On the factors controlling the reversibility of DNA denaturation

J. Mol. Biol. (1962) 4, 467-487

On the Factors Controlling the Reversibility of DNA Denaturation E. PETER GEIDUSCHEK Oommittee on Biophysics, University of Ohicago, Ohicaqo, Illinois, U.S.A. (Received 17 February 1962) The experiments described in this paper distinguish two processes which lead to a reversal of DNA denaturation and to the re-formation of Iong-etaoked arrays of nucleotide pairs. Type I reversibility is a very rapid intramolecular process which occurs in DNA preparations from viral, bacterial and animal sources, in solvents varying greatly in ionic strength. Type II reversibility ("renaturation", Marmur & Lane, 1960; Doty, Marmur, Eigner & Schildkraut, 1960; Marmur & Doty, 1961) has the properties of an intermolecular process, is much slower, and occurs only in relatively homogeneous DNA preparations at moderately high ionic strength. Data on the dependence of type I reversibility upon DNA size and composition are consistent with the proposal that type I reversibility is controlled by GC·rich nucleotide pair sequences which act as "nuclei" for the re-establishment oflong-range order. Under the conditions of these experiments, such nuclei control the re-stacking of extremely long sections of polynucleotide chain. Consequently, type I reversibility is strongly affected by molecular weight changes in the range 0·5 to 10 x 108 • GC·rich "nuclei" may, however, be dissociated by heating. The loss of type I reversibility is accordingly associated with the final stages of the dissociation of a double helix. However, if complementary polynucleotide chains of DNA are linked covalently, type I reversibility is permanent. (Such material is called "reversible" DNA.) The interpretation of experiments on the stability of DNA secondary structure, especially those involving bacterial transformation assays, is discussed in the light of these results.

1. Introduction The phenomenon of irreversible denaturation in nucleic acids is extremely restricted. It appears to be a property of polynucleotides of complex sequence such as DNA and complementary RNA (Gulland, Jordan & Taylor, 1947; Geiduschek, Moohr & Weiss, 1962). The available information is consistent with the following widely held interpretation of irreversible denaturation:(a) when totally disordered or dissociated DNA is brought to conditions in which the helical secondary structure is thermodynamically stable, it is possible for short, imperfectly ordered sequences of polynucleotide from different polymeric chains or different portions of the same chain to interact. Most frequently such bonded sequences will not be "in register". t

t Short nucleotide sequences of two DNA chain segments are "in register" if, by forming complementary base pairs, they order their respective polynucleotide strands so as to permit the re-formation of long completely ordered double helical arrays. In the simplest case, such a situation will involve the complementary strands of a single molecular species of DNA (Doty et al., 1960; Marmur & Lane, 1960). However, two complicating factors may be envisaged: (a) when the DNA sample consists of mechanical degradation products of a very much longer parent chain (Cavalieri, 1957; Davison, 1959), a uniquely definable set of molecular species no longer exists, and the above process does not lead to complete re-formation of ordered sequences; (b) if long, nearly-homologous regions of DNA exist, and if these are located in different sections of the (bacterial) chromosome, then the above process could lead to a peculiar type of mismatching, in which long, almost perfectly ordered polynucleotide regions would still be formed; however, at either end of such a region, non-homologous DNA strands would be attached. 467

468

E. PETER GEIDUSCHEK

(b) Owing to the imperfection of this type of secondary structure, many nucleotide segments are unable to form secondary structure links. These configurations are therefore thermodynamically unstable. (c) To repair such imperfect, denatured configurations it is necessary to break entire sequences of secondary structure bonds. The concomitant free energy of activation requirement restricts finite equilibration rates to environmental conditions close to those of the helix-coil transition. The kinetic barrier to reversible denaturation arises from this requirement. (d) Substantially imperfect ordering is possible only in copolynucleotides of sufficiently complex sequence. For instance, the perfectly regular polyribo-A + polyribo-U complex and the perfectly alternant polydeoxyribo-AT undergo completely reversible denaturation. The kinetic barrier to reversibility of total denaturation has been overcome only in the more intermolecularly homogeneous DNA's under restricted conditions. The term "annealing" aptly describes the procedures required to achieve this equilibration. On the other hand, there is ample evidence to indicate that partial denaturation of high molecular weight DNA can generally be easily and rapidly reversed (Cavalieri & Rosenberg, 1957; Geiduschek, 1958; Doty, Marmur & Sueoka, 1959). As a result of these complex kinetic properties, denaturation experiments on DNA fall into two distinct categories: (a) those which explore equilibrium properties and the dissociation of base pairs, and which we designate as "d-assays", and (b) those which measure irreversibility of denaturation, which we designate as "i-assays". In this communication we investigate the properties of DNA which determine this distinction. The experiments described below explore the relationship between reversible partial denaturation and irreversible total denaturation in DNAs of varying molecular weight and average composition, as well as the effect of chemical modification on denaturation reversibility.

2. Material and Methods (a) DNA Samples

DNA has been isolated from a variety of animal, bacterial and viral sources by various methods. Salmon testis and sea urchin (Strongylocentrotus drobacbieneie) testis DNA were isolated by the detergent method of Simmons (1957). E. coli, Ae. aeroqenes, S. marcescens and Ps. fiuorescens DNAs were isolated by a method based on the work of Simmons (unpublished experiments, 1956) and Jones (1953), involving lysis by detergents, acid precipitation of protein, detergent deproteinization and cetyltrimethylammonium bromide fractionation of RNA from DNA and polysaccharides. A detailed description has been given elsewhere (Hamaguchi & Geiduschek, 1962). One preparation of B. subtilis DNA (sample 8, Table 1) was made by a method adapted from the work of others (Nester & Lederberg, 1961; Mandell & Hershey, 1961), involving isolation of "bacterial nucleoprotein" followed by phenol deproteinization. This method is described in further detail below. Bacteriophage T2r+ DNA was prepared by phenol deproteinization according to Mandell & Hershey (1961), and dialysed against 0·01 M-NaCl. M. lysodeikticus DNA, isolated according to the method of Marmur (1961) which involves detergent disruption of bacteria followed by chloroform-isoamyl alcohol deproteinization, RNase digestion and alcohol precipitation, was the generous gift of W. F. Dove. B. subtilis (Table I, sample 9) and E. coli DNAs (Table 1, sample 11) were made by the method of Marmur (1961) modified to include an additional deproteinization with phenol, and removal of enzymatically digested RNA by dialysis. Bacteriophage T7 DNA was the generous gift of D. M. Freifelder. It had been prepared according to the phenol procedure of Mandell & Hershey (1961). Several of the DNA samples had been stored for varying lengths of time as airdried fibers. T2 and T7 DNA, on the other hand, were never precipitated from solution

