procedure (20). cent freshly prepared solutions in distilled water unless otherwise stated. Since the ratio of nitrogen to phosphorus in nucleic acids is a fixed quantity, any increase in TABLE 1 the ratio found upon analysis of the precipitate EFFECTOFMIXINGBPP ANDDRNA formed upon interaction of nucleic acid and the basic polypeptide would be due to combination BPP DRNA (mg.) (mg.) Precipitate with protein and, therefore, addition of protein 1.25 2.5 None; viscous nitrogen. Nitrogen to phosphorus ratios were thus 1.25 1.25 Slight used throughout the work to characterize the 1.25 0.675 Fibrous 1.25 0.337 Fibrous synthetic nucleoprotein obtained. 1.25 0.167 Fibrous 2.5 1.25 Slight RESULTS 1.25 1.25 Slight 0.675 1.25 None INTERACTIONOFBPP ANDDRNA 0.337 1.25 None A preliminary experiment was performed to de 0.167 1 25 None termine the effect of mixing solutions of BPP and B. Ribonucleic and (RNA).â€”Commercial RNA DRNA. Two series of tubes were set up; in the (Schwarz), purified by the method of Kunitz (11), first, varying amounts of DRNA solution were was used in 0.5 per cent solutions in distilled water. added to a given volume of 0.5 per cent BPP. In This likewise was prepared just before use. This the second the same amount of DRNA was added preparation contained 13.30 per cent nitrogen and to each tube, but the amounts of BPP used were 8.01 per cent phosphorus. varied. The results may be seen in Table 1. It C. Desoxyribonucleic acid (DRNA).â€”This was is at once apparent that the weight of BPP present prepared from the nucleoprotein fraction isolated must equal or exceed that of DRNA in order for from fresh calf thymus by the method of Mirsky precipitation to occur. Analysis of a number of and Pollister (16). The protein was removed by precipitates formed by the addition of the same chloroform extraction carried out according to amount of DRNA to a given amount of BPP the directions of McCarty (12). The DRNA con showed that they contained the same proportion tained 12.68 per cent nitrogen and 6.88 per cent by weight of phosphorus and nitrogen. phosphorus. Because of the high viscosity of its An experiment was then conducted to deter aqueous solutions, DRNA was used in 0.25 per mine whether the nitrogen/phosphorus ratio cent solution in distilled water. varied when increasing amounts of BPP were used with a given quantity of DRNA. The results METHODS shown in Table 2 indicate that, when more than In general, the method of observation of the re a certain minimum of BPP is present, the nitro action of BPP with nucleic acids was as follows: gen/phosphorus ratio of the precipitate formed A solution of BPP was placed in a number of remains constant, no matter how the relative con centrifuge tubes, and to each was added a solution centration of the two materials may vary. This is of nucleic acid. The tubes were allowed to stand also shown graphically in Fig. 1 from additional at 2Â°to 5Â°C. for 24 hours and then were examined data. In other words, a stoichiometric relationship

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1949 American Association for Cancer Research. LEWIS et al.â€”Recombination of Nucleic Acids 105 exists between the two reactants at these ranges of As with DRNA, experiments were conducted to concentration. ascertain whether or not the nitrogen/phosphorus ratio was constant at a given concentration of INTERACTIONOFBPP ANDRNA BPP and of RNA and also if the nitrogen/ A preliminary experiment was performed to phosphorus ratio varied when the same amount of determine the effect of BPP on RNA, as in the RNA was added to increasing quantities of BPP. case of DRNA. In the first series of tubes the The results, presented in Table 4, indicate not amount of BPP was held constant, and the quan only that the nitrogen/phosphorus ratio varies at tity of RNA varied in each tube. The results are a given concentration but also that it varies widely shown in Table 3. As observed above with DRNA, as the relative concentrations of BPP and RNA there must be one part or more of BPP to one of change. These findings are shown graphically in RNA for the formation of a precipitate. Fig. 1, which also presents the results obtained TABLE 2 with DRNA and BPP at the same concentrations. ANALYSISOFPRECIPITATESRESULTINGFROM DISCUSSION MIXINGBPPANDDRNA ANALYSISOF PRECIPITATE The formation of a precipitate when DRNA DRNA(110.)3.044.883.782.953.505.564.333.243.724.15S.183.883.06BPP(HO.)1.524.8818.9029.5070.005.5621.6532.4074.404.1515.9038.8061.20BBP/DRNA0.5105.010.020.01.05.010.020.01.05.010.020.0N(mg.)0.35421and BPP come into contact may be taken as TABLE3 04630.92870 EFFECTOFMIXINGBPPANDRNA .70830.82251.03501.12740.62580.95710.65720.79480.83821.4147P(mg.)0.12510.28140.22130.16840 RNA Precipitate* .14830.28620.26750.14500.23070 BPP (mg-) (mg.) 5.0 5.0 Noticeable 5.0 10.0 None 5.0 20.0 None 5.0 40.0 None 5.0 5.0 Noticeable 17060.18010.21440.3309N/P2.833.724.194.215.723.624.214.324.153.854.413.914.2710.0 5.0 Fair amount 20.0 5.0 Rather large 40.0 5.0 Trace * All precipitates were participate.

