Reprinted from the PRoceEDINGs OF THE NaTIONAL ACADEMY OF SCIENCES Vol. 51, No. 2, pp. 351-859. February, 1964. DNA CIRCULARITY AND THE ALECHANISM OF STRAND SELECTION IN THE GENERATION OF GENETIC MESSAGES* By AL. Hayasar, M. IN. Havasu, anp 8. SpreGELMAN DEPARTMENT OF MICROBIOLOGY, UNIVERSITY OF ILLINOIS Communicated by I’. AI. Sonneborn, December 9, 1963 A potentially informative paradox exists in the literature of genetic transcription. Experiments'~* with preparations of purified DNA and transcribing enzyme (the DNA-dependent RNA polymerase, for brevity referred to as “transcriptase’’) have yielded RNA complementary to both strands of the DNA employed as a template. On the other hand, analyses‘ of transcription in the intact cell revealed that primarily only one of the two complementary DNA strands generates RNA messages. Resolution of this riddle could conceivably illuminate the mecha- nism operating to restrict in vive transcription to one of the two strands. As a departure for a further analysis of the difference between the in vire and tn yw oe we BIOCHEMISTRY: HAYASHI ET AL, Proc, N. ALS. vitro results, the following three possibilities can be listed for the strand selection mechanism of transcription. (1) The control device functioning in the cell is not a structural component of either the DNA or the transcribing enzyme. An ob- vious example is an enzyme which would selectively destroy one of the two RNA complements. Others can be readily devised. (2) A structural feature of the transcribing enzyme controls selection and is rendered nonfunctional during puri- fication. (3) The selection device is a built-in feature of the DNA which is de- stroyed during its isolation. The experimental material and information currently available permits a direct test of the third alternative. It is known that, due to its inordinate length, the most likely injury suffered by DNA during isolation is fragmentation. The ques- tion immediately arises whether this is sufficient to explain the absence of strand selection exhibited in test-tube experiments. Two requirements must be satisfied for an interpretable experiment. One is a source of nonfragmented DNA. The other is a preparation of the transcriptase sufficiently free of DNAase to avoid introducing breaks in the time period of RNA synthesis. To provide the first requisite, attention was focused on the replicating form (RF DNA) of the bacteriophage ¢X174 which infects /. coli. Our previous ex perience with RF DNA suggested that it possessed a number of obvious advantages for the purposes at hand. We have already shown‘ that messages are generated in the cell only from one of its two complements. The molecular weight of RI DNA (3.4 X 10) is small enough to minimize fragmentation during purification. A chromatographic method has been developed* permitting the isolation of pure RF DNA virtually free of contamination with fragments of host DNA or the single strands (hereafter called vegetative DNA) found in mature virus particles. Chro- matographically purified preparations were shown® to be composed almost exclu- sively of circular DNA, thus providing another objective criterion of intactness. In addition, the possibility arose that the circularity, which had also been ob- served by Sinsheimer and his colleagues,’ !° might, be related to the selective mechanism. The necessity for a pure transcriptase was satisfied by purification to the point where no DNAase or RN Aase could be detected by a sensitive assay detailed below. It is the purpose of the present paper to describe the experiments made possible by the availability of intact DNA and nuclease-free transcriptase. The results are satisfyingly clear-cut. When intact circular DNA is employed as the template, only one strand is transcribed, as shown by both hybridization tests! and nearest, neighbor analysis of the RNA product. However, when the DNA circles are broken, RNA complementary to both strands is found. The results clearly estab- lish that the strand selection mechanism of genetic transcription is a feature of intact double-stranded circular DNA. Materials and Methods.