CONTROL OF DNA REVERSIBILITY

469

and were treated with the special precautions appropriate to very high molecular weight DNA preparations (Davison, 1959). Properties of the DNA samples are summarized in Table l. TABLE

I

Properties of DNA preparations Source

Nucleotide Sample composition number Mole % Ref. GC

Bacteriophage T2 Prep I Prep 5 Prep 7

1 2 3

35

a

Strongylocentrotus drobachiensis

4

37

5 6 7

43

8 9

44

10

51

Salmon ST·l C H·Il B. subtilis Prep 1 Prep 4

Sedimentation coefficient Cone. % S20,W

[7]]

dl./g

Isolation procedure

39·0 e 504 55·7 f

0·002 0·002 0·002

detn,

23·3 f

0·002

55

h

c

21·7f 22·W 25·8 f

0·002 0·003 0·002

61 68

h h h

b

404 26·6 f

0·002 0·002

b

16·1f 31'3f

0·004 0·002

33

k

294 22·1f 174 20·1 f 25·1 f

0·002 0·002 0·002 0·003 0·002

g g g

own

E. coli

Prep 2 Prep 3 Bacteriophage T7 Prep 28A Ae. aeroqenes S. marcescens Ps. fiuorescens M. lysodeikticus

11

12 13 14 15 16

52 57 59 64 71

d b b b a

j

52 34 41

g k k k 1

Nucleotide composition: (a) Josse, Kaiser & Kornberg (1961); (b) Belozherski & Spirin (1959); Chargaff (1955); (d) Sinsheimer (1959). Sedimentation coefficients determined at 42,040 rev.rrnin (except samples 1,2, 3 and 12 which were run at 31,410 rev.rmin) in two different solvents; (e) 0·1 M-NaCI; (f) 0·9 M·NaCI. Isolation procedures: (II) Mandell & Hershey (1961); (h) Simmons (1957); rn Nester & Lederberg (1961) modified; (j) Marmur (1961) modified; tk) Hamaguohi & Geiduschek (1962); (1) Marmur (1961). (e)

(b) Preparation of DNA from B. 8ubtilis (sample 8, Table 1) The "bacterial nucleoprotein" from 30 g (wet weight) of bacteria was prepared exactly as described by Nester & Lederberg (1961). It was then dispersed in 100 ml. 0·15 M-NaCI, shaken with an equal volume of redistilled, water-saturated phenol and centrifuged. The aqueous phase was extracted with ether, precipitated with 1 vol. of ethanol and the precipitate dispersed in 0·01 M-NaCl. NaCI0 4 was added to a final concentration of 1·5 M. This solution was once more treated with phenol, extracted with ether and finally dialysed against 0·01 M-NaCl. (c) Mechanical degradation of DNA

DNA was degraded by sonic irradiation and shear degradation. One series of salmon DNA samples was prepared by irradiation of deoxygenated DNA solutions (0,1 %), containing 10-4 M-AET (S-2,isothiouronium bromide hydrobromide; Doty, McGill & Rice, 1958) in a Raytheon 9 kc/s 150 w sonic oscillator, followed by alcohol precipitation of DNA. Details are given elsewhere (Freifelder, Davison & Geiduschek, 1961). A sample of bacteriophage T2 DNA was irradiated as a 0·025% solution in 0·01 M-NaCI in a Raytheon 10 kc/s 250 w sonic oscillator, after it had been repeatedly degassed and flushed with 31

E. PETER GEIDUSCHEK

470

alkaline pyrogallol-washed N 2 • Samples of sheared T2 DNA were prepared by repeatedly forcing 0·025% DNA solutions in 0·01 M-NaCl by hand, from 1 ml. syringes through no. 350'5 in., no. 30-0,5 in., no. 24-1 in. and no. 20-1·5 in. hypodermic needles. Median sedimentation coefficients and intrinsic viscosities of the degraded salmon DNA samples are given in Table 2; sedimentation coefficients of bacteriophage T2 DNA samples are given in Table 3. TABLE

2

Properties of sonically degradedYI salmon DNA Sample

S20,W

(2)

['7)](3)

dl.Jg C (parent)(4) C-12 C-18 C-70 C-160

22·6 20·7 15·0 9·7 9·4

61 52 33 13 10

Irradiated in 9 kcJs oscillator in presence of 10-' M-AET. Measured at e = 0·002 to 0·003% in 0·9 M-NaCl. (3) Measured at 25°C in 0·1 M·NaCl. (4) M = 6·0 X lOa by light scattering. (1)

(2)

TABLE

3

Type I reversibility of degraded T2 DNA Sample

Degradation

7 5 5-1 7-1 7-2 5-2 7-3 5-3

(parent) (parent) shear shear shear shear shear sonic

S20,W

(1)

55·0 50·0 26·7 23·2 22·0 20·6 18·6 H·9

f"T

5·8 5·9 3·2 4·5 4·0 3·6 3·5 1·5

(ll Measured at c = 0·002% in 0·9 M-NaCl, 0·01 M·tris pH 7. Samples 7-3 and 5-3 run at 42,040 rev.Jmin, rest at 31,410 rev.Jmin.