COMPOSITION OF PPT. ( N/P RATIO) IN RELATION TO RELATIVE AMOUNTS OF BPP AND RNA OR DRNA

l 2 4 8 FIG. 1.â€”Romannumerals refer to individual experiments

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1949 American Association for Cancer Research. 106 Cancer Research indication of a physical or chemical reaction. nucleic acids, one might anticipate the same type When the two reactants are mixed in varying pro of combination with BPP. This, however, is not portions and any precipitates formed are analyzed the case, as is evident from Fig. 1, which shows the for nitrogen and phosphorus, the analyses show reactions of the two nucleic acids to differ marked- the ratio of nitrogen to phosphorus in the pre ly. cipitates to be constant. This is suggestive evi While the results are not explicable on the basis dence for the stoichiometric combination of the of our present knowledge of nucleic acid structure, reacting substances. they may lead to a clue to some of the biological This should not be surprising, since thymo- differences observed in the behavior of the two nucleoproteins have been known as chemical nucleic acids. The stoichiometric combination be entities for some time and samples extracted under tween the nuclear, DRNA, and basic constituent similar conditions (2, 15) give the same nitrogen/ would seem likely if these components have genet phosphorus ratio, showing that they are repro ic implication, as random combination of the ducible entities. Studies of the protein-DRNA substances would not lead to a clear concept of bond in nucleoproteins have indicated that it is of autosynthesis or reduplication. the polar variety (14, 18, 19). If this is correct, re- From this study it would appear that the polymerization of the DRNA would be the im TABLE 4 portant regulating factor in the nature of the final compound. McCarty's recent work on the influ ANALYSISOFPRECIPITATESRESULTINGFROM MIXINGBPPANDRNA ence of the DRNA of bacterial cells on the trans ANALYSISorPBECIPITATE formation of cell types would add further evidence RNA(no.)5.0fl.O5.05.05.05.05.05.05.05.0505.0BPP(UG.)5.010 on this point, as the DRNA appears to be one of the major controlling factors. If, as Stedman (20) 4016.8947.9560.9211 020.040.05.010.020.040.05010.020.040.0BPP/RNA194811481248N(mg.)1has suggested, the basic protein is one of the regu lating mechanisms of cellular division, the study of the basic protein-nucleic acid bond may eluci 4642.8338.0520.2361.6057.0837.9820.188P(mg.)0.1190.2700.2830.0840.1020.2620.32703510.8124.623.1115.2927.0328.312.68date certain of the regulatory factors concerned in the reproduction of the cell. Studies with other 0760.1050.2620.2820.070N/P11.7729.9728.1110.9614basic proteins, polypeptides, and amino acids may reveal further evidence which will help in the understanding of the nucleic acid-protein struc ture thought to be important. Furthermore, this technic may be useful in clarifying the differences in chemical behavior of the two nucleic acids, one action between the two components in vitro might difference having been shown in the manner in take place by the same mechanism. The results which they combine with a basic polypeptide iso obtained with DRNA and BPP were, therefore, lated from normal tissue. This combination may not unexpected. It was anticipated that similar possibly be similar to that in naturally occurring findings might be obtained when RNA and BPP nucleoproteins. were brought together in solution. As shown in Fig. 1, that was not the case. As the reactive mix SUMMARY ture became richer in BPP, an increasing amount A precipitate is formed when solutions of BPP of BPP was present in the precipitate until finally are mixed with solutions of RNA or of DRNA. a peak in the nitrogen/phosphorus ratio was These precipitates have been analyzed for nitro reached, after which successive additions of BPP gen and for phosphorus, and the N/P ratio is caused a decrease in the amount of BPP reacting. found to behave characteristically for each of the This phenomenon is extremely difficult to explain. nucleic acids when varying proportions of the The two nucleic acids, UNA and DRNA, differ reactants are employed. With the desoxy type the in several respects: pyrimidine bases, carbo ratio remains fixed, while in the case of the ribose hydrate, and size of molecule. However, presum form it varies markedly. ably only a difference in the phosphate group This might suggest that in the cell nucleus, would be significant in this case. Even in Gulland's only one type of nucleoprotein (desoxyribonucleic postulated structure of RNA (7), three of the four acid+basic protein) is formed, regardless of the phosphate groups are comparable to those found relative amounts of nucleic acid and protein in the generally accepted structure of DRNA. Be present. The differences in this nucleoprotein cause of this similarity in structure of the two would thus appear to depend upon the type