—(a) Nucleic acid preparations: @X174 RF DNA and the correspond- ing single-stranded DNA were isolated and purified chromatographically as described by Hayashi etal. The columns used for RF DNA had a height to surface ratio of 1.55. All DNA preparations were tested for nuclease (§ ¢) and those employed in the experiments described showed no activity. RNA was isolated and purified according to the protocol of Hayashi and Spiegelman." Purity and uniformity of RF DNA was monitored by constant specific radioactivity on repeated chromatography, banding in CsCl density gradients, and densitometer tracings of sedimentation VoL. 51, 1964 BIOCHEMISTRY: HAYASHI ET AL, 353 cpm/ TUBE PX RE DNA 2000 Fig. 1.—Kinetics of RNA synthesis. Each (0.25 ml) reaction mixture contained the components in the concentration described in §(e) of Materials Px Single oNA = and Methods. 150 cpm corresponds to one mymole of GMP incorporated in RNA. RF DNA #1 and RF single-strand were used with enzyme #1. When < sonicated RF was used, a curve similar to those ; shown was obtained but at a rate approximately 1/, that shown with intact RF-DNA as a template. 1000 0 20 40 60 80 100 Min. patterns using UV optics in the analytical ultracentrifuge. Such preparations are highly infectious in the protoplast test.” (b) Transcriptase purification: The DNA-dependent RNA polymerase was isolated? from E. coli (C-122), harvested in log phase, and stored at —15°C. The last step involves an elution from a DEAE column with 0.28M KCI collected in fractions of about 2 ml. Each fraction was assayed, for 20 min at 36.5°C for R NA polymerase activity using calf thymus DNA and the supplement detailed in (§¢). Regions showing activity were then assayed for the presence of RNAase and DNAase. Only those fractions devoid of detectable nuclease activity were used in the ex- periments described. Purified enzyme was stored at 0°C. On isolation the three preparations used had the following specific activities in the units of Chamberlin and Berg,? #(1)-6000; # (3-1)-4000; # (4)-4,700. (c) Assay for DN Aase and RNAase: Each assay (1 ml) contained 90 ug of CDNA (4,800 cpm/zg) or 20 ug of P*-ribosomal RNA (3,500 cpm/yg) from E. coli. Incubation was carried out for 20 hr at 37°C under conditions suitable for RNA synthesis (§ e) except for omission of the nucleoside triphosphates. Negligible activity was assumed if less than 5% of the DNA and less than 8% of the RNA was converted to acid-soluble form, In examining the transcriptase, 100 ug of purified enzyme were tested. (d) GTP* preparation: GTP labeled with P® in the nucleotide phosphorus was prepared ac- cording to Haruna et al,‘ The initial specific activity was about 3.3 X 10° epm/uM of GTP. All counting was done in a Packard liquid scintillation spectrometer. Acid-precipitable material was washed with TCA and dried on Millipore membranes. Acid-soluble material was dried onto plastic planchets which were inserted into the vials. (e) RNA synthesis: The reaction mixture (1 ml) contained 24 yg of template DNA, 40 «M of tris buffer pH 7.9, 1 uM MnCh, 4 uM MgCh, 46 uM KCl, 12 uM of 8-mercaptoethanol, 40 ~ 100 ug of enzyme, 500 muM ATP, CTP, UTP, and GTP* labeled in the nucleotide phosphorus. Reaction temperature was at 36.5°C, The kinetics of RNA synthesis with circular RF DNA and mature DNA as templates are shown in Figure 1. The kinetics with disrupted circles are quite similar to those shown but at a rate corresponding to about 25% of the rate obtained with intact circles. (f) Analysis of nearest neighbor to guanosine: On termination of the synthesis with GTP* as the only labeled component, the contents were precipitated and washed five times with cold 3% PCA. Four mg of £. coli C bulk RNA were added to each tube and the contents hydrolyzed with 0.3 N KOH at 37°C for 15 hr. Chromatographic separation of 2’-3’ nucleotides and distribution of the radioactivity among them was carried out as described previously.™! (g) Sonication of DNA: DNA (50 y/ml) dissolved in SSC (0.15 M NaCl; 0.015 M Na citrate) was sonicated with a Raytheon sonic oscillator at 4°C for 6 min. SY of sonicated RF was about 8. (h) Hybridization: The details of formation and detection of DNA-RNA hybrids are as de- scribed by Hayashi, Hayashi, and Spiegelman.‘ * Denatured DNA and RNA are mixed in 2 X SSC buffer and incubated at 42.5°C for 16 hr. The reaction mixtures are treated with pancreatic 354 BIOCHEMISTRY: HAYASHI ET AL, Proc. N. ALS. Fie. 2.