(d) Preparation of "reversible" DNA

"Reversible" DNA was made from E. coli DNA by cross-linking with bis(,B-chloroethyl)-methylamine-hydrochloride at pH 7·1 by a procedure described previously (Geiduschek, 1961b). (e) Preparation of DNA 80lutions

Stock solutions of DNA were made by dissolving Na-DNA in 0·01 M-NaCl at 1 to 5°C with stirring, and were stored at these temperatures. Solutions of DNA in 7·2 M-NaCI0 4 or 50% methanol were made from these stocks in such a way as to avoid any possibility of irreversible DNA denaturation by transient inhomogeneities of denaturant (i.e, N aCIO 4 or methanol) during mixing. With bacteriophage T2 and T7 DNA, additional precautions were taken to avoid mechanical degradation (Davison, 1959). (a) DNA was never precipitated. (b) NaCIO. was added to DNA dropwise, with gentle swirling, at - 2°C. For transfer of DNA solutions we used volumetric pipettes whose tips had been sawed off. (c) DNA (0,002%) was delivered into the analytical centrifuge cell by flowing under gravity, through a no. 180·75 in. hypodermic needle.

CONTROL OF DNA REVERSIBILITY

471

(f) Vitlcosity and sedimentation

The intrinsic viscositi es listed in Tables 1 and 2 were m easured in four -bulb sus pe nded level capillary viscometers covering a range of average velocity gra dients of 50 to 200 sec- 1 in water at 25°C (Schneider, 1953). Viscosities are extrapolated to zero shear and concentration. Sedimentation velocities wer e measured at 23 to 25°C in a Spinco model E ul tracentrifuge at low DNA concentration using the conve ntional u. v. absorption came ra with an auxiliary Corning no . 9863 filt er. Densitometric t races wer e made on a JoyceLoebl recording mi croden sitometer. The values of 8 recorded in Tables 1 to 3 correspond to t he rat e of migration of t he midpoint of absorbance boundaries (often denoted as 8&0)' Values of 8 have be en corrected to the v iscosity and buoyancy of wa ter at 20°C, assuming all DNAs to have a partial specific volume of 0'55 cm 3jg . (g) Denaturation exp eriments

Denaturation experiments wer e performed in the following manner. A DNA solution was heated to a given temperature in the thermostated cell compa rt men t of a spectrophotometer and the absorbance (A) at 259 mft was measured after equilibrium was attained. The cuvette was then bri efly plunged into ice (quenched) and re-equilibrated in a second spectrophotometer maintained at 25°C, when the absorbance was measured a gain. This heating and qu enching cycle was repeated at successively higher temperatures. Although an entire denaturation curve could be performed on a single sample, a certain saving of time was achieved by using two cells simultaneously. Appropriate control measurements were made to verify that the properties of the DNA at the highest temperatures (in our experiments) were not substantially affected b y previous cycles of h eating. Absorbances measured at t he elevated temperat ures y ield d-assay denaturation curves whil e 25°C measurements of qu enched solutions y ield i-ass ay d enaturation curves (e.g, Figs. 3, 4 and 9). In several instances (viral and bacterial DNA in 7·2 M-NaClO.) i-ass ay exper imen ts are complicated by the time-dep endent absorbance of quenc he d solutions . In these cas es, heated cuvettes wer e plunged into ice for 40 sec, transferred to water at 25°C for 20 sec, then rapidly dried and t ransferred to the 25°C· thermostated spect r op hot ometer . Absorbances were then measured as a function of t ime and ex trapolated to zero time at 25°C. Owing to the fini te rate of t he rmal equilibration, t h is zero ti me is no t precisely established. However, under the cond itions of our exp erimen ts , the rates of t ype II d enaturation re versal were sufficien tly slow (F ig. 2) t o permit such ex t r apolation to be made in a simple manner, without introducing an error of more than ± 0·02 in ju (itl is defin ed in section 2(h)). Absorbances have not b een corrected for thermal expansion of solven t. (h) Presentation oj data We define jj, the transition fraction, as

where AT is the absorbance (at 259 mft) for the temperature T, A o is the absorbance at a temperature outside the transition region-usually 25°C in these ex perim en t s- for which jt is therefore zero; .6.AmM is the m aximum absorbance change upon complete denaturation. We further designate ju andjtd as the transition fractions for [, and d-aasays respectively. The tem pe rat ure at whichjtd or iu '= 0·5 is called the midpoint transition t emperature and denot ed as T"d or T"I respectively . W e also define .6.T = T"I- T' .d' The tem peratu re range over which jtd changes from 0·25 to 0·75 is ca lled t he transition half-wid th and denoted by S. In the t heory of polyp ep t id e helix-eoil t rans itions, this quantity is determined b y a paramet er wh ich descr ibes t he tende ncy of helical segments to be clustered t ogethe r, and by the entha lpy of t ransferring a peptide unit fro m a disordered t o a helical array (Flory, 1961). (j) Other materials R ea gen t grade NaCIOe, t ris, EDTA and sp ectre-grade methanol were used without further purification.

E. PETER GEIDUSCHEK

472

3. Experimental Results (a) DNA thermal denaturation in 7·2 M-NaOlO4 and 51% methanolr-

type I and type II reversibility In this section, experimental evidence for the existence of two entirely distinct types of denaturation reversibility is presented. One of these processes has been described in detail by others (Marmur & Lane, 1960; Doty et al., 1960; Marmur & Doty, 1961): it has the characteristics of an intermolecular reaction, occurs under restricted conditions of ionic strength and temperature in bacterial and viral DNA samples which contain a limited variety of DNA molecules, and is a relatively slow process. We designate this as type II reversibility; Marmur, Doty & co-workers call it "renaturation". The other process, which we call type I reversibility, is a relatively fast process which occurs at high and low ionic strength in heterogeneous and homogeneous DNAs (Geiduschek, 1961a).

u

0"

$

FIG. 1. Dependence of thermal stability (Pi,d) on average nucleotide composition of DNA. (a) 0,7,2 M-NaCI0 4 , EDTA pH 7. (b) e, 51 % (by vol.) methanol, 10-3 M-NaC!, 10- 3 M-tris pH 7.

To demonstrate the relationship between these two types of helix re-formation we have chosen two denaturing solvents, the suitability of which became apparent in connection with experiments on the stability of the DNA helix (Geiduschek & Herskovits, 1961; Hamaguchi & Geiduschek, 1962). One of these solvents is 7·2 Maqueous NaCI04 , buffered at pH 7 with EDTA. The other is 51 % by vol. methanol containing 10-3 M-NaCl and 10-3 M-tris at pH 7. These two solvents lower the denaturation temperature of DNA relative to 0·15M-NaCl-citrate pH 7 by 30 to 50°C. Both solvents are essentially transparent at 260 m/-,. The dependence of the midpoint denaturation temperature, Tt,d' on DNA average nucleotide composition in the two solvents is shown in Fig. 1. The dependence of Tt,d is greater in 7·2 M-NaCl0 4 (0'56°Cjmole % guanine +cytosine) than in 0·15 M-NaCl-citrate pH 7 (0'41°Cjmole % guanine + cytosine; Marmur & Doty, 1959); in 51 % methanol it is slightly lower (0'35°Cjmole % guanine + cytosine).

