ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 144, 650-656 (1971)

The Double-Stranded Polyadenylic Acid: Poly-L-Lysine Complex’

A Conformational Study and Characterization

BETTY DAVIDSON2 AND GERALD D. FASMAN3

Gradute Department of Biochemistry, Bran&is University, Waltham, Massachusetts 02164

Received January 4, 1971; accepted March 15, 1971

Double-stranded poly A associates with poly-n-lysine at pH 4.2 to form a complex with an altered circular dichroism (CD) spectra ([81%% N 100,000; [IY]~~~~~‘~~ N 57,000). Maximum complex formation, as measured by the change in ellipticity at

x max , is attained at a lysyl/adenyl residue ration of unity, r = 1, and is not de-

pendent on ionic strength. The shape and position of the final CD spectrum is very similar whether poly A is added to poly-n-lysine or the order of addition is reserved. However, the magnitude of the spectral change is variable, depending on conditions

of mixing. The CD spectrum of double-stranded poly A at pH 4.2 has also been found to be variable. Complex formation is associated with extensive aggregation, even at lysyl/adenyl ratios well below equivalence, and it is probable that the extent of ag-

gregation determines the extent of the CD spectral change. It is concluded that complex formation between double-stranded poly A and

poly-n-lysine is accompanied by a change in conformation of the polynucleotide, as seen by circular dichroic changes. These spectral changes might reflect changes in

the position of the adenine bases relative to the polyphosphate backbone induced by poly-n-lysine binding or due to formation of a large aggregate of poly A: poly-L-

lysine.

Early studies of the possible role of his- tones in genetic expression have focused on the specificity of the polynucleotide : poly- peptide interaction (1-8). The thermal sta- bilization of native DNA by bound histones or histone-like materials has also been extensively studied (3-6, 8-11). Recently many investigators have considered the question of whether native DNA is con-

1 This investigation was generously supported by research grants from the National Science Foundation (GB-8642), the National Institutes of Health of the U. S. Public Health Service (GM

17533-09)) the American Heart Association (69-739) and the American Cancer Society (P-577). Publi- cation No. 774 from the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154.

2 Present address : Thyroid Research Labora- tory, Massachusetts General Hospital, Boston,

Massachusetts. 3 To whom correspondence should be addressed.

formationally altered when associated with histone. Both “model” systems (12-15) and DNA with histones and purified histone frac- tions have been studied for their conforma- tional effect on native DNA (10, 16-22).

The present study, a model system for histone : DNA interaction, describes a con- formational change associated with inter- action of double-stranded poly A4 with poly-L-lysine (at pH 4.2, 0.01 M NaOAc). This is a continuation of work from this laboratory described in a previous paper (13) on the interaction of single-stranded poly A with poly-L-lysine (at pH 7.0, 0.01 M sodium phosphate). The work herein demon- strates that changes in the CD4 spectrum of double-stranded poly A occur as a result of combination with poly-n-lysine. It should be noted that a major difference between this

4 Abbreviations used: Poly A, poly adenylic acid; CD, circular dichroism.

650

POLY A(DOUBLE STRANDED) : POLY-L-LYS COMPLEX 651

system and histone:DNA systems is that double-stranded poly A is not stable at pH 7, but only at lower pH and, therefore, this study was conducted at pH 4.2 where poly A has essentially zero charge.

MATERIALS AND METHODS

Twice distilled water was used for all work. The second distillation was performed in an all-glass

apparatus. Poly-t-lysine.HCl was synthesized as previ-

ously described (23). The molecular weight esti-

mated by viscosity measurements was about

55,000. Poly A was purchased from Miles Chemical Co.

Almost all of the experiments reported herein were done wit,h lot No. 11-58-301. In later experiments,

poly A was treated to remove any residual protein. A soltttion of this poly A in 0.007 M phosphate buf-

fer, Na, pH 7.1 was extracted three times with an equal volume of phenol, saturated with the phos-

phat,e buffer. Residual phenol was removed by ext traction with ether followed by dialysis agains-

water (24). The resulting stock solution at pH 7.0 (water) of poly A was 2.55 X 1W3 M.

