The DNA-Acridinedye Complexes Have Been Studied

VOL. 52, 1964 BIOCHEMISTRY: ISENBERG ET AL. 379

substrate, fructose-6-P, to an equilibrium mixture with glucose-6-P, it was necessary to apply a correction to the concentration of fructose-6-P added in order to obtain its actual concentration in the reaction mixture. The Km thus obtained for fructose-6-P is in fairly good agreement with the value of 3.8 X 10-4 M reported by Ghosh et al.'" for a purified preparation of rat liver enzyme which was free of isomerase. 23 Winzler, R. J., in The Plasma Proteins, ed. F. W. Putnam (New York: Academic Press, 1960), vol. 1, p. 314. 24 Wyman, J., in Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 483. 26 Gerhart, J. C., and A. B. Pardee, in Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963). p. 491. 26 Steiner, D. F., and R. H. Williams, J. Biol. Chem., 234, 1342 (1959). 27 Lowry, 0. H., J. V. Passonneau, F. X. Hasselberger, and D. W. Schultz, J. Bzol. Chem., 239, 18 (1964). 28 Strominger, J. L., Physiol. Revs., 40, 55 (1960).

DELA YED FL UORESCENCE IN DNA-ACRIDINE DYE COMPLEXES* BY I. ISENBERG, R. B. LESLIE, S. L. BAIRD, JR., R. ROSENBLUTH, AND R. BERSOHN

INSTITUTE FOR MUSCLE RESEARCH, THE MARINE BIOLOGICAL LABORATORY, WOODS HOLE, MASSACHUSETTS, AND DEPARTMENT OF CHEMISTRY, COLUMBIA UNIVERSITY Communicated by Albert Szent-Gyorgyi, June 22, 1964 The DNA-acridine dye complexes have been studied extensively by a variety of techniques. Such studies are of importance for a number of reasons. A prin- cipal biological interest centers around the attempt to understand the powerful mutagenic activity of many acridine dyes on T2 and T4 bacteriophages. Two different types of complexes have been distinguished (Steiner and Beers, 1961; Bradley and Wolf, 1959; Bradley and Felsenfeld, 1959). The first, termed Complex I, occurs at high dye to nucleotide ratios. Another type of complex, Complex II, occurs at low dye to nucleotide ratios. This paper will deal exclusively with certain phosphorescence properties of Complex II. Lerman (1961, 1963) has presented evidence indicating that Complex II has a configuration in which a dye molecule is intercalated between the base pairs of DNA. Work on the quenching of DNA phosphorescence by Mn++ ions has indicated that an excited triplet in DNA is delocalized (Bersohn and Isenberg, 1964). Such a delocalization suggests that a phosphorescence study of DNA-acridine dye complexes might be fruitful. While singlet transitions from one molecule to another may occur via long-range dipole-dipole coupling, triplet migration presumably occurs only via wave-function overlap. It might be expected, therefore, that an intercalated dye molecule would interfere with the migration of triplet excitation. Conversely, any interference or interaction that could be shown might serve as a verification of the intercalation model. As will be seen, not only was the expected interaction observed, but this took the unexpected form of an energy transfer from an excited triplet of DNA to an excited singlet of the dye. An analysis of the data shows that the observed energy transfers are reasonable on the assumption of an intercalation model and may be taken as a verification of this model. Downloaded by guest on September 30, 2021 380 BIOCHEMISTRY: ISENBERG ET AL. PROC. N. A. S.