CONTROL OF DNA REVERSIBILITY

473

In 7·2 M-NaCI04 , T2 DNA has an anomalously low stability, probably due to the replacement of cytosine by partly glucosylated hydroxymethylcytosine, HMC (glu) (Hamaguchi & Geiduschek, 1962). T4 and T6 show the same effect. The stability of T4 DNA in this solvent, on the other hand, is normal. In 51% methanol both viral DNAs have anomalously high stability but for an entirely different, and trivial, reason. As prepared by the phenol method, T2 DNA is contaminated with polyamines, and T7 DNA contains some Mg2+. Rather than submit these DNA preparations to the further manipulations necessary for the removal of multivalent cations, we have used them in this form. In addition to lowering the thermal transition temperature, NaCI04 and 51% methanol have the following advantages for our experiments. (1) In both solvents at 25°C, the absorbance of irreversibly denatured salmon, sea urchin and phage T2 DNA is only slightly lower than that of the totally disordered polynucleotide at high temperatures (Figs. 2 and 3). This is due to the instability of

-100

Time (min)

FIG. 2. Time dependence of absorbance of solutions of bacteriophage T2 DNA (Table 1, sample 1) at 25°C after heating to "complete" denaturation (i.e, to itl = 1,0). Curve a, DNA (A o = 0,43) in 7·2 M.NaCI0 4 (Tl,d = 35°C). Curve b, DNA (A o = 0,39) in 51 % methanol (Tl,d = 49°C).

denatured DNA secondary structure in these conditions. If one realizes that it is Tt.d - T, rather than temperature alone, which mainly determines the absorbance of disordered DNA and the stability of its secondary structure, then the above result is entirely in keeping with the lower Tt.d encountered in these two solvents; the absorbance of denatured DNA is similarly elevated in 0·15 M-NaCI at 65°C or in 0·001 M-NaCI at 25°C. Large, reversible absorbance changes in these solvents must therefore be associated with the destruction and re-formation of long-range ordered secondary structure. (2) In general, DNA acts in a closely similar manner in 7·2 M-NaCI04 at 25°C, and in 0·15 M-NaCI at about 60 to 65°C; in fact type II reversibility ("annealing") experiments are performed at room temperature in concentrated perchlorate (Fig. 2, curve a). On the other hand, type II reversibility does not occur in the low ionic strength methanol-water solvent (Fig. 2, curve b). Consequently, the use of these two solvents provides one criterion for distinguishing type I and type II reversibility. Another criterion is provided by the relatively slow rate, at these DNA concentrations, of type II reversal. We turn now to a series of thermal denaturation experiments which illustrates the distinction between type I and type II denaturation.

E. PETER GEIDUSCHEK

474

0.150

0.100

A)4o 1.30

1.20

m

-2 "'I

0.050

1.10

0 25

45

55

65

T (°el

(a)

AMo 0.200 lAO 1.30 c,

d

~ 0.100

1.20 1.10

0

60

A/Ao 1.4 0.200

l1;

.( 0.100 "'I

o 30

40

nOel

50

60

(e)

FIG. 3. d-assay and i-assay denaturation curves of intermolecularly heterogeneous DNAs. (a), sea urchin DNA (sample 4, Table 1) in 7·2 M-NaClO•. (b), sea urchin DNA (sample 4, Table 1) in 51 % methanol, 10-3 M.Nael, 10-3 M-tris pH 7. (c), salmon DNA (sample 5, Table 1) in 7·2 M· NaClO•. 0, i-assay. e, d-assay. Ll.A269 , change in absorbance at 259 miL' A/A., relative absorbance at 259 miL' A. is the absorbance at 25°C.

475

CONTROL OF DNA REVERSIBILITY

It is clear that both in heterogeneous salmon or sea urchin DNA (Fig. 3) and homogeneous T2 DNA (Fig. 4) the complete reversibility of partial denaturation can be demonstrated. For example, heating a sample of sea urchin DNA to 44°C in 51% methanol causes a 30 % increase in ab sorbance at 259 mt-' (Fig. 3(b), curve d). If this solu ti on is cooled in ice and re-equilibrated at 25°C, the relative absorbance , A/A o,

35

45

T (OCl

(a)

A/AO 1.4

0.150

1.3

0.100

1.2

:1: .( "'l

0.050 1.1

0

1.0

40 (b)

FIG. 4. d -assayand i-assay denaturation curves of bacteriophag e T 2 DN A. (a) , T 2 DNA (sa mple 2, Table 1) in 7·2 M-NaCIO(. i-ssasy absorbanees ex trapolated to zero t ime at 25°C, to eliminat e absorbance changes caused by t ype II reversibilit y. (b) , T 2 DNA (sample 1, Table 1) in 51 % m ethanol. DNA doe s not exhibit t ype II re ve rs ibilit y . 0, i-assay . e, d-assa y,