—Electron microphoto- graphs of ¢X-RF (left) and sonicated @X-RF (right), The method of Kleinschmidt eé al.24 was used. Prep- arations were shadowed with 12 mg of V30 at a distance of 11 em and an angle of 6° while grids were rotating. Pictures were taken with a Siemens Elmiskop Ib at 50 kv by Barbara Chandler, Department of Zoology, University of Wisconsin. RN Aase and the resistant DNA-RNA hybrids separated on columns of methylated albumin coated on kieselguhr. (i) Electron microphotographs: Electron microphotographs of intact and sonicated RF DNA were kindly taken by B. Chandler, University of Wisconsin, according to the procedure detailed elsewhere.® Experimental Resulls—Purity of RF DNA: As had been done previously,’ the RF DNA was labeled with H*thymidine during its synthesis in the infected complex. This permits monitoring the progress of the purification by means of specific activity. Chromatography is repeated until constant specific activity of the RI DNA peak is achieved. Such preparations exhibit a homogeneous band in equilibrium centrifugation in CsCl gradients at a position corresponding to a p of 1.708 using Ps. aeruginosa NY-DNA as a density marker at 1.746. Further, densi- tometer tracings of sedimentation profiles indicate that more than 95 per cent of the DNA sediments with an SY of 21.0 + 0.5, the value found originally’ for the RF DNA. Finally, electron microphotographs show that more than 90 per cent of the material is in the form of intact circles. Figure 2 compares one of the purified RF DNA preparations used in the present study before (left) and after (right) sonic disruption. Purification of transcriptase: In our experience, the Chamberlin-Berg procedure? used with /. coli (strain C) yielded DNA-dependent RNA polymerase remarkably free of both DNAase and RN Aase as tested with radioactive substrate by the 20-hr assay described under §(c) above. Figure 3 shows the elution profile of enzyme (#3) and identifies two fractions used in experiments described below. The ap- parent splitting of the activity into two fractions is not uncommon, and its signifi- cance remains for elucidation. Specific Activity 0.23M KCI + 7 : op Fic. 3.—Chromatography of transcriptase. 280 mu | Enzyme 3-1 About 5 mg of fraction 3 of Chamberlin and Berg? 0.3 4 Speciti was put onto a DEAE column (7 em X 1 cm apecitie enayme 3-2 diameter), and washed with 0.16 Mf KCl in buffer B (0.002 M KPO, buffer pH 8.4, 107? M MgCh, 107? M B-mercaptoethanol, 10-4 M EDTA) until O.D.° became less than 0.02. The enzyme was eluted with 0.23 4 KCl in buffer B. Every 2 ml of effluent was collected. Enzyme activity was measured as described. Specific activities are expressed in arbitrary units. TUBE NUMBER Vou. 51, 1964 BIOCHEMISTRY: HAYASHI ET AL. 355 TABLE 1 Frequency or Nearest NEIGHBOR TO G IN SYNTHESIZED RNA Theoretical CpG ApG UpG GpG (A) On mature strand (single)* 22 34 24 20 (B) On complementary strandt 18 21 39 22 (C) On both strandst 20 28 32 20 Experimental - GMPs Template ineorp Incubation No. INA Enzyme (mpM) time (min) 1 RF-1 1 26.8 0-20 21.1 21.4 37.4 20.1 2 RF-1 1 24.0 20-90 22.2 20.8 37.7 19.3 3 RF-1 1 52.0 0-90 20.8 20.8 38 .2 20.2 4 RF-2 1 40.0 0-90 22.1 20.8 37.5 19.6 5 RF-2 3-1 20.0 0-30 21.9 21.3 36.8 20.0 6 RF-3 4 24.0 0-30 22.2 21.2 37.6 20.0 Average 21.7 21.0 37.5 19.8 7 RF sonic-1 1 10.0 0-90 23.1 25.4 30.0 21.5 8 RF sonic-2 3-1 4.2 0-30 22.5 27.7 30.0 19.8 9 RF sonic-3 4 3.8 0-380 21.9 26.0 31.0 21.1 Average 22.5 26.3 30.3 20.8 10 Mature-1 1 11.0 0-20 22.5 35.9 23 .0 18.6 iL Mature-1 1 9.0 20-90 23.9 34.7 22.4 19.0 12 Mature-1 1 18.7 0-90 22.0 34.3 23.8 19.9 Average 22.8 34.9 23.0 19.1 13 RF-2 3-2 3.6 0-30 21.7 25.1 33.3 19.9 * ted from limited replication of ¢X174 assuming the RNA synthesized is complementary and anti- parallel. } Limited replication data are converted into a complementary antiparallel template for the formation of an antiparallel RNA complementary to it. t Calculated from extensive replication of Table 2 of Swartz ef al.16 Details of RNA synthesis and nearest neighbor frequencies to G are as described in Methods. The muM of GMP incorporated are those found and are not normalized to the amount of enzyme used, which varied from 40-100 ug. In experiments 2 and 11 the reaction was run for 20 min with unlabeled nucleoside triphosphates and P32-GTP put in for the last 70 min. ‘‘RF-sonic’’ 1, 2, and 3 are sonicated aliquots of RF 1, 2, and 3. In all cases 24 yg of the corresponding DNA were used. Theoretical calculations employ the data of Swartz et al.}6 Numbers repre- sent ‘‘moles-per cent.”’ Nearest neighbor analysis of RNA synthesized on intact and disrupted DNA circles: The elegant studies of Swartz et al.