If the [@IL, nm of the diluted poly A at pH 4.2 was less than 90,000, the solution was heated at 95-100” for 5 to 10 min, then gradually cooled to room

temperature. This treatment restored maximum ellipticity (95,000-105,000) and the heated and

cooled poly A was then used for further experi- ments.

Concentration of poly-L-lysine. Concentrations

of stock solutions of poly-L-lysine.HCl were deter- mined either by a modified microbiuret procedure (25) ttsing poly-r,-lysine.HCl dried to constant

weight as a standard or by Nessler micro-Kjeldahl nitrogen analysis (26).

Concentration of poly A. Poly A concentrations were det,ermined spect,rophotomet)rically at abottt

pH 4.2, 0.01 M sodium acetate or cit,rate buffer, by relating maximum absorbance at, 252 nm to the molar residtte ext,inction coefficient, enrax = 8.7 X

lo” l:/(mole. cm) (27). Polymer concentrations are expressed as mole

residues per lit,er. pH Measurements. Routine measurements of

pH were made with a Sargent Model DR pH meter equipped wit.h a combination glass-AgCl micro- electrode, 30070-10. Standard pH buffers at pH 4.0 and 7.0, from the Fisher Scientific Co. were Itsed.

Circcrlur dichroism. Measurements were made

wit.h a Gary Model 60 recording spectropolarimeter

with a 6001-circular dichroism accessory (slit-

widt.h programmed to maintain a 15-K half-band

widt,h). A fused quartz optical cell of l-cm path

length was used. Optical cells were purchased from

the Opt,ical Cell Co., Brentwood, Md. Data are re-

ported as residue molar ellipticity, (per adenyl

residue) [e] (deg cm2/dmole) calculated using [t?] = 10 f&/l c, where &bs = ellipticity (degrees), 1 = path length (decimeters), c = residue concentra-

tion (M). The temperature was maintained at 22”. Ultraviolet absorption. Ultraviolet absorbance

was measured with a Cary 14 spectrophotomet,er. Standard matched l-cm rectangular quartz cells

were used. UltracentriflLgation. A Spinco model 1~ ult,racen-

trifuge equipped with UV optics was used.

Addition of poly-L-Eysine.HCl to poly A. Poly- L-lysine.HCl (5.7 X IOW M) in wat,er (pH z 5) was added in 5-J aliquots to a l-cm path length optical

cell containing 3.0 ml of 5.1 X 1O-5 M poly A in 0.01 M acet,at,e buffer, pH 4.2. Additions were made

with disposable micropipettes supplied by Bor117s

Laborat)ories, Reading, Mass., and mixing was ac- complished by drawing the solut,ionsinto a pipette. The circttlar dichroic spectrum (310-230 nm) was

recorded after each addition of poly-n-lysine. The reference base line was the solvent. The volume

ranged from3.000 to 3.035 ml. Concentrations were corrected accordingly in this and all ot,her experi-

ments. Ilesidue ratios, r, are calctdated as lysyl residtres/adenyl residues. In the experiments de- picted in Figs. 1 and 2, the residtte ratio ranged

from 0 to 1.3. Addifion of poly A to poly-L-lyrine. Poly A

(2.6 X lo-” M, wat,er) was added in lo- and 5-~1 aliquots (t,he first two additions were 10 ~1) to a l-cm cell containing 3.0 ml of 5.7 X IOy5 M poly-L-

lysine in 0.01 M acetate buffer, pH 4.2. The 01,. served rotation of the reference base line, 3.0 ml of

5.7 X lOA M poly-n-lysine, was zero over the wave- length range studied (310-240 nm). Cl) spectra

were recorded after each addition of poly A. In the experiment shown in Fig. 3, the residue ratio ranged from 6.7 to 0.49, but since opalescence be-

came discernible at r = 0.95, the data are shown

only to this residtte ratio.

The eflect of ionic strength on complezjormation,.

Aggregation of poly A at ionic strengt#h above 0.01

was minimized by adding the desired amount, of

NaCl t,o t.he poly-n-lysine rather than t,o the

poly A.

This poly-r-lysineeNaC1 solut,ion was then

added to a solut,ion of poly A in either 0.01 NI ace-

tate or H20 at pH 4.2 (curve A, Fig. 4). Alterna-

tively, a solution of poly A in Hz0 was added to

the poly-L-lysine-NaCl solution, at pH 4.2, 0.01 M

acetate bttffer (curve B, Fig. 4). The complexes

were formed at t,he ionic strengths indicated and

their CD spectra recorded from 310 t,o 240 nm.