476

E. PETER GEIDUSCHEK

returns within seconds to a value of 1·03 (curve i). The experiment shown in Fig. 4(b) affords an even more striking example. When a phage T2 DNA solution in 51 % methanol is equilibrated at 51·3°0 (Fig. 4(b), curve d) the absorbance increases 37%, yet this absorbance change is completely reversible upon rapid co~ling ("quenching") and re-equilibration at 25°0. We call this process type I reversibility. The reversal must involve essentially complete recovery of long-range helical order since the intrinsic viscosity returns to its original value (Geiduschek & Herskovits, 1961), and since, as has been pointed out above, disordered DNA secondary structure is unstable in this solvent at 25°0. If T2 or T7 DNA is heated in 7·2 M-NaOlO.. to a temperature sufficiently high to complete the transition shown in Fig. 4(a) and then quenched and re-equilibrated at 25°0, the absorbance decreases with time as shown in Fig. 2, curve a. The rate of this change depends on the DNA concentration and ultimately the absorbance returns to its original value. This is the "renaturation" of Marmur, Doty & co-workers (Marmur & Lane, 1960; Doty et al., 1960) which we have designated as type II reversibility. In order not to complicate the consideration of the very rapid type I reversibility, the absorbances for the i-assay shown in Fig. 4(a) have therefore been extrapolated to zero time at 25°0 (i.e, to zero type II reversibility). As Fig. 2, curve b, shows, type II reversibility does not occur in 51% methanol, at low ionic strength. Neither does it occur in heterogeneous, animal DNAs. Yet Figs. 3(b) and 4(b) show that type I reversibility occurs in 51 % methanol; we have already pointed out that it occurs with sea urchin and salmon DNA in 7·2 M-NaOlO.. (Figs. 3(a) and 3(c)). (b) A thermal denaturation experiment using a mixture of two DNA samples

The intramolecular nature of type I reversibility is shown in the following experiment. The thermal denaturation of a mixture of bacteriophage T2 DNA with Ps. ftuorescens DNA in 7·2 M-NaOlO.. is shown in Fig. 5 (points) and compared with the

T(O()

FIG. 5. d-assay and i-assay denaturation curves of a mixture of P8. fiuoreecens DNA (Table I, sample no. 15; A 259 = 0'273) and bacteriophage T2 DNA (Table I, sample 3; Am =.0'248) in 7·2 M-NaClO,. Solid lineB, transition curves calculated from experiments on the separate DNA samples. Pointe, thermal transition of the mixture. e, d-assay. 0, i-assay,

CONTROL OF DNA REVERSIBILITY

477

i-assay and d-assay denaturation curves calculated from experiments on the separate DNAs. The two DNA samples were chosen so that both i-assay and d-assay thermal transitions might be well separated. The experiment shows that the denaturation of the mixture of samples is well predicted by the behavior of the separate mixtures of T2 and Pseudomonas DNA molecules. This must be the consequence of three conditions being fulfilled at these DNA concentrations: (I) the equilibrium properties of DNA molecules are not substantially affected by the presence of other DNA molecules, whether these be helical or denatured; (2) the type I reversibility of T2 DNA is not substantially affected by the presence of ordered Pseudomonas DNA molecules; (3) the type I reversibility of Pseudomonas DNA is not substantially affected by the presence of completely denatured T2 DNA molecules. (c) Representation of type I reversibility experiments

Two ways of describing the results of type I reversibility experiments have been found useful. The first is a "reversibility plot" (Hamaguchi & Geiduschek, 1962) in which the transition fractions for i- and d-assays, ft! and ftd (see section 2(h) for 100,..--,---,---,---,-----.." /

1.0r------,r-----r----,---,----,

/ / /

80

/ / /

/ /

60

/

0.6

/ /

/ /

40

/ / /

/

/

20

0.2

/ / /

/

o

0.2 (a)

0.6

f;d (b)

FIG. 6. (a) Reversibility plot of T2 DNA (Table I, sample no. 1) in 7·2 M-NaCI0 4 and 51 % methanol. 1td and1t1 are the transition fractions in d- and i-assays (for definition see section 2 (h)). Data taken from Figs. 4(a) and (b). 0,7,2 M.NaCI0 4 • • ,51 % methanol. (b) Reversibility plot of T7 DNA (Table I, sample 12) in 7·2 M-NaCI0 4 •

definitions), are plotted against each other. Figs. 6(a) and 6(b) show this representation for experiments on the denaturation of bacteriophage T2 and T7 DNA. The dotted diagonal line in Fig. 6(a) shows the result that would be given by a collection of molecules undergoing all-or-none transition and having no type I reversibility. The actual result for T2 and T7 DNA is vastly different. In fact, with the exception of materials degraded by the action of DNase or radiation, all DNA samples examined by us have been found to fall between the limits shown in Fig. 6(a), that is, all show some type I reversibility. One problem ofthis type of representation is that ft refers to the entire DNA sample. The breadths of the i- and d-transitions are both obviously affected by the intermolecular heterogeneity of a DNA sample (cf. Figs. 3(a) and 4(a)). Consequently, the shape of a reversibility plot depends not only on the extent of type I reversibility but also on the intermolecular heterogeneity of the sample. Another quantity which characterizes the type I reversibility is !1T = Tl,i-Tt,d' the average temperature

478

E. PETER GEIDUSCHEK

interval separating denaturation and irreversibility. In view of the experiment described in the previous section (Fig. 5) on the type I reversibility of dilute DNA mixtures, we expect the quantity!:J.T for a single molecular species to be independent of intermolecular heterogeneity. It is therefore used in the subsequent experiments to describe the average degree of type I reversibility of entire DNA samples. (d) The dependence of type I reversibility on DNA nucleotide composition

Experiments in i- and d-assay denaturation have been performed on the DNA samples listed in Table 1 in 7·2 M-NaCl04 and 51 % methanol. We have found the following correlation between AZ' and the nucleotide composition in both solvents: the lower the guanine-cytosine content, the greater !:J.T. Results are shown in Figs. 7(a)

8

4 ,. ,

6

., 7 I "6

},

,".......

JO.

2 30 (a)

....14 15 ............... 16 13 ~ ..

50 mole %GC (b)

FIG. 7. Dependence of type I reversibility on average composition of DNA. Samples are identified in Table 1. The straight lines terminating at the bottom right-hand comer of each figure have a slope equal to dT1,d/dGC in the appropriate solvent. (a) AT as a function of average GO content in 7·2 M.NaCI0 4 • (b) AT as a function of average GO content in 51 % methanol.

and (b). It is also clear that there is considerable scatter of the !:J.T-GC content data in both solvents. Since the correlation between Tl,d and GC content is, in general, excellent (Fig. 1, curves a and b), the irregularity must arise from additional variables which affect the behavior of DNA preparations in i-assays. The experiments described in the next section show that molecular weight is one of these variables. (e) The dependence of type I reversibility on molecular size

Two series of experiments have been performed on the relationship between average molecular size and !:J.T. For one experiment, a series of sonically degraded salmon DNA samples was used. The parent preparation had a weight-average molecular weight of 6·0 million as determined by light scattering (Geiduschek & Holtzer, 1958). Applying the equations of Doty et al. (1958; Eigner, 1959) to the sedimentation and viscosity data, we estimated that the sonically degraded preparations range down to M = 0·7 million (Table 2). In both 7·2 M-NaCl0 4 (Hamaguchi & Geiduschek, 1962) and in 51 % methanol, the equilibrium properties

CONTROL OF DNA REVERSIBILITY

479

are little affected by this change in molecular weight. On the other hand, Fig. 8 shows that Ttl> and concomitantly tiT, change considerably. Neither the i- nor the dtransitions are broadened by this extent of degradation.