400 500 .X My FIG. 1.-Emission of proflavine. Salmon sperm DNA complex 3.3 X 10-3 M in DNA phosphate, 1.1 X 10-4 M proflavine. (A) Emission with rotating shutter in place. (B) Total emission. Experimental Methods.-All measurements were made at 770K in a medium containing 50 per cent glycerol and 50 per cent water. The measurements were made using salmon sperm DNA (Calbiochem.), calf thymus DNA (Worthington), and T2 DNA. T2 DNA was prepared from purified phage by osmotic shock (Anderson, 1950) followed by alternate deproteinization (Sevag, Lackman, and Smolens, 1938) and ethanol precipitation. Proflavine hydrochloride (Mann Res. Lab.) was dis- solved in 50-50 ethanol-water, the free base was precipitated by 0.1 N NaOH; the precipitate was collected, washed, and recrystallized from 90 per cent ethanol. Acridine orange (National Aniline Div.) was recrystallized twice from 65 per cent ethanol. Quinacrine (Mann Res. Lab.), acriflavine (Nutritional Biochem. Co.), and acridine yellow (Allied Chemical) were used without additional purification since these dyes were used simply to test for the general phenomenon of delayed fluorescence and not used for extensive or quantitative studies. Measurements were made on an Aminco-Keirs spectrophosphorimeter used with a photomultiplier photometer manufactured by the Hruska Radio Co., Lutherville, Md. No corrections have been made for wavelength variations of the exciting light or the photomultiplier detector. Results.-Native DNA, protected against denaturation by the presence of 1.8 X 10-2 M NaCl and activated at 290 mjg, shows a phosphorescence peaking about 458 m~i. In the presence of proflavine the DNA phosphorescence is diminished, and two new peaks appear. One of these peaks is at about 560 mjh. This can be identified as the phosphorescence of the dye. The other peak is at about 495 mu. This peak is at the wavelength of the fluorescence of the dye-DNA complex. In using an Aminco-Keirs spectrophosphorimeter, phosphorescence is custom- arily detected by means of a rotating shutter which alternately permits exciting light to reach the sample, shuts off the exciting light, and permits the light emitted by the sample to fall on the detecting system. The rotating shutter may be re- moved while holding the sample in place, thus permitting the total emission to be observed. In the present case, the total emission is mainly fluorescence with an intensity some two orders of magnitude greater than the delayed light intensity. The fluorescence can be fitted on the same recorder scale by decreasing the amplifica- tion of the photomultiplier signal. When this is done, it can be seen (Fig. 1) to be Downloaded by guest on September 30, 2021 VOL. 52, 1964 BIOCHEMISTRY: ISENBERG ET AL. 381

40O 500 X m/l 600 FIG. 2.-Emission of acridine orange. Salmon sperm DNA complex 3.3 X 10-3 M in DNA phosphate, 1.1 X 10-4 M acridine orange. (A) Emission with rotating shutter in place. (B) Total emission.

FIG. 3.-Emission of quinacrine. Calf thymus DNA complex. 3.3 X 10-3 M in DNA phosphate, 2.5 X 10-6 M quinacrine. (A) Emission with rotating shutter in place. (B) Total emission.

similar both in position and shape to the new peak observed in the delayed emission spectrum. If an acridine orange-DNA system is used, the results are similar (Fig. 2). Again a delayed emission is observed that is similar in position and shape to the fluorescence. Quinacrine-DNA complexes were also studied. These provide a fairly critical test since the fluorescence shows a characteristic structured emission in the region 450-500 m,0. Again it was observed that the new peaks in the delayed emission were similar to those occurring in fluorescence (Fig. 3). Similar results were obtained with acriflavine and acridine yellow complexes. There can be little doubt that delayed fluorescence does occur in DNA-acridine dye complexes. Activation spectrum of the delayed fluorescence: The delayed fluorescence is activated in a way that is identical to the activation of DNA phosphorescence (Fig. 4). It in no way resembles the absorption spectrum of the dye. It cannot, for example, be activated by absorption in the visible region of the spectrum. We therefore conclude that the exciting light must be absorbed by the DNA in order for the delayed fluorescence of the dye to occur. Double light beam activation: The spectrophosphorimeter was modified to permit simultaneous activation by two light beams, one from the phosphorimeter mono- Downloaded by guest on September 30, 2021 382 BIOCHEMISTRY: ISENBERG ET AL. PROC. N. A. S.