DAVIDSON AND FASMAN

.

I I I I I I I

240 260 260 300

Wavelength, “m

FIG. 1. The circular dichroism spectra observed

upon the addition of poly-L-lysine to poly A, pH 4.2: Poly-L-lysine (5.7 X 10e3 M) was added in 5 ~1

aliquots to 3.0 ml of 5.1 X lW6 M poly A in 0.01 M

acetate buffer, pH 4.2. The optical path length was

1 cm. Spectra were recorded after each addition of poly-L-lysine and correspond to the following resi-

due rat,ios: Spectrum 1, 0 (poly A alone) ; 2, 0.185; 3, 0.370; 4, 0.555; 5, 0.740; 6, 0.925; 7, 1.11; 8, 1.30.

RESULTS

The change in the circular dichroism of double-stranded poly A on addition of poly- A-lysine. The addition of poly-L-lysine to maximally protonated poly A, (28) pH 4.2, causes a change in the double-stranded heli- cal conformation of the polynucleotide which is reflected in an altered CD spectrum. Figure 1 is a tracing of the CD spectral data obtained from a typical experiment in which microliter volumes of 5.7 X 10e3 fi1 poly-L- lysine are added to 3.0 ml of 5.1 X 10M5 M poly A. Details of the experimental condi- tions are given in the figure legend. As com- plex formation proceeds, the positive peaks of the CD spectrum of poly A diminish in magnitude, particularly in the region of the 262 nm maximum [@‘$z,” -96,000; [~]gigexJ=l = 56,500]. The 275-nm shoulder shows a lesser change and finally, a new optically active transition develops at around 285-290 nm. The 242-nm trough increases slightly in magnitude and the entire spectrum is shifted to longer wave- lengths b? about 3 nm. The addition of poly-L-lysme beyond a, lysyl/adenyl residue

ratio of 1.0 results in no further change in the CD spectrum. In the experiments re- ported herein no precipitation was noted at residue equivalence.

The above described spectral changes have been observed in all experiments but the rotational magnitudes of the final 1: 1 complex have varied as much as 30 %. The problem of reproducibility is certainly re- lated to the extreme sensitivity of complex formation to the conditions of mixing (e.g., rate of mixing, order of addition of com- ponents, total volume of system), many of which are hard to control. In addition, it, is probable that the conformation of the start- ing material, poly A, at, pH 4.2 is not com- pletely defined. This double-strand poly- nucleotide is known to be aggregated at ionic strengths above 0.1 (29). Furthermore, we have noted wide variation in the CD parameters of acid poly A, depending on the sample. Ellipticities at X,,, vary from [0] = 67,000 to 105,000. Heating at 95 to 100° restores maximum ellipticity (95,000- 100,000) to those poly A samples of initial low ellipticity (see the experimental section). This indicates that there may be more than two acid forms of the polynucleotide at ionic strengths well belowO.1, in agreement with the suggestion of Dr. S. Hanlon.5

The stoichiometry of complex formation. The maximum stoichiometry of complex formation is 1: 1 lysyl/adenyl, as depicted in Fig. 2. In Fig. 2a, the residue ellipticity at x max (262 nm for poly A, 265 nm for the complex), [e]Amax is plotted against r, the mole residue ratio lysyl/adenyl, as poly-L- lysine is added to poly A. The figure shows three different experiments to illustrate the variations encountered. No change in the ellipticity is seen at residue ratios 0 to 0.2, although the spectrum is slightly red shifted and the 285-290-nm shoulder is appearing (see Fig. 1). The major decrease in [B]xmax occurs at ratios 0.2 to 0.7. At residue equiva- lence (r = 1.0) no further change in [O]hmax is seen upon addition of poly-L-lysine. It is concluded that under these experimental conditions, pH 4.2 the [poly A + poly-L- lysine] complex contains one lysyl residue