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1949 American Association for Cancer Research. LEWIS et al.â€”Recombination of Nucleic Acids 107 polymer of nucleic acid and not upon the nucleic 9. CULLANO,J.M., BARKER,G. R., and JORDAN,D. O. The acid-protein bond. On the other hand, the cor Chemistry of the Nucleic Acids and Nucleoproteins. Ann. Rev. Biochem., 14:175-206, 1945. responding combination in the cytoplasm might 10. HAMMERSTEN,E.Zur Kenntnis der biologischen Bedeu give rise to a series of compounds containing tung NucleinsÃ¤ureverbindungen. Biochem. Ztschr., 144: differing proportions of the components. 383-466, 1924. 11. KÃœNITZ,M.Crystalline Ribonuclease. J. Gen. Physiol., 24:15-32, 1940-1941. REFERENCES 12. McCARTY,M. Purification and Properties of Desoxyribo- nuclease Isolated from Beef Pancreas. J. Gen. Physiol., 1. BLOOM,W. L., WATSON,D. W., CROMABTIE,W.J., and 29:123-139, 1945-1946. FREED, M. Studies on Infection with Bacillus anthracis. 13. MILROY,T.H. Ueber die Eiweiss-Verbindungen der Nucle- IV. Preparation and Characterization of an Anthracidal Ã¯nsaure und ThyminsÃ¤ure und ihre Beziehung zu den Substance from Various Animal Tissues. J. Infect. Dis., Nucleinen und Paranuclem. Ztschr. f. physiol. Chem., 22: 80:41-52, 1947. 307-326, 1896. 2. CARTER,R. O., and HALL,J. L. The Physical Chemical 14. MIRSKY,A. E. Chromosomes and Nucleoproteins. Adv. Investigation of Certain Nucleoproteins. I. Preparation iÂ»Enzymol., 3:1-34, 1943. and General Properties. J. Am. Chem. Soc., 62:1194-1196, 15. MIRSKY,A. E., and POLLISTER,A. W. Fibrous Nucleo 1940. proteins of Chromatin. Biol. Symposia, 10:247-260, 1943. 3. FISKE, C. H., and SUBBAAOW,Y.The Colorimetrie De 16. . Nucleoproteins of Cell Nuclei. Proc. Xat. Acad. termination of Phosphorus. J. Biol. Chem., 66:375-400, Sc., 28:344-352, 1942. 1925. 17. SEVAG,M.G., and SMOLENS,J.Studies on the Conjugation 4. GREENSTEIN,J. P. The Colloid Osmotic Pressure of of Streptococcal Nucleoprotein. J. Biol. Chem., 140:833- Mixtures of Protein and Thymus Nucleate. J. Biol. 845, 1941. Chem., 160:107-112, 1943. 18. SEIBERT,F.B. Removal of the Impurities, Nucleic Acid and 5. . Chemical Studies on the Components of Normal Polysaccharide, from Tuberculin Protein. J. Biol. Chem., and Neoplastic Tissues. I. Viscosity and Streaming Bire 133:593-604, 1940. fringence of Sodium Thymonucleate. J. Nat. Cancer Inst., 19. SEIBEHT,F. B., and WATSON,D. W. Isolation of the 1:77-90, 1940-1941. Polysaccharides and Nucleic Acid of Tuberculin by 6. GHEENSTEIÃŽÂ»,J.P., and JENRETTE, W. V. Physical Electrophoresis. J. Biol. Chem., 140:55-69, 1941. Changes in Thymonucleic Acid Induced by Proteins, 20. STEDMAN,E.,and STEDMAN,E.Chromosominâ€”a Protein Salts, Tissue Extracts, and Ultra-violet Irradiation. Cold Constituent of Chromosomes. Nature, 162:267-269, 1943. Spring Harbor Symp. Quant. Biol., 9:236-254, 1941. 21. WAGNER,E. C. Titration of Ammonia in Presence of 7. GULLAND,J. M. The Structures of Nucleic Acids. Cold Boric Acid. In the Macro-, Semimicro-, and Micro- Spring Harbor Symp. Quant. Biol., 12:95-103, 1947. Kjeldahl Procedures, .using Methyl Red Indicator and 8. . Some Aspects of the Chemistry of Nucleotides. J. the Color-matching End Point. Indust. & Engin. Chem. Chem. Soc., pp. 208-217, 1944. (Anal. Ed.), 12:771-772, 1940.

Cancer Res 1949;9:103-107.

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