AlL 4i4/ N

2150 3100 X M/.L so FIG. 4.-(A) Activation spectrum for delayed fluorescence of dye, in this case a DNA proflavine sample. (B) Activation spectrum for phosphorescence of DNA. The maxi- mum occurs at 290 mu rather than at 260 mi/ probably because the lamp intensity increases with increasing wavelength. chromator set in the ultraviolet for optimum activation of DNA emission, and the other from a tungsten lamp whose output is not absorbed by DNA. The beams irradiated the samples from opposite sides. Samples of DNA-acridine orange were examined in thin tubes of about 1.0-mm diameter in order to make certain that both beams irradiated the same portion of the sample. Energy from the tungsten lamp enhanced the dye phosphorescence without increasing the delayed fluorescence. For example, when using a sample of 3.3 X 10-3 M in DNA phosphates and 1.7 X 10-5 M in acridine orange, upon turning on the auxilary light the dye phosphorescence increased by a factor of 33, while the delayed fluorescence peak did not increase at all. Concentration dependence of delayed fluorescence: The height of the delayed fluorescence peak increases rapidly with added proflavine or acridine orange. How- ever, at a mole ratio of about 1 dye molecule to 50 DNA phosphates, further addition of dye leads to a diminution of the delayed fluorescence. This decline can be understood when one realizes that, at this concentration, the dye begins to absorb an appreciable fraction of the exciting light and we know that only light that is absorbed by the DNA is effective in producing delayed fluorescence. If, instead of considering the absolute height of the delayed fluorescence, we consider the height of the delayed fluorescence relative to the height of DNA phosphorescence in the same sample, a simple saturation phenomenon is evident, as will be seen in Fig. 5. The time decay of the delayed fluorescence of the DNA-proflavine and the DNA- acridine orange complexes is dependent on the concentration of the dye. All of the decays deviate markedly from simple exponentials. It is therefore, at present, impossible to present meaningful decay times as inverse rate constants. However, to present some idea of the order of magnitude of this effect it may be said that at 200 DNA phosphates per proflavin the half life is about 100 msec; at 100 to 1, it is about 80 msec, while at 50 to 1 it is about 50 msec. Downloaded by guest on September 30, 2021 VOL. 52, 1964 BIOCHEMISTRY: ISENBERG ET AL. 383

2 *'60

0~~~~~~

a s o4O@

.4z. ~~~~~~~~~~~~~~~~~~20

0 0.02 0.04 0.06 0.08 D o.lo 0 20 40 60 DNA phosphorescence FIG. 5.-Ratio of delayed fluorescence to DNA phosphorescence (R) versus ratio of dye molarity to FIG. 6.-Variation of delayed fluo- DNA phosphate molarity (D). Upper curves for rescence versus DNA phosphorescence native DNA; lower curve is for denatured DNA. in the absence of dve. The exciting Dye is acridine orange. light intensity is the altered parameter. See text for explanation. Dependence of delayed fluorescence on exciting light intensity: Several rather crude experiments were run to determine the variation of the delayed fluorescence intensity with changes in exciting light. Quartz "filters" were placed between the exciting light and the sample chamber. For each "filter" arrangement the phosphorescence of a sample of DNA alone and the delayed fluorescence of a DNA- acridine orange complex were observed. The results (Fig. 6) indicate that the delayed fluorescence intensity varies linearly with the DNA phosphorescence. We may conclude that the number of quanta emitted as delayed fluorescence is pro- portional to the number of triplet excitations that would be produced by the exciting beam in the absence of dye. Dye-denatured DNA complexes: DNA was denatured by holding stock solutions at 950C for about 20 min and rapidly chilling the solutions to 00C. The extent of the denaturation was checked by observing the optical density at 260 my as a function of temperature. The denatured samples showed only small rises in optical density upon heating. It is likely that almost all of the DNA was denatured by our treatment, but it is possible that small fragments could have been left in native form. Denatured DNA-dye complexes also show the phenomenon of delayed fluorescence. However, we have observed two striking differences from the results obtained using native DNA complexes. Relative to the amount of DNA phosphorescence, the height of the delayed fluorescence is an order of magnitude smaller (Fig. 5). Furthermore, the decay times do not vary with the concentration of the dye in the denatured DNA-dye complexes. We have recorded time decays on samples having concentrations between 1 dye to 200 DNA phosphates and 1 dye to 10 DNA phosphates. In contrast to the native DNA complexes, these decay curves are all the same and show no concentration dependence. They show a half life of about 100 msec. Downloaded by guest on September 30, 2021 384 BIOCHEMISTRY: ISENBERG ET AL. PROC. N. A. S.