5 Personal communication from Dr. S. Hanlon.

POLY A(DOUBLE STRANDED) : POLY-L-LYS COMPLEX 65X

,O- 0.2 0.4 0.6 03

Residue Ratio, l.ysyl/Adrnyl

‘b

FIG. 2a: Stoichiometry: The change in [0]+,,,, of poly A, pH 4.2 with addition of poly-L-lysine in three experiments. Curve 1: 5- and 2-~1 aliquots of

6.2 X 1W3 M poly-L-lysine were added to a l-cm cell containing 3.0 ml of 5.1 X IF5 M poly A in

0.01 M acetate buffer, pH 4.2. The CD spectrum (31@235 nm) was recorded after each addition.

Acetate buffer (0.01 M, pH 4.2) was used as the reference base line. Curve 8: As in curve 1, at a poly-L-lysine concentrat.ion of 5.7 X lO+ M. Curve 3: As in curve 1, at a poly A concentration of 5.5 X lO+ M. (Zb): Stoichiometry: the change in

A PI h,,,([fI]ziz/ - [O]~~~~A+comp~ex with addition of poly-L-lysine. This figure is an average of 6 ex-

periments which were performed using the same conditions of volume and concentration as de- scribed in 2a.

for every adenyl residue, which is involved in a double-stranded helix.

The precision of the data is indicated by the bars in Fig. 2b at 1’ = 0.6 and at T h 1.0.

The CD spectra of uncomplexed poly A pH 4.2, ionic strength 0.01 exhibit variations (f6 %), as shown by dif’ferent (19)~~~~ values at ?’ = 0 in Fig. 2a. These are reflected in CD spectra of the final 1: 1 complexes, which vary by fl4 %. However, if the data are nor- malized, they can be readily compared.

If A[e] ([fJ]f$” -[ ,j];;=xpO’y * + coml”ex)

is plotted vs residue ratio, as in Fig. 2b, the “endpoint” A[0]x,,, values of each experi- ment are in good agreement (h;) %), although the shape of the individual curves still varies widely (see error bar at 1’ = 0.6). The data have been presented as molar ellipticities, [e] (Fig. 2a) and as A[01 (Fig. ‘Lb) in order to indicate the range of the absolute values of [0] for poly A and the complex and also to illustrate the experimental difficulties encountered.

The circular dichroism spectrum of the 1: 1 complex, addition of poly A to poly-L-lysine. If poly A is added to a mole-residue excess of

-10

I 240 260 280 300

Wavelength. nm

FIG. 3. The circular dichroism spectra observed upon the addition of Poly A to Poly-L-lysine, pH 4.2. Poly A (2.6 X 10d3 M) was added in lo- and 5-J

aliquots to a l-cm cell containing 3.0 ml of 5.7 X 1W5 M poly-L-lysine in 0.01 M acet,ate buffer, pII 4.2. The poly-L-lysine solution, which had zero ro- tation over the wavelength range studied, was used as the reference base line. CD spectra were

recorded after each addition of poly A. Residue ratios corresponding to spectra l-12 were, respec- tively, 1,6.7;2,3.35;3,2.69;4,2.23;5, 1.91;6, 1.67; 7, 1.34; 8, 1.11; 9, 0.955.

654 DAVIDSON AND FASMAN

FIG. 4. The ellipticity of the complex, [t?]~~~~ex a function of ionic strength. for T = 0.5, 2.9 and - 5.8 complexes. The complexes were formed as de-

scribed in the methods section. Curve A is an av- erage of the results of four experiments. Poly-L- lysine (-6 X 10-z M) in varying concentrations of

NaCl was added to a l-cm cell containing 3.0 ml of ~5 X W6u poly A.The CD spectraofthe re- sulting T = 0.5 complex were recorded at the indi-

cated ionic strengths. Curve B. Poly A (0.025 ml of

3.85 X KY4 M) was added to a l-cm cell containing 1.0 ml of 5.4 X 1W5 M poly-L-lysine containing the desired concentration of NaCl and 0.01 M acetate

buffer, pH 4.2. CD spectra were recorded for each ionic strength, at T = 5.8. An additional 0.025 ml of poly A was then added to the samples at each

ionic strength, decreasing T to 2.9, and t,he CD spectra were recorded again. No change in [B] was

observed at the two different values.