8 -e o•

..?I o•

..?-

NaCI04

MeOH

0

FIG. 8. Dependence of type I reversibility on molecular size. b.T of sonically degraded salmon DNA as a function of molecular weight. Molecular weight ratios are calculated from the equation (Doty et al., 1958; Eigner, 1959)

where the subscript zero refers to the parent sample. Properties of DNA preparations are summarized in Table 2.

The second series of experiments was performed with degradation products of two relatively homogeneous T2 DNA preparations (Table 3). The range of molecular weights covered partly overlaps that of the salmon DNA samples; preparations 5 and 7 contain particles that comprise the entire DNA content of T2 and are of molecular weight 1 to 1·4 X 108 (Davison, Freifelder, Hede & Levinthal, 1961; Rubinstein, Thomas & Hershey, 1961), while preparations 5-3 and 7-3 are in the size range of the salmon DNA samples. For this molecular weight range the equilibrium properties are, once again, little changed; Tl,d of samples 7 and 7-3 in 7·2 M-NaOl04, differ by only 0·2°0. On the other hand, tiT does decrease with the average molecular size, even among these longer DNA chains. It is clear from these experiments that tiT is molecular-weight sensitive, in the range of sizes represented by our samples. It might be expected that "hidden" single-strand breaks, such as those introduced by irradiation or by the action of endonucleases, would produce similar effects on tiT. In fact, we have found that attack by pancreatic DNase, which only slightly decreases the viscosity and S, almost abolishes type I reversibility. A similar result may be observed when single-strand breaks are introduced into dye-photosensitized DNA by irradiation with visible light (Freifelder et al., 1961). (f) The denaturation of "reversible DNA" in 7·2M-NaGlO4,

The peculiar properties of DNA after treatment with nitrous acid or HN2 (fJfJ' -bischloroethyl-aminomethane) have been described previously (Geiduschek, 1961b). Fig. 9 shows another such experiment in which HN2-treated E. coli DNA has been

480

E. PETER GEIDUSCHEK

denatured in 7·2 M.NaCI0 4 • A transition occurs which is almost completely reversible, and the rate of equilibration is extremely rapid. Thus "reversible" DNA demonstrates type I reversibility even when the transition, as judged by absorbance measurements, is complete.

0.100

0.050

o 40

SO

60

70

T (oe)

FIG. 9. Thermal denaturation of "reversible" DNA, made by reacting E. coli DNA (sample no. 10, Table I) with the bifunctional nitrogen mustard HN2. Solvent: 7·2 M-NaCIO,.

4. Discussion The experiments which have been described (section 3(a)) clearly demonstrate the distinction between two types of denaturation reversibility which, in order to avoid a profusion of descriptive terms, have been labeled type I and type II. This is by no means the first evidence for type I reversibility. Its occurrence has been pointed out in partial denaturation by acid (Cavalieri & Rosenberg, 1957; Geiduschek & Holtzer, 1958) and heat (Rice, 1955). However, this is, to our knowledge, its first systematic investigation. As a result of the existence of type I reversibility, experiments on helix-coil equilibria (d-assays) and experiments on irreversible denaturation (i-assays) involve different properties of DNA. This poses a severe problem for the interpretation of those experiments which are obligatory i-assays, such as CsCI density gradient centrifugation and bacterial transformation. As an example, we compare the d-assay thermal stability of D. pneumoniae DNA with the thermal stability of transformation by various genetic markers. The appropriate data are collected in Table 4. Ti,d of this DNA in 0·15 M.NaCI, 0·015 M-citrate pH 7 is 85·3°C (Doty et al., 1959). The CsCI density data of Rolfe & Ephrussi-Taylor (1961) and the results of Marmur & Doty (1959) on the dependence of Tt,d on average GC content are readily combined to yield the expected Tt,d for four genetic markers (Table 4, column 3). The observed T t for these and other markers in the transformation assay are listed in columns 4 to 6. There is a considerable spread of results, which cannot be accounted for solely on the basis of differences in solvent composition. In part, this is undoubtedly due to the problem of separating inactivation by denaturation from inactivation by depurination (Ginoza & Zimm, 1961). Part of the difference may also arise from the dependence of Tt,1 on molecular weight (see section 3 (e)). In fact, Guild (1962) has shown just such a molecular weight dependence for TI as measured in the transformation assay. What

CONTROL OF DNA REVERSIBILITY

481

is quite clear is that thermal stability in the transformation assay is not merely a measure of the composition of single DNA molecules (or sections of molecules) but also reflects all the other properties of DNA molecules which determine irreversibility of denaturation. It is entirely possible for the thermal stability of transformation by a genetically marked DNA molecule to be determined by a particularly stable nucleotide sequence located in the same DNA molecule, but outside the polynucleotide sequence constituting the marker. Striking support for this interpretation is given by TA.BLE

4

Thermal stability of D. pneumoniae DNA Drug resistance marker Whole sample d = 1·700 Whole d = 1·698 Novobiocin Micrococcin Bryarnicin Erythromycin Optochin Streptomycin Aminopterin Amethopterin

Equilibrium properties Pi,d

a

Pi,d

Transformation assay b

Pi,i

Til

Til

85·3 (I) 87·8 (III) 89·0 (II) 84·7 (I) 85·0 (I) 85 ·2 (I)

87·7 (I) 87·9 (I) 88 ·15 (I)

88·6 (III) 88·9 (III) 89·95 (II)