If deoxyadenosine or deoxyguanosine, mixed with acridine orange, is examined in the phosphorimeter, a small peak is observed at the dye fluorescence position. This peak is seen only at relatively high dye concentrations and it decays exponentially with the time constant of the phosphorescence of whichever purine is used. (De- layed fluorescence becomes evident at phosphate to dye concentrations of 1500:1, 300 :1, and 40:1 for complexes of native DNA, denatured DNA, and purines.) It is probable that this, too, is an example of delayed fluorescence. It should be noted that the delayed fluorescence here could arise via dipole-dipole coupling. It has been pointed out (Fbrster, 1959; Ermolaev, 1963) that, while a triplet to singlet transfer has a low probability, this low probability is compensated by the long lifetime of the energy donor state. The result is that the probability of transfer per lifetime of the donor is the same for triplet -* singlet transfer and singlet -> singlet transfer. However, it appears likely that at least part of the transfer is due to a trivial mechanism, namely, an energy transfer via the radiation field rather than radiationless transfer. Such a possibility has been generally ignored in energy transfer studies because it is usually negligibly small. To test this, we placed a solution of 2.5 X 10-3 I deoxyadenosine in a capillary of 1.5-mm diameter. The capillary in turn was placed within a sample tube of 3-mm diameter containing 1.2 X 10-4 M acridine orange. In the phosphorimeter we observed a small emission at the dye fluorescence position. It is, therefore, still an open question as to how much of the transfer in purine-acridine orange mixtures is due to the radiation field and how much is radiationless transfer. Summary of Results. Delayed fluorescence occurs in complexes of acridine dyes with native or denatured DNA and may occur in complexes with adenosine and guanosine. The delayed fluorescence is activated by absorption of light by the DNA. The results obtained on native DNA complexes differ from the denatured DNA complexes in several respects. Relatively small concentrations of dye are needed to observe delayed fluorescence. The ratio of dye-delayed fluorescence to DNA phosphorescence is an order of magnitude higher in native DNA complexes than in denatured DNA complexes. The delayed fluorescence observed in native DNA complexes has time decays that are dependent on the concentration of the dye. The emission decays faster as the concentration of dye increases. Discussion.-Triplet migration: In the DNA-acridine dye complexes excitation energy is absorbed by DNA, and part of the energy is subsequently emitted as a delayed fluorescence of the dye. In the process the energy must be transferred from the DNA bases to the dye. This transfer cannot be via singlet-singlet resonance coupling since this must occur within the lifetime of the singlets of the bases, a time much too short to account for the decay time of the delayed fluorescence. Furthermore, the transfer cannot involve passage through the triplet state of the dye, for, if it did, the delayed fluorescence could be excited in the absorption bands of the dye, a process that does not occur. Delayed fluorescence has previously been observed in naphthalene and phenan- threne crystals (Sponer, Kanda, and Blackwell, 1958; Blake and McClure, 1958), and benzene crystals (Sternlicht, Nieman, and Robinson, 1963). The mechanism responsible for it appears to be different, however, involving triplet-triplet annihilation (Sternlicht, Nieman, and Robinson, 1963) rather than a conversion of the triplet excitation of one molecule to the singlet of another. It appears unlikely Downloaded by guest on September 30, 2021 \VOL. 52, 1964 BIOCHEMISTRY: ISENBERG ET AL. 385