poly-L-lysine at pH 4.2, the CD spectrum which develops should be that of the [poly A + poly-L-lysine] complex, free of any spectral contributions of uncomplexed poly A, i.e., the spectra should be similar to those observed at residue equivalence in Fig. 1. This expectation is supported by the data of Fig. 3, a tracing of a typical experiment in which aqueous poly A (epH 7.0) is added to poly-L-lysine at pH 4.2. The resulting set of CD spectra are similar to that of the 1: 1 complex of Fig. 1. Both have crossover wavelengths at 252.5 nm and both show a broad maximum at 262-265 nm and the new component at 285-290 nm. In many experi- ments, resolution of the 262-265-nm maxi- mum into two peaks at 267 and 273 nm, has been observed. The observed rotations are proportional to the poly A concentration from r = 13.0 to r = l-l.5 depending on the experiment, showing that no precipitation has occurred.

volume = 1 ml instead of 3 ml), the [O]o,oG~~~x was 35,000-39,000.

The conditions of mixing the components obviously affect the final CD spectra even more in this system than in the experiments in which poly-L-lysine was added to poly A. This is because observed rotations are small and any conditions which cause incomplete association along a molecule of poly A will result in a large spectral contribution from the uncomplexed portion of the polynucleo- tide. Also perhaps there is binding of single- stranded poly A, before it converts com- pletely to the double stranded form, pre- venting maximal complex formation.

Dependence of complex formation on ionic strength. Association of poly A with poly-L- lysine appears independent of the ionic strength of the medium, over the range 0 to 0.5 M NaCl, provided that conditions are chosen to minimize prior aggregation of the polynucleotide and/or insolubility of the complex, as described in the methods section. The dependence of [O]chmz$ex on the molarity of NaCl in the medium at residue ratios far from equivalence is seen in Fig. 4 (Curve A at r = 0.5, poly-L-lysine added to poly A; curve B, r = 2.9 and 5.8, poly A added to poly-L-lysine). The complex was formed at each designated ionic strength, as described in Fig. 4 and the methods section. For each mixing procedure the resulting CD spectra were the same, over the ionic strength range studied. The CD spectrum of a poly A control was also invariant ([13],,,,, N 100,000) from 0 to 0.2 M NaCl. Measure- ments of poly A were limited to Iow ionic strengths because of its aggregative proper- ties (29). Turbidity accompanied complex formation at residue ratios near equivalence, CC > 0.03, and was a problem even at ratios far from equivalence, particularly around 0.06 to 0.10 M NaCl. The reasons for this are not known. The insolubility of the 1: 1

complex at high ionic strength is probably related to its state of aggregation combined with its relative lack of charge.

The state of aggregation of the poly A:Poly- L-lysine complex. The complex of double- stranded poly A and poly-L-lysine is highly

In the experiment depicted in Fig. 3, aggregated, even at residue ratios less than

PI OOmpleX 265 nm = 75,000. In other experiments 1.0, as shown by the following experiments.

performed with slight variation (total Ultracentrifugation. The complex at r =

POLY A(l>OUBLE STIIANL)ED) : POLY-L-LYS COMPLEX 6%

0.5 and T = 1.0, pH 4.2,O.Ol 11 citrate buffer is totally sedimented in a Model E ultrs- centrifuge before the rotor attains a final speed of 33,450 rpm. Double-stranded poly A alone and an I’ = 0.5 complex of single- stranded polg A with poly-r&sine (0.01 RI phosphate buffer, pH 7.2) are just beginning to sediment after 30 min at 33,450 rpm. Thus the aggregates have a molecular weight corresponding to greater than 325.

Tmdipeed ceutrifuyatio)l. The poly A: yoIy-n-Iysine complex, Y = 0.5 to 1.1, pH 4.2, 0.002 to 0.0’20 31 citrate or acetate is optically clear (absorbance - 0.02, 1 cm, 370 nm). However, if centrifuged for 15 mm in a table-top clinical centrifuge (-5OOq) t,he complex separates as a gel. So ultraviolet absorbing material remains in the supernatant solution. Under these con- ditions, neither of the known double- stranded forms of poly A (form A, pH 4.2 and form B, pH 5.8) show any diminution in ultraviolet spectra as a result of centrifuga- tion.

Filtration throucqh glass wool. If filtration through a plug of glass wool is substituted for low-speed centrifugation the same ultra- violet spectral results are obtained as in the centrifugtvtion experiments, the material is held back.