88 ·85 (III) 89·4 (III)f

85 ·2 (I) 92-5 (II)

Solvents: 1= 0·15 M-NaCI , 0 ·015 M·citrate pH 7; II = 0·14 M-NaCI, 0·02 x-pbospbate pH 6·8 ; III = 0·1 M-phosphate pH 7. la) Calculated from density data of Rolfe & Ephrussi-Taylor (1961), Sueoka, Marmur & Doty (1959) and value of dT/dGC in this solve nt (Marmur & Doty, 1959). lb) Data of ~Iarmur & Lane (1960). (c) Data of Roger & Hotchkiss (1961). ld) Data of Ginoza & Zimm (1961) . Ie) The average Pi,d of the whole DNA sample used by Rolfe & Ephrussi-Taylor is recalculated on the assumption that the value of p (1'698) differs from that reported by Marrnur & Lane (1'700) as the result of a small difference in base composition (2 mole % CG). However, such a difference lies at the limits of the precision of the buoyant density and nucleotide content analyses. If) DNA prepared by two different methods, from different streptomycin-resistant strains.

the 7·0°C difference between the calculated Tt,d of pneumococcus DNA and the Ti.1 of the amethopterin resistance marker. Such a temperature difference in Ti,d would correspond to a difference of 17 mole % guanine-cytosine content in DNA composition. No transforming activity can be recovered from a location in a CsCI density gradient corresponding to such a composition difference (Guild, 1961; Rolfe & Ephrussi-Taylor, 1961). In fact, it is clear that this temperature difference arises, not from a variation of average composition among different molecules, but from the fact that we are comparing d- and i-assay thermal stabilities. Several properties of type I denaturation have been demonstrated: (1) it is a rapid process even at low ionic strength, where electrostatic repulsions block "renaturation", (2) it is an intramolecular process (at least at the concentrations of these experiments; Fig. 5), (3) it is strongly affected by the molecular weight of the DNA preparation (Fig. 8, Table 3), (4) it can be drastically reinforced by forming covalent crosslinks between complementary DNA chains (Fig. 9).

482

E. PETER GEIDUSCHEK

On the basis of this information one may construct the following model of type I reversibility. According to this model, helical portions of partly denatured DNA molecules maintain the "registration" of complementary sequence in a pair of polynucleotide chains and provide "nuclei" from which helix reformation may propagate readily by accretion of single nucleotide pairs. The portion of the i-assay denaturation curve in whichltl changes deals with the rupture of these "nuclei". The strong influence of molecular weight on irreversibility of denaturation is a reflection of the ability of a small fraction of nucleotide pairs to nucleate type I reversal of denaturation. When nuclei or other points of reference are not subject to thermal, acid or other dissociation, then the helix-coil transition becomes completely reversible (Geiduschek, 1961b). The other experiments presented in this paper are an attempt to determine the nature of these residual elements of the native structure or "nuclei".

-6

1.0

-4

0.8

..:£'

Is 0.6 <.1l Is 0.4

~

l.

0.2 0

1.10 s

FIG. 10. Comparison of the thermal transition in "homogeneous" DNA and a homopolynueleotide. Curve a, fraction of dissociated base pairs, 1 - 8 in an infinitely long homopolynucleotide as a function of 8; 0'0 = 0,10, t1h = - 5·5 kcal. (cf. Zimm, 1960, Fig. 3; numerical data kindly provided by Dr. B. H. Zimm). Curve b, temperature dependence of ftd for T2 DNA (sample 2, Table 1) in 7·2 M-NaCIO•. Curve o, temperature dependence of ft. of the same sample in the same solvent.

Two extreme possibilities may be envisaged as an aid. to focusing the subsequent discussion: A. A DNA molecule behaves like a homogeneous polynucleotide of its average composition. The helix-coil transition occurs over a range of temperatures. Not until every secondary structure link (or some very high fraction of these) is broken, does denaturation become irreversible. AT is then approximately the average temperature range over which ltd changes from 0·5 to some value close to 1'0, in the various molecular species of a DNA preparation. B. The "nuclei" represent regions of DNA molecules having greater stabilities, possibly due to the inhomogeneous distribution of nucleotide pairs within a single DNA molecule. The existence of such inhomogeneities in DNA is known (Shapiro & Chargaff, 1957, 1960; Burton, 1960). The first proposal can be tested by comparing the sharpness of the equilibrium transition (d-assay), and of AT, in the very homogeneous T2 DNA, with Zimm's (1960) calculated equilibrium transitions of homopolynucleotides. In theory, two parameters suffice to describe the transition. One is the equilibrium constant for the formation of a secondary structure link (i.e. a nucleotide pair), 8, and the other is a stacking parameter, 0'0' which determines the tendency of secondary structure links to be clustered. In Fig. 10 we compare the measured temperature dependence of ltd, with

CONTROL OF DNA REVERSIBILITY

483

the calculated dependence of the fraction of disordered nucleotide pairs (1- e), upon the equilibrium constant (s) for the formation of a secondary structure link. The s and T scales are simply related by Van't Hoff's law dlns dT

D.h RT2

e

where we can estimate D.h = -5,5 kcal/rnole.] The relationship between ltd and 1is less readily established. The experiments of Rich & Tinoco (1960) suggest that helix-associated hypochromicity:j: is maximal for ordered nucleotide pairs more than 5 steps removed from the terminus of a helical array; other measurements lead to even lower estimates (Khorana, personal communication, 1961). For very short, stacked arrays of nucleotide pairs the helix-associated hypochromicity should, however, decrease. Accordingly, as the limit of complete secondary structure dissociation is approached, ltd, as measured by absorbance and optical activity, will tend to underestimate the existence of short, residual ordered segments, i.e. lim e-+ 0, ltd> 1- e. Consequently, our simplification of equating ltd with 1- will be most satisfactory for the initial and middle regions of the helix -+ coil transition. Comparison between the calculated (curve a) and T2 DNA (curve b) transitions is made in Fig. 10. The sharpness of the two transitions has been matched at the midrange, ltd = 0·4 to 0,6, by a suitable choice of the stacking parameter for the theoretical transition, Go = 0·10. From what follows it will be clear that this choice of Go does not describe the stacking tendency of nucleotide pairs in T2 DNA, since that transition is broadened by intramolecular compositional heterogeneity. It will be noted that the two transitions have different shapes, b being broader than a for 0 < 1- e< 0·4 and sharper for 0·6
e

e

t Data on heats of DNA helix formation are limited. Experiments on the acid denaturation of salmon DNA at 25°C yield tJ.h= - 5 kcal/nucleotide pair (Sturtevant & Geiduschek, 1958) and the heat of combination of poly A with poly U has been found to be - 6·8 kcal/nucleotide pair (Ross & Sturtevant, 1961). A suitable average is - 6·0 kcal. If we also assign the entire lowering of the transition temperature in NaClO, and methanol to a lowering of Sh, then we estimate tJ.h = - 5·5 kcal/mole.