that an annihilation mechanism is operative in the production of DNA-dye-delayed fluorescence for two reasons: (1) the linear dependence of the intensity of the delayed fluorescence on the triplet density in the absence of dye, and (2) the failure of increased dye triplet density to increase the delayed fluorescence intensity. In crystals where triplet migration has been observed, the transfer of triplet excitation is rapid. Nieman and Robinson (1963) obtained an exchange integral for triplet migration in benzene of 12 cm-', which means a hopping rate of about 1012 sec-1. There are reasons for believing that the migration of triplets in benzophenone crystals is not as efficient as in some other crystals, such as those of benzene or naphthalene (Hochstrasser, 1964). Yet, even for benzophenone the migration rate is greater than 1010 sec'1. In DNA, however, it may be expected that the migration rate, on the average, will be much smaller. Let us assume, for example, that a guanine is in an excited triplet state. If this guanine is adjacent to another guanine, then the energy may transfer to it in a time we will call T. If, however, the energy must pass over an intervening adenine to transfer to another guanine, then the transfer time increases sharply by a factor that may be estimated to be about 103 (Isenberg and Bersohn, 1963). (The transfer of energy from a guanine triplet to an adjacent guanine on the opposite strand should be almost as efficient as the transfer to an adjacent guanine on the same strand. In the order of magnitude estimates considered here, we therefore refer to adjacent purines without regard to whether or not they are on the same strand.) If the energy must pass over two intervening adenines, the time for transfer is 106T; for three adenines, 109r. Thus it may be seen that even though r is very small, 106r or 109r may be of the order of tens of milliseconds. If we then consider the time for migration of triplet energy from one part of the DNA helix to another part, we should expect times of the order of perhaps 10-4 to 10-1 sec rather than times of 10-10 to 10-12 sec that might be expected in pure crystals. It is, therefore, not unreasonable to ascribe at least part of the decay times observed for delayed fluorescence to the time needed for the triplet energy to wander along the helix until it is adjacent to a dye molecule. This offers a simple interpretation of the observation that the decay time decreases with increasing concentration of dye. The average time for energy migration from an excited purine to a dye molecule is shorter if there are more dye molecules present. The data, as a whole, are therefore consistent with the following interpretation. Purines are excited to the first excited singlet which are delocalized (Weil and Cal- vin, 1963). (Pyrimidine excitation may be ignored in this discussion since pyrimi- dines do not phosphoresce.) Part of the singlet excitation is converted to triplet excitations. These triplets may wander among the purines until a triplet excitation reaches the neighborhood of a dye molecule. Part of this energy may then be transferred to the dye where it is emitted either as dye fluorescence or as dye phosphorescence. This interpretation is, of course, tentative. It is not proved by the data but is simply consistent with it. It should be noted, however, that the data presented in this report verify the interpretation that the triplets in DNA are delocalized (Bersohn and Isenberg, 1963 and 1964). There also appears to be evidence for delocalized triplets in synthetic polynucleotides (Rahn, Longworth, Eisinger, and Shulman, 1964). Triplet to singlet conversion: It might be expected that the conversion of a purine triplet excitation to a dye singlet would be an event of extremely low proba- Downloaded by guest on September 30, 2021 386 BIOCHEMISTRY: ISENBERG ET AL. PROC. N. A. S. bility. However, as has been pointed out previously, this is not necessarily the case. In a dipole-dipole resonance transfer, the low probability per unit time will be compensated by the long lifetime of the triplet (F6rster, 1959; Ermolaev, 1963). In the case of DNA-dye complexes, what must balance the low probability of transfer is not the phosphorescence lifetime of the purine, but the time during which a purine excitation is in the vicinity of a dye molecule. F6rster (1960) has given plots of expected transfer rates for singlet-singlet resonance transfer as a function of distance of separation for random distributions (F6rster's Fig. 4) and reasonable coupling parameters. As a crude approximation, we will assume F6rster's values are valid for our case. We may estimate the transfer rate for triplet -* singlet transfer from that for singlet -* singlet transfer by

kTS = (rs/TT)kss. We will assume as a reasonable ratio, Ts/TT = 10-7, so that krs = 10-7 kis. k,, and hence kTs is a sensitive function of the separation between the purine that is excited and the dye molecule. For large separations these parameters vary as the inverse sixth power of the separation. From Forster's plot we obtain, at 3.4 A separation, k88 = 2 X 1014 sec- and kTs = 2 X 107 sec'. While these numbers are crude, they do indicate that, on an intercalation model for DNA-acridine dye, there can be efficient transfer of excitation from a purine triplet to a dye singlet provided the excitation is next to a dye for a time of the order of magnitude of 5 X 10-8 sec. This is a reasonable length of time. As indicated previously, if r is the time of transfer of triplet excitation from a guanine to an adjacent guanine, then the time for transfer of the triplet over an intervening adenine may be estimated to be 10Tr. If we now consider an excited guanine triplet bounded on one side by an adenine and on the other by a dye molecule, we will have a reasonable probability for conversion to a dye singlet, if Xr 10-10 sec which is a reasonable value. If the guanine is bounded by two adjacent adenines on one side and a dye on the other, the excitation will be adjacent to the dye for a time of the order of 106r, and the transfer effi- ciency to excite a dye singlet will be excellent. On the other hand, if the guanine is bounded by a dye on one side and one or two guanines on the other, the probability of exciting a dye singlet is reduced, but only by a factor of two or three. It should also be realized that at 3.4 A separation overlap of charge distribution will also as- sist in triplet-singlet conversion. We therefore see that an intercalation model makes it reasonable to have good triplet to singlet conversion and the efficient transfers that have been observed experimentally may be taken as a verification of such a model. The authors would like to thank Dr. Martha B. Baylor and Dr. Helen Van Vunakis for advice on the isolation and purification of T2 DNA. * This work was supported by grants from the National Institutes of Health (GM-10383 and CA-07712), and the National Science Foundation (G-5836). Anderson, T. F., J. Appl. Phys., 21, 70 (1950). Bersohn, R., and I. Isenberg, Biochem. Biophys. Res. Commun., 13, 205 (1963). Bersohn, R., and I. Isenberg, J. Chem. Phys., 40, 3175 (1964). Blake, N. W., and D. S. McClure, J. Chem. Phys., 29, 722 (1958). Bradley, D. F., and G. Felsenfeld, Nature, 185, 1 (1959). Bradley, D. F., and M. K. Wolf, these PROCEEDINGS, 45, 944 (1959). Downloaded by guest on September 30, 2021 VOL. 52, 1964 ASTRONOMY: H. E. SUESS 387