In general, an interesting aspect of all of these experiments is that they show that there are no free poly A molecules, even at 1‘ = 0.5. Furthermore, at 1’ = 0.5 the com- plex is already in an aggregated state.

1)ZSCUSSION

At pH 4.2,O.Ol 11 acetate or citrate buffer, poly A exists as a double-stranded helix (29-30). Under these conditions the X1 adenine ring nitrogen is essentially fully protonated (29931). The resulting electro- static attraction between the X1 nitrogen on one polynucleotide strand and the phos- phate groups on the opposite strand are believed to be a major factor in the stabili- zation of the double-helical conformations of poly A. The net charge of poly A at pH 4.2,O.Ol M acetate is zero, or very close to it. Complex formation with poly-L-Iysine in- volves association of a positively charged random polypeptide with an uncharged helical polynucleotide. It is expected that

such interactions should not be very sensi- tive to ionic strength, and the experimental data verify this hypothesis.

It is curious that charged poly-L-lysine binds to a polymer which is electrostatically neutral. Thus it would appear that forces other than electrostatic are involved in this interaction. However, upon binding, the lysyl residues must be associated with the charged phosphates (e.g., 1: 1 stoichiometry) possibly bringing about a redistribution of charge within the polynucleotide double helix. Perhaps these events might well con-

tribute to the conformational change of the poly A. As described above the complex would possess a high positive charge, which would not t,end to aggregate. However, aggregation occurs readily. This might sug- gest that upon binding of poly-L-lysine to poly A, an equivalent number of protons arc released per lysyl residue, maintaining new trality and consequently aggregation of thr: neutral polymer occurs. Such considerations might likewise play an important, role in histone: DKA interactions.

Complex formation is accompanied by a cooperative change in the CD spectrum of poly A. The stoichiometry is 1.0 but there is no free poly A discernible at residue ratios less than 1 (0.5-l). Therefore, complex formation with poly-L-lvsine does not in- volve complete association of some mole- cules of poly A and no association of others, as has been suggested for complexes of DNA with histones or histone-like materials (3-0, 8, 9). Furthermore, when poly-L-lysine is added to poly A at low ionic strength (0.00% 0.020 nr ?;aCl), there is no unaggregated complex present between r = 0.5 to 1: 1. Thus, aggregation seems to occur simul- taneously with complex formation under these conditions. It should be noted that the cooperativity of the spectral changes indi- cates that the aggregative phenomenon is not simply a random crosslinking of polynu- cleotide chains by a.dded poly-n,-lysine.

Because the complex is an aggregate its CD parameters are not well quantitated but are influenced by many factors; e.g., the total mixing volume of the components affects the final CD spectrum of the com- plex. This situation has its count*erpart in studies of the interaction of DNA with

656 DAVIDSON AND FASMAN

histones. It has been found that repro- ducible CD spectra can be obtained only by very gradual formation of the histone: DNA complexes (17-32). In this case the method used was gradient dialysis from a high salt medium (2 ~1) where there is minimal com- plex formation, to a salt concentration of 0.14 M. This particular technique cannot be used in the poly A:poly-L-lysine system be- cause poly A is itself aggregated at high ionic strength. In fact, the conformation of double-stranded poly A is probably more complicated than previously believed. We have found the CD spectrum of poly A at pH 4.2, 0.01 acetate to vary widely ([e],,,, from 67,000 to 105,000) with the particular sample chosen. Most often heating the pH 4.2 material at 90-100” for 10 min brings the CD spectra of all of these samples to the same maximum value ([0]~,5,,,~ = 95,000- 100,000).

Occasionally a single sample of poly A will display a maximal CD spectrum on 1 day but will require heating on another day. These unknown factors related to the con- formation of poly A alone must also have a bearing on the parameters of the [poly A: poly-L-lysine] complex. It is clear that further characterization of the polynucleo- tide is necessary.

These experiments have demonstrated complex formation between double-stranded poly A and poly-L-lysine, as illustrated by changes in the CD spectrum of the poly A, probably indicating conformational changes in the double-stranded poly A. Under maxi- mal complex conditions, one lysyl residue is bound per adenyl residue. It is not known definitely whether the accompanying CD changes are a result of aggregation producing an ordered anisotropic arrangement of molecules within the complex (liquid crys- tals) (15), or whether they reflect a change in the position of the adenine bases relative to the helix axis within the individual com- plex molecules.

