HELIX FORMATION BY GUANYLIC ACID
`BY IARTIN GELLERT, MARIE N. LIPSETT, AND DAVID R. DAVIES
`
`NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES, NATIONAL INSTITUTES OF HEALTH,
`BETHESDA, MARYLAND
`
`Communicated by Norman Davidson, October 25, 1962
`In 1910, Bang1 reported that concentrated solutions of guanylic acid formed a gel.
`We have also observed that concentrated solutions (25.0 mg/ml) of guanylic acid
`(GMP) at pH 5 are extremely viscous and, if cooled, form a clear gel.
`Less con-
`centrated solutions also gel on cooling but assume a more normal viscosity at
`room temperature. From examination of the optical properties of the gel and in-
`vestigation of the structure of fibers obtained from the gel by drying, we have con-
`cluded that, at least in the case of the 5' isomer, the phenomenon may be explained
`as being due to helix formation by the guanylic acid. A possible structure is pre-
`sented for this helix.
`Materials and Methods.-Ultraviolet absorption was measured with a Cary Model 15 spectro-
`photometer, using an 0.1 mm cell. A water-jacketed cell housing was used to maintain tempera-
`ture. To measure optical densities greater than 2.0, calibrated neutral density screens were placed
`in the reference beam. In this way, optical densities up to at least 3.5 could be accurately meas-
`ured, the instrumental stray light level being sufficiently low.
`Optical rotation measurements were performed on a Rudolph Model 80 spectropolarimeter,
`Specific rotations are uncertain by 4 100.
`using a water-jacketed 1-cm cell.
`Fibers were obtained from concentrated solutions at pH 5 by suspending a drop of the material
`over the ends of a U-shaped piece of paper clip and allowing it to dry slowly at 40C. The material
`dried into highly negatively birefringent fibers 5 mm long by 0.1 to 0.3 mm wide. X-ray diffraction
`patterns were recorded in a modified Philips fiber camera at several different relative humidities.
`The materials examined were obtained from the following sources: 5' GMP, Pabst and Schwarz:
`Since the chromatographic
`guanosine, Schwarz; mixed 2'-, 3 '-GMP, disodium salt, Sigma.
`separation of 2'- and 3'-GMP was found to require different conditions from those previously
`described for hydrolysates of crude RNA,' the separation is described.
`The commercial isomeric mixture of GMP was found by paper chromatography in the system
`(saturated (NH4)2SO4: 1 M Na Acetate: isopropanol: :80:18: 2)3 to contain 5 per cent guanosine,
`15 per cent 2'-GMP, and 80 per cent 3'-GMP. One hundred milligrams were dissolved in 400
`ml water, brought to pH 8.2 with NaOH, and loaded on a 2.3 X 12.8 cm column of Dowex-1-
`formate, which had been prepared by suspending 40 gm Dowex 1, 8 per cent cross-linked, 200-400
`mesh, chloride form, in 1 M HCOONa, packing, washing with this solution until chloride-free,
`Five per cent of the optical
`and then washing with water until neutral to bromothymol blue.
`density added to the column came off immediately with the guanosine peak using 0.05 M
`HCOONH4-0.01 M HCOOH, as was expected, but less than 1 per cent of the 2' and 3'-GMP were
`removed in the next step, using 0.1 M HCOONH4-0.1 M HCOOH. The salt concentration had
`to be raised to 0.5 M HCOONH4-0.1 M HCOOH before the remainder of the material came off,
`in two peaks. The fractions were lyophilized and identified chromatographically as 2'- and 3'-
`GMP respectively, with no detectable cross-contamination. More than 98 per cent of the material
`was recovered from the column.
`The materials obtained by this method (preparation I), while they were chromatographically
`free of other nucleotide materials, were found to contain appreciable amounts of ammonium
`formate, even after lyophilization and prolonged storage in vacuQ over KOH and P2OF.
`This
`Purification by barium
`contamination may have amounted to as much as 50 per cent by weight.