° on DNA synthesis with single-stranded DNA of ¢X174 provides the nearest neighbor frequencies in it, as well as its complement. From these data one can readily caleulate the nearest neighbors of RNA synthe- sized on the original single strand, its complement, or both. Examination reveals that nearest neighbors to guanosine (G) provide the most sensitive discrimination among the three possibilities. The corresponding numbers are given as the theo- retically expected values in Table 1. To decide whether intactness of circularity plays a role in strand selection, nearest neighbor frequencies to G were determined on RNA synthesized with intact circles, ruptured circles, and as an added control, on the mature single-stranded DNA. From the last, only one outcome is pos- sible. To make certain that any differences observed could be ascribed without ambiguity to one of the two reactants, independent preparations of RF DNA and transcriptase were made and interchanged. The frequencies of nearest neighbors to G found in the various experiments are recorded in Table 1. Experiments 1-6 involved three RF DNA preparations com- bined with three independent enzyme purifications. The results obtained are in- distinguishable and independent of the particular pair used. Further, it will be noted that experiments 1~3 used the same pair and compared the nearest neighbors ob- served during different periods of the synthesis. It is clear from a comparison of expected and observed values that the best agreement has been obtained with the calculation which assumes that only one of the two strands of the RF DNA is tran- 356 BIOCHEMISTRY: HAYASHI ET AL. Proc. N. A. 8. scribed. Further, the values obtained specify that it is the strand complementary to that found in the virus which determines the base sequence of the RNA synthe- sized. These conclusions are further strengthened by the results of experiments 7-9, which employed the same enzyme preparations tested in the first six experiments, but used sonicated RF DNA as templates. Here it is evident that agreement is with line C of the theoretical values, indicating that both strands of the RF DNA are functioning virtually equally in determining the base composition of the RNA synthesized. Finally, experiments 10-12 show that the enzyme has no difficulty in synthesizing RNA complementary to the mature strand if it is the only tem- plate in the reaction mixture. Experiment 13 is reproduced since it yielded a result of possible significance. The enzyme used was #3-2, which came from the second peak of activity in Figure 3. Although free of detectable nuclease activity, it yielded a product with RI DNA suggesting that 30-40 per cent of the RNA is generated from the mature strand and the remainder from its complement. These results imply the possibility that the transcriptase in this peak is contaminated with an enzyme which can cleave the circular elements without further degradation of the double-stranded structure. Exploitation of this suggestive clue would appear to be worthy of further effort. Hybridization tests with RNA synthesized on intact and disrupted circles of RF DNA: The analyses of nearest neighbors to G are consistent with the inference that intact circles generate complementary RNA copies from one strand, whereas with broken circles both strands of the DNA are involved. Hybridization tests can subject this conclusion to a definitive test. figure 4 shows that RNA synthe- sized on either intact or disrupted circular RF DNA hybridizes to heat-denatured RF DNA. This result is expected since RF DNA contains both complements and establishes that both preparations contain hybridizable RNA. Iigure 5 shows the decisive experiment in which the two types of preparations are challenged with single-stranded DNA from the virus particle. Here we see (I'ig. 58) that the RNA synthesized on broken circles hybridizes readily, whereas the preparation generated by the intact circles forms RN Aase-resistant complexes (Fig. 5A) very poorly (less than 10% of 5B). This is precisely the outcome predicted from the nearest neigh- bor analysis. The RNA synthesized on circular templates should be composed principally of only one complement and, since its base composition is not comple- mentary but similar to the mature strand, hybridization between the two should not occur. Finally, RNA generated from disrupted circles should contain both complements, one of which should be complementary to the mature strand. As a consequence, successful hybridization should occur. Discussion.