Ermolaev, V. L., Soviet Phys.-Usp. (Russian) 80, 333 (1963). Forster, Th., Discussions Faraday Soc., 27, 7 (1959). Forster, Th., in Comparative Effects of Radiation, ed. M. Burton, J. S. Kirby-Smith, and J. L. Magee (New York: John Wiley & Sons, 1960). Hochstrasser, R. M., J. Chem. Phys., 40, 1038 (1964). Isenberg, I., and R. Bersohn, in Electronic Aspects of Biochemistry, Ravello Symposium, ed. B. and A. Pullman (New York: Academic Press, 1964). Lerman, L. S., J. Mol. Biol., 3, 18 (1961). Lerman, L. S., these PROCEEDINGS, 49, 94 (1963). Nieman, G. C., and G. W. Robinson, J. Chem. Phys., 37, 2150 (1962). Ibid., 39, 1298 (1963). Rahn, R. O., J. W. Longworth, J. Eisinger, and R. G. Shulman, these PROCEEDINGS, 51, 1299 (1964). Sevag, N. G., D. B. Lackman, and J. Smolers, J. Biol. Chem., 124, 425 (1938). Sponer, H., Y. Kanda, and L. A. Blackwell, J. Chem. Phys., 29, 721 (1958). Steiner, R. F., and R. F. Beers, Polynucleotides (New York: Elsevier, 1961). Sternlicht, H., G. C. Nieman, and G. W. Robinson, J. Chem. Phys., 38, 1326 (1963). Weil, G., and M. Calvin, Biopolymers, 1, 401 (1963).

ON ELEMENT SYNTHESIS AND THE INTERPRETA TION OF THE ABUNDANCES OF HEAVY NUCLIDES BY HANS E. SUESS UNIVERSITY OF CALIFORNIA (SAN DIEGO AT LA JOLLA) Presented before the Academy, April 29, 1964, by invitation of the Committee on Arrangements for the Annual Meeting* The relative abundances of the elements, derived from analyses of meteorites and from spectral analyses of the sun, combined with the isotopic composition of the elements, give the data for the abundances of the individual nuclear species. These data, sometimes called cosmic abundance data, although they may perhaps apply only to our solar system, follow certain rules.' The most important of these rules postulates a smooth dependence of abundances on mass numbers. The rule holds for a wide range of mass numbers, if one considers even and odd mass- numbered nuclear species separately. The rule appears to be valid for the odd mass- numbered nuclear species at mass numbers A > 40, and for even mass-numbered species at A > 90. In certain mass ranges, and in a most pronounced way in the mass range 70 < A < 90, an additional rule holds, saying that the abundances of nuclei with the same neutron excess number show a smooth dependence on mass number. Exceptions to these rules occur primarily where the nuclides contain a closed shell of neutrons. It had been noted already by Goldschmidt2 in 1938 that the data for the abundances of the nuclear species show clear correlations with nuclear properties; conspicuous maxima exist at mass numbers where the nuclei contain a closed shell of neutrons. The abundances of the nuclear species were no doubt determined by the nuclear properties which defined their yield in nuclear processes that led to their formation. In addition to discontinuities in the abundance lines that can be correlated with closed shells of neutrons, irregularities exist in the mass region around 102 and 122. Downloaded by guest on September 30, 2021