`precipitation proved impractical because of the relative insolubility of barium formate.
`There-
`fore, 300 mg of the 3'-GMP in 300 ml 0.01 Al NH4HCO3, pH 8.6, was put on a 2.2 X 9.5 cm
`DEAE-cellulose column, washed free of formate with 200 nil of the same buffer, and eluted with
`50 ml 0.3 M NH4HCO3. Lyophilization and subsequent storage in vacuo over KOH and P205
`2013
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`removed the last traces of NH4HCOs (preparation II). Treatment of a solution of this ammonium
`guanylate with solid Dowex 50- Na+ yielded a clean preparation of sodium 3'-guanylate for
`study (preparation III).
`Results.-1. Gel formation: When solutions of 5'- and 3'-GMP at a concentra-
`tion of 10 mg/ml in 0.01 M acetate buffer at pH 5 and 0.2 M NaCl are cooled to
`00C, they form clear viscous gels. At pH 7 or pH 2, no gel formation is observed;
`the pK of the secondary phosphate of the guanylic acids is 6.0, and the protonation
`This implies that the form which is capable of
`of the guanine occurs at pH 2.4.4
`gel formation is that which has a single negative charge on the phosphate and the
`neutral form of the guanine. 2'-GMP does not appear to form a gel under the con-
`Guanosine is not soluble
`ditions in which gelation occurs for the 5' and 3' isomers.
`at this concentration; there appears to be a slight increase in viscosity of saturated
`guanosine solutions, but the effects are too faint to be conclusive.
`Figure 1 shows the difference spectrum produced by
`2. UV absorption studies:
`Figure 2 shows the change in optical
`warming a 5'-GMP gel from 10C to 400C.
`
`3.0 1\7
`
`1.6-
`
`2.0 2.528-0030__
`
`we1.0~~~~~~~~~~~~~~~~~~~~~~~~.
`
`1A-
`
`1.2
`
`300 320Iu l<'
`0
`
`0 240
`
`260
`
`280
`X(mp)
`FIG. 1.-Difference spectrum between
`Conditions:
`5'-GMP at 40'C and 1VC.
`0.025 M GMP, 0.2 M NaCl, 0.01 M Na
`Acetate, pH 5.0.
`
`10
`
`30
`
`40
`
`20
`TCT)
`FIG. 2.-Melting curves of 3'-GMP and 5'-
`GMP gels. The optical density at 275 mu is
`Figure
`in
`plotted. Same conditions
`as
`1.
`o, 5'-GMP; X, 3'-GMP, preparation I.
`
`density at 275 mA upon gradually increasing the temperature of solutions of 5'-GMP
`It can be seen that there is a substantial increase in optical density
`and 3'-GMP.
`on warming, whose magnitude is comparable to that found on denaturation of
`helical polynucleotides. By this criterion, the gels are completely "melted"
`at 400C, the optical density coinciding with that of dilute solutions of GMP.
`Optical density changes were found to be only moderately reproducible in
`matched experiments, due in large part to a considerable time dependence of the
`optical density in the melting region. The melting curves also showed a clear
`hysteresis; the steepest optical density change of a solution which is being cooled
`lies several degrees below that of a solution being warmed.
`The 2'-GMP under similar conditions showed only a small change in optical den-
`sity upon cooling (-5 per cent).
`3. Optical rotation changes upon gelformation: The optical rotation changes upon
`It can be seen that a large change in
`gel formation are summarized in Table 1.
`specific rotation occurs upon cooling the 5'-GMP, The magnitude resembles that
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`TABLE 1
`OPTICAL ROTATION OF GUANYLIC ACIDS
`10C
`-2000
`-1 1700°
`
`5'-GMP
`[a]D
`[Cd3"s
`3'-GMP (preparation I)
`[aID
`[at365
`3'-GMP (preparation III)
`[aID
`[Ct ]365
`2'-GMP
`[ai D
`[i ]365
`(Same conditions as in Figure 1.)