—F requency of one- and two-strand transcription: It is of interest to com- pare the purity of the components used with the quantitative results obtained. Tracings of the sedimentation patterns and random field examinations of electron microphotographs both indicate that the RF DNA employed consisted of at least 90 per cent unbroken circles. This is a minimal estimate, since breakage could have occurred, for example, in preparation of grids, etc. Comparison of the theoretical and observed ApG and UpG values listed in Table 1 provides an estimate of the relative frequency with which each strand of the DNA duplex is employed in the transcription. The data obtained with intact ‘circular Vot. 51, 1964 BIOCHEMISTRY: 43,p32 Naci cpm (mM) 2000t P22. RNA oncircular DNA x H® heat denatured qs RF DNA NaCl conc. ~. ~~ 1000 10 18 20 25 30 os H3 p32 NaGi cpm (My P32 RNA on sonic.DNA x H® heat denatured RF DNA 2000 ahs 8 2 os é NaCl conc. uo f \. i» 1000 10 Os 5 lo 15 20 25 30 TUBE NUMBER Fig. 4.—Hybridizations of RF DNA and RNA synthesized on intact (A) and disrupted (B) circles. The P?2-RNA was synthesized on intact (A) and disrupted (B) circles. H*RF DNA was sonicated, dialyzed against 0.1 X< SSC, heated to 97-98 °C for 10 min, and quickly cooled to 0°C. Prior sonication was necessary for complete irreversible heat denaturation. Each hybridization mixture contained 108 pg of denatured DNA and0.1 ugof RNA. Incuba- tion was in 0.3 M NaCl, 0.03 M Na citrate at 42.5°C for 16 hr. Subsequently, the mixture was subjected to RN Aase (free of DNAase) at 30 pg/ml at 26°C for 30 min. The mixture was then loaded on a MAK column and eluted as detailed by Hayashietal.4 The H? identifies the DNA, and the P#? the RNA in the RNAase- resistant hybrid structures. HAYASHI ET AL, 357 0.0.,P32 Nol cpm P32 RNA on circulor DNA x Single DNA (o.D) (M) 0.2} 2000 4 16 ® NeCl cone mee ° a : + | OIF 1000 é aa \'° 2 0.0 naa i ao ps2 8 ° Sx * = £9 40.5 5 10 15 20 25 30 35 Om P** RNA on sonicated DNA x Single DNA (o Db) a | 0.27 2000 Abs NaCI cone. a oe | » | ’ I {ho ~ a5 5 10 15. 20 25 30 35 TUBE NUMBER Fie. 5.—Hybridization tests of RNA synthe- sized on intact (A) and disrupted (B) circles with mature single-stranded DNA. The conditions of hybridization and subsequent treatment are the same as described in Fig. 4. Each hybridizing mixture contained 100 ug of mature DNA and 0.1 xg of P?~RNA., The QO.D.?® profile identifies the DNA, and the P#? the RNA in these RNAase-resistant hybrid structures. RF DNA indicate that 90—100 per cent of the RNA synthesized has been directed by only one of the two DNA components, the complement to the mature strand. This frequency range is clearly consistent with the hybridization tests carried out with the mature strand in Figure 5A. Similar calculations with disrupted circles as templates indicate that both strands are transcribed with equal frequencies. The quantitative data agree, therefore, with the estimations of purity of RF DNA for circularity. They further indicate that except for the minor fraction 3-2, the preparations employed are not contaminated with impurities which can de- stroy circularity. One preparation (#2) had a comparatively low specific activity (1200) and was not used in the experiments reported. Conclusions on the nature of the strand seleciton mechanism: It is clear from the experiments described that we have succeeded in reconstructing in the test-tube the conditions leading to a strand selection mechanism which makes the same choice 358 BIOCHEMISTRY: HAYASHI ET AL. Proc. N. A. 8. observed! in genetic transcription of the intact cell. The necessary components are a nuclease-free transcriptase and intact circular double-stranded DNA. The data cannot be explained in terms of a quantitative limitation on RNA synthesis with circular DNA. In point of fact, twice as much RNA (corresponding to 3 times the weight of the template DNA) is produced in equivalent incubations with cir- cular DNA as with ruptured material. The data of Table 1 clearly establish that it is the intactness of the RF DNA which determines whether one or both strands are transcribed. Each of the three enzyme preparations used gave both results, the outcome being determined by the DNA included in the reaction. These results argue against a control mechanism