REFERENCES

1. FELSENFEI~D, G. AND HUANG, S., Biochim. Bio- phys. Acta 34, 234 (1959).

2. AKINRIMISI, E., BONNER, J., AND Ts6, P. 0. P., J. Mol. Biol. 11, 128 (1965).

3. LENG, M., AND FELSENFELD, G., Proc. Nat. Acad. Sci. U. S. 66, 1325 (1966).

4. SZER, W., Biochem. Biophys. Res. Commun. 22,

559 (1966) . 5. SZER, W., J. Mol. Biol. 16, 585 (1966).

6. TSUBOI, M., in “Conformation of Biopoly- mers” (Ramachandran, G. N., ed.), Vol. 2, p. 689. Academic Press, New York (1967)

7. LATT, S. A., AND SOBER, H. A., Biochemistry

6, 3293, 3307 (1967). 8. OLINS, D. E., OLINS, A. L., AND VON HIPPEL,

P. H., J. Mol. Biol. 33,265 (1968).

9. OLINS, D. E., OLINS, A. L., AND VON HIPPEL,

P. H., J. Mol. Biol. 24, 157 (1967).

10. OLINS, D. E., J. Mol. Biol. 43,439 (1969).

11. SHIH, T. S. AND BONNER, J., J. Mol. Biol. 48, 469 (1970).

12. COHEN, P. AND KIDSON, C., J. Mol. BioZ. 36,

241 (1968). 13. DAVIDSON, B. AND FASMAN, G. D., Biochemis-

try 8,4116 (1969).

14. SHAPIRO, J. T., LENG, M., AND FELSENFELD,

G., Biochemistry 8, 3219 (1969).

15. HAYNES, M., GARRETT, R. A., AND GRATZER,

W. A., Biochemistry 9, 4410 (1970).

16. LEES, C. W. AND VON HIPPEL, P. H., Biochem- istry 7, 2480 (1968).

17. FASMAN, G. D., SCHAFFHAUSEN, B., GOLD-

SMITH, L., AND ADLER, A., Biochemistry 9, 2814 (1970).

18. SHIH, T. S. AND FASM~N, G. D., J. Mol. Biol. 62, 125 (1970).

19. SHIH, T. S. AND FASMAN, G. D., Biochemistry

20.

21.

22.

23.

24.

25.

26. 27.

28.

29.

30.

31.

32.

(in press) (1971). PERMOGOROV, V. I., DEBABOV, V. G., SLAD-

KOVA, I. A., AND REBENTISH, B. A., Biochim. Biophys. Acta 199, 556 (1970).

SIMPSON, R. AND SOBER, H., Biochemistry 9, 3103 (1970).

SPONAR, J. BOUBLIK, M., FRIC, I., AND SOR-

MOVA, Z., Biochim. Biophys. Acta 209, 532 (1970).

FASM.~N, G. D., IDELSON, M., AND BLOUT,

E. R., J. Amer. Chem. Sot. 83, 709 (1961). STEVENS, C. L. AND ROSENFELD, A., Biochem-

istry 6, 2714 (1966).

ZAMENHOF, S. AND CHARGAFF, E., Methods En- zymol. 3, 702 (1963).

LANG, C. A., Anal. Chem. 30,1692 (1958). HOLCOMB, D. N. END TINOCO, I., Jr., Biopoly-

3, 121 (1965).

ADLER, A. J., GROSSMIN, L., AND FASMAN,

G. D., Biochemistry 8, 3846 (1969). FRESCO, J. R. AND KLEMPERER, E., Ann. N. Y.

Acad. Sci. 81, 730 (1959). RICH, A., DAVIES, D. R., CRICK, F. H. C.,

AND WSTSON, J. D., J. Mol. BioZ. 3,71 (1961). HOLCOMB, D. N. .~ND TIMASHEFF, S. N., Bio-

polymers 6, 513 (1968). HUANG, R. C., BONNER, J., AND MURRAY, K.,

J. Mol. BioZ. 8, 54 (1964).