`
`-30
`-180°
`
`-600
`-2800
`
`-200
`-1000
`
`400C
`-200
`-100°
`
`-200
`-900
`
`-200
`-900
`
`-200
`-900
`
`observed for the formation of helical polyribonucleotide complexes, but the ordered
`5'-GMP solution is laevorotatory, as opposed to the dextrorotation observed for
`the helical polyribonucleotides.6
`The changes in the 3'-GMP solutions are smaller but occur with the same sense.
`The 2'-GMP shows no changes in optical rotation upon cooling to 1'C.
`4. X-ray diffraction studies: X-ray diffraction patterns from fibers of the 5'
`isomer and of two salts of the 3' isomer are shown in Figure 3a, b, and c, respectively.
`Figure 3a is a characteristic helix diffraction pattern with a layer line spacing of
`13.0 A and a strong meridional reflection at 3.25 A.
`This is indicative of a helical
`structure with four units per turn of the helix, each unit being spaced 3.25 A apart
`The equator contains a strong reflection at 24.2 A, which
`along the helix axis.
`may be regarded as a measure of the distance between neighboring molecules.
`However, because of the lack of other sharp reflections on the equator, it is not pos-
`sible to assign a lattice and hence to determine the distance unambiguously.
`For
`instance, if this were the first-order reflection from a hexagonal lattice, then the in-
`termolecular separation would be 27.9 A. At 33 per cent relative humidity, the
`diffraction pattern remains essentially unchanged, but the spacing of the strong
`equatorial reflection is reduced to 21.7 A.
`Although the two diffraction patterns of the 3'-GMP are quite different, they both
`
`*
`
`.j.............
`
`, *_ i~
`
`_''.............
`
`FIG. 3.-(a) X-ray diffraction pattern of 5'-GMP.
`(b) X-ray diffraction pattern of 3'-GMP
`The fiber axis i.
`(c) X-ray diffraction pattern of 3'-GMP (preparation II).
`(preparation I).
`approximately vertical in all cases.
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`jet
`
`c
`N
`c><p--4t_ is
`
`<
`
`Thus, both
`have reflections on or very near to the meridian at a spacing of 6.73 A.
`structures have asymmetric units which repeat along the fiber axis every 6.73 A.
`In this respect, the structures differ from the 5'-GMP and from the ordered poly-
`nucleotides, where the first meridional reflection occurs with a spacing of between
`3 and 4 A.
`In Figure 3b, there are, in addition to the equator, only two layer lines, with spac-
`ings of 6.73 A and 3.4 A.
`This indicates that the structure forms an exact repeat
`every 6.73 A with a unit screw rotation of 00. The equatorial reflections may be
`indexed on a hexagonal lattice with a spacing of 31.4 A.
`In Figure 3c, the distribution of layer line intensity may be interpreted in terms
`of helical structure with z axis repeats of 6.73 A and 12.1 A for the asymmetric unit
`and the pitch of the helix respectively.
`The structure is, therefore, a helix with a
`6.73 A unit translation and + 200'/n unit rotation, with n-fold rotational symmetry
`along the helix axis. The very sharp equatorial reflections may be indexed on a
`hexagonal lattice with a = 25.5 A, and the sharpness of the reflections is consistent
`with a nonintegral screw.
`The considerably larger intermolecular separation for the material in preparation
`I is presumably the direct result of the presence of large quantities of ammonium
`Patterns similar to Figure 3c were obtained for the
`formate in this preparation.
`sodium salt of 3'-GMIP (preparation III) as well as for the sodium salt plus one
`equivalent of sodium chloride.
`Discussion.-Recent work by Ralph, Connors, and Khorana6 has shown that
`tri- and tetranucleotides of deoxyriboguanylic acid are capable of forming or-
`It is perhaps not too surprising,
`ganized macrostructures of remarkable stability.