in- volving a component which is separable from the DNA and the transcriptase. They are equally inconsistent with identifying the control element as either a structural or contaminating component (e.g., a nuclease which selectively destroys one RNA strand) of the transcriptase. All the data support the thesis that the strand selec- tion mechanism is to be identified as a unique feature of circular double-stranded DNA. DNA intactness—circularity: The experiments described do not prove that only circular DNA will retain the strand selection device. It is conceivable that any DNA molecule, open or closed, will exhibit this property, providing it has never experienced fragmentation. ‘This possibility may soon be testable with ma- terial now available. In any event, at the present writing intact circularity has been shown to be a key controlling factor. This leads one to entertain the concept that circularity may turn out to be a general feature of strand selection in tran- scription. It will be recalled that the concept of a circular DNA is not a new one. It first emerged as a brilliant deduction in Jacob and Wollman’s” formal model of their data on recombination in FH. colz. Subsequently, genetic evidence for cir- cularity was provided for the bacteriophages T4!* and Al. Recently, direct evi- dence for ring structures came from autoradiographic experiments with FE. coli” and electron microphotographs of viral DNA.*: * 71.22 Finally, note should be made of Stahl’s?? ingenious ring-chain model for chromosomes, which serves to ex- plain a number of paradoxes which grew out of fine structure genetic analysis. Tn conclusion, it would not be surprising to find that circularity is not confined to bacteria and viruses. Further, it may be that a circular element will be found to constitute the unit of transcription (i.e., the transcripton). Such circular DNA molecules are likely to contain a unique region of closure, which may also serve to start and direct the relative movement of the transcribing enzyme. The RI DNA of ¢X174 is an obvious choice for attempts at the chemical identification of such unique elements, if they exist. Summary.—The experiments described were designed to resolve the following paradox; genetic transcription in the cell generates RNA strands complementary to only one of the two components of the DNA, whereas in vitro experiments show that both strands of the DNA are transcribed. The possibility was considered that the inconsistency could derive from the fact that test-tube experiments invariably use fragmented DNA. The experimental material consisted of a purified prepara- tion of “replicating form’? DNA of ¢X174 which was shown to be composed of intact double-stranded circles. The RNA synthesized on intact and disrupted circles was compared by nearest neighbor analysis and by hybridizations with the Vo. 51, 1964 BIOCHEMISTRY: HAYASHI ET AL. 359 mature single-stranded DNA. The data clearly established that intact circles generated RNA complementary to only one strand, the complement of the mature strand in the duplex. Disrupted circles produced strands complementary to both DNA components. It appears that the strand selection mechanism which characterizes normal genetic transcription is a unique feature of circular double-stranded DNA. It can be reproduced and studied outside the cell. * This investigation was aided by grants-in-aid from the U.S. Public Health Service and the National Science Foundation. 1 Geiduschek, E. P., T. Nakamoto, and S. B. Weiss, these Proceepinas, 47, 1405 (1961). ? Chamberlin, M., and P. Berg, these ProceEpinas, 48, 81 (1962). ’ Hayashi, M., M. N. Hayashi, and S. Spiegelman, Science, 140, 1313 (1963). 4 Hayashi, M., M. N. Hayashi, and S. Spiegelman, these Proceepines, 50, 664 (1963). 5 Greenspan, C., and J. Marmur, Sczence, 142, 387 (1963). 6 Tocchini-Valentini, G. P., M. Stodolsky, A. Aurisicchio, F. Graziosi, M. Sarnat, 8. B. Weiss, and E. P. Geiduschek, these ProcrEpinas, 50, 935 (1963). 7 Sinsheimer, R. L., B. Starman, C. Naglar, and S. Guthrie, J. Mol. 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Vogt, these ProceEpines, 50, 236 (1963). 2 Weil, R., and J. Vinograd, these ProceEprnas, 50, 730 (1963). 3 Stahl, F. W., in Deoxyribonucleic Acid, Proc. 11th Annual Reunion Soc. de Chim. Phys. (Pergamon Press, 1962). *4 Kleinschmidt, A. K., D. Lang, D. Jacherts, and R. K. Zahn, Biochim. Biophys. Acta, 61, 857 (1961).