`therefore, to find that guanylic acid itself can also form a regular structure.
`There are four ways in which two guanines may be paired to form satisfactory
`hydrogen-bonded dimers, as originally pointed out by Donohue7 (structures 9, 10,
`11, and 12). None of these dimers accounts for the remarkably stable structures
`If, however,
`formed by guanylic acid.
`two pairs of Donohue's structure 10 are
`brought together, they can form the hy-
`drogen-bonded arrangement shown in
`Figure 4, in which the four guanines
`are related to each other by the opera-
`tion of a fourfold rotation axis.8
`In
`such an arrangement, there are now
`two hydrogen bonds per base compared
`with one for each of the dimers, and
`one would expect this to be a particu-
`The existence
`larly stable structure.
`in solution of planar tetramers of this
`kind could then result in the formation
`Cof linear aggregates formed by stacking
`N=Ax
`the tetramers on top of each other, since
`C,;> the large planar surfaces would result in
`strong van der Waals attractions.
`FIG. 4.-Proposed arrangement of the bases
`Such aggregates would be roughly cy-
`in GMP gels.
`
`N
`
`C~>
`
`\
`
`Ng
`
`C
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`lindrical in appearance and would contain a hole in the middle in which it might
`be possible to place one water molecule per tetramer.
`This hypothesis has been tested in the case of 5'-GAIP, whose diffraction pattern
`may be simply interpreted in terms of such a structure.
`Each group of four bases
`would then have a similar group above and below it with a screw rotation of
`t22.50
`and an axial translation of 3.25 A.
`In order to reconcile the helix repeat of 3.25
`A with a base separation of 3.36 A, the bases have to be tipped by a small amount.
`Models have been constructed with these dimensions; it is clear that the model with
`right-handed rotation of 22.50 cannot be constructed because of close contacts be-
`tween neighboring ribose-phosphate groups. With the left-handed rotation, how-
`ever, such a model can be constructed, and it becomes apparent that the phosphate
`group is then located in a position which is very favorable for hydrogen bonding.
`Hydrogen bonds can be formed from the 2-amino group to one of the oxygens of a
`phosphate one layer below; the other oxygens can form hydrogen bonds with neigh-
`boring phosphates and with the 2'-hydroxyl group of a neighboring ribose, two
`Preliminary calculations of the Fourier transform of the model show
`layers above.
`a satisfactory agreement with the observed intensity distribution.
`It should be noted that there exists a possible alternative structural explanation
`for the 5'-GAIP diffraction pattern.
`If the bases are uniformly tilted so that the
`fourth pair of hydrogen bonds is used, not to close the ring, but to bond to a fifth
`guanine situated 3.25 A above the first, then by proceeding in this manner a con-
`tinuous connected helix can be formed.
`Such an interpretation would require that
`the Bessel function on the fourth layer line be J1 instead of a J0.
`Although the
`present diffraction patterns indicate that this is indeed a Je, it is possible that the
`meridional minimum is obscured by the relatively poor orientation of the fiber, so
`that this alternative hypothesis cannot be ruled out.
`In the case of the 3'-GMiP, the diffraction patterns may be discussed in terms
`of the same general hydrogen bonding scheme.
`Here, the better orientation of
`the diffraction pattern gives one more assurance that the reflection at 3.36 A is
`truly meridional, thus favoring the planar tetramer hypothesis.
`However, both diffraction patterns of the .3' isomer, i.e., with and without an
`excess of ammonium formate, require a repeating unit with an axial translation of
`This requirement may be the result of some relatively minor periodic dis-
`6.73 A.
`tortion of the helix in such a way as to couple adjacent tetramers together to form
`a repeating unit with a thickness of 6.73 A.
`If such diffraction patterns were ob-
`tained from a covalently linked polynucleotide, then it would be necessary to in-
`voke such a hypothesis to account for the observed layer line distortion.
`However,
`when no covalent backbone exists, it is no longer obligatory to stack the bases in
`a regular manner such that the face of one base is in contact with the back of the
`It is then possible for the base stacking to occur in cou-
`next base along the helix.
`ples formed by stacking two adjacent tetramers face to face. The next couple may
`then be stacked on top of this, and so on, giving rise to helical aggregate ABAB-
`ABAB. .. with a repeating unit (AB) of 6.73 A.
`We propose this mode of alternating base stacking as a possible interpretation
`For the material of Figure 3b, containing
`of the observed diffraction phenomena.
`an excess of ammonium formate, the stacking of adjacent couples occurs without
`rotation about the helix axis, whereas in the case of Figure 3c the rotation would be
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`=1 500. The rotation between the two tetramers A and B is, however, not defined
`by the dimensional features of the diffraction patterns, and it is, therefore, not pos-
`sible to verify this hypothesis in detail.
`The differences between the structures of the 3' and 5' isomers indicate clearly
`that the position of the phosphate group plays an important role in determining the
`This is presumably because of the ability of the
`structure of the linear aggregates.
`phosphate group to form hydrogen bonds with atoms on neighboring molecules, thus
`adding to the stability of particular configurations, as well as being due to the
`The further differences
`electrostatic repulsion of the charge on the phosphates.
`between the two preparations of the 3' isomer indicate that the structures can also
`vary according to the nature of the environment of the aggregate.
`The large difference in optical rotation of gels of the 3' and 5' isomers is also con-
`sistent with the X-ray results. The 5' isomer forms a regular helix and would be
`expected to give a large rotation in the helical form,9 whereas the 3' isomer consists
`of pairs of planar tetramers stacked on top of each other and would be expected to
`have a considerably different helix contribution to the optical rotation. The differ-
`ences in optical rotation between preparations I and III of the 3'-GMP are not
`unexpected in light of the observed differences in the diffraction patterns.
`I Bang, I., Bioch. Ztschr., 26, 293 (1910).
`2 Cohn, W. E., and E. Volkin, Nature, 167, 483 (1951).
`3Markham, R., and J. D. Smith, Bioch. J., 49, 401 (1951).
`4Jordan, D. O., in The Nucleic Acids, ed. E. Chargaff and J. N. I)avidson (New York: Academic
`Press, 1955), vol. 1, p. 459.
`5Doty, P., H. Boedtker, J. R. Fresco, R. Haselkorn, and M. Litt, these PROCEEDINGS, 45,
`482 (1959).
`6 Ralph, R. K., W. J. Connors, and H. G. Khorana, J.A.C.S., 84, 2265 (1962).
`7Donohue, J., these PROCEEDINGS, 42, 60 (1956).
`8 A four-stranded model containing a similar arrangement of the bases, but with one hydrogen
`bond per base, was considered by A. Rich (Biochim. Biophys. Acta, 29, 502 (1958)) as a possible
`structure for polyinosinic acid.
`9 Moffitt, W., D. D. Fitts, and J. G. Kirkwood, these PROCEEDINGS, 43, 723 (1957).
`
`STREPTOMYCIN AS A MUTAGEN FOR NONCHROMOSOMAL GENES
`BY RUTH SAGER
`DEPARTMENT OF ZOOLOGY, COLUMBIA UNIVERSITY
`Communicated by M. Demerec, October 1, 19)62
`Stable hereditary determinants segregating in a non-Mendelian manner were
`first described in 1908 by Correns.'
`In the following decades, some hundred or
`more well-established examples of non-Mendelian heredity were reported,2 3 but
`few attempts have been made to integrate them into a general theory of genetics.
`The principal difficulty blocking a systematic study has been the rarity of their oc-
`currence and their failure to respond to mutagenic agents.
`The chance isolation, some years ago, of a mutant of the alga Chlamydomonas
`reinhardi, exhibiting nonchromosomal inheritance of streptomycin resistance
`(sr-500),4 provided a new material with which to reinvestigate the role and origin
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