l The helix-associated hypochromicity is defined as 1- Ah/A c• Ah/A c is the ratio of absorbances of ordered and thermally disordered (ft = 1) polynucleotide. A c is usually less than the sum of absorbances of isolated nucleotides. § The thermal transition profile of poly A+poly U, on the other hand, has the same form as curve 8 (Haselkorn, 1959; Doty, 1961).

484

E. PETER GEIDUSOHEK

values of AT are shown in DNAs of low GC content. The magnitude of the variation of AT with DNA composition is not compatible with interpretation in terms of the properties of a homogeneous polynucleotide, i.e. the GC dependence of AT cannot arise solely from an increasing sharpness of the transition as the guanine-cytosine content increases. t This variation of AT with GC content is not confined to the two solvent systems used here. It also occurs in the more familiar dilute aqueous buffers, as shown by a comparison of previously published data on the dependence of i- and d-transition temperatures on DNA composition: in 0·1 M-NaCI, Tt,l increases 0·21°C/mole % GC (Bunville & Geiduschek, 1960), while Tt,d increases 0'41°C/mole % GC in 0·15 M-NaCI, 0·015 M-citrate (Marmur & Doty, 1959); although the ionic strengths are slightly different, this does not obscure the trend to lower Tt,l- Tt,d as the GC content increases. There is a considerable variability of the data shown in Fig. 7 with regard to the GC dependence of AT. Part of this is due to the size dependence of type I reversibility, but this does not entirely account for the scatter. The discrepancy of the AT values for the very high molecular weight T2 DNA (Fig. 7(a), 1-3), relative to sea urchin (4) and salmon (5-7) DNA in 7·2 M-NaCI04 , deserves further comment. The low value of AT for T2 DNA is, in fact, entirely consistent with the notion of GC-rich "nuclei": (1) T2 DNA contains partly glucosylated HMC rather than C; (2) the thermal stability of G-HMC (glu) base pairs appears to be anomalously low in 7·2 M-NaCI0 4 as shown by the low value of Tt,d for T2 (Fig. 1, open circles), T4 and T6 DNA (unpublished results). The highATofthe animal DNA's maybe due to one of two factors: (1) greater intramolecular heterogeneity of the DNA of higher animals, or (2) presence of DNA·bound peptide or protein contaminants. The difficulty of removing peptides from sperm DNA has been commented upon recently (Borenfreund, Fitt & Bendich, 1961). It is probable that such contaminants could change AT. However, the high values of AT show up not only in low ionic strength media (Fig. 7(b)), but also in concentrated NaCI04 which is an excellent protein-DNA dissociating agent, and in buffered saline. This suggests that protein-DNA interaction is not the primary cause of the comparatively great type I reversibility of salmon and sea urchin DNA. In this discussion, the fast, type I reversibility of DNA denaturation has been interpreted in terms of the variation of secondary structure stability within one molecule. It is appropriate to compare this interpretation with the recent and current studies of others. Beer & Thomas (1961) have shown disordered and ordered regions coexistent in single molecules ofT2 DNA partly reacted with formaldehyde. However, this does not constitute a demonstration of a variation of secondary structure stability within one molecule. The reaction of DNA with 1 % formaldehyde goes to completion (Berns & Thomas, 1961) and the above result does not necessarily have any significance for the equilibria of DNA molecules. On the other hand, Freifelder & Davison (1962) have shown that the dissociation of 14N_15N E. coli DNA molecules does not

t Two supporting arguments can be presented. (a) The sharpness of the transition may change with average nucleotide composition, but the variation of d8jdT, at T i •d , should be less than 10% for the entire range of DNA composition covered in these experiments. (b) The transition halfwidth, Il, of bacteriophage T7 DNA (50 mole % GO, Il = 3-8°0) in 7·2 M-NaOIO. is actually greater than that of T2 DNA (35 mole % GO, Il = 3-2°0). If the breadth of the transition were entirely controlled by the average composition, one would expect exactly the opposite result.

CONTROL OF DNA REVERSIBILITY

485

begin until one attains temperatures at which the equilibrium transition (d-assay) of the entire mixture of DNA molecules is well advanced. This latter result is entirely consistent with the experiments and interpretation presented in this paper. If the existence of "nuclei" leads to type I reversibility, then linking complementary strands of DNA should produce the same effect. Indeed, it has been shown previously that a bifunctional nitrogen mustard, HN2, and nitrous acid so modify the denaturation of DNA that even heterogeneous salmon DNA undergoes completely type I reversible thermal denaturation (Geiduschek, 1961b). The reversible transition of a sample of HN2-treated "reversible" E. coli DNA in 7·2 M-NaOl0 4 is shown in Fig. 9. In addition, Marmur & Grossman (1961) have demonstrated that u.v. irradiation crosslinks DNA and have produced DNA that is also partly type I reversible. That the effects of "nuclei" in determining re-formation of secondary structure extend over relatively long distances along a DNA molecule is suggested by the ease with which hydrolytic cleavages in single polynucleotide strands affect type I reversibility (and !1T). The experiments with shear-and sound-degraded DNA chains (Fig. 8 and Table 3) show that molecular weight changes in the range 106 to 107 also affect reversibility of denaturation. This suggests that under the conditions of our experiments, nuclei may propagate their reordering effect over chain lengths of corresponding magnitude, i.e. of the order of a micron. To determine the properties of "nuclei" it is therefore advisable to use DNA preparations that meet the requirements of (1) containing only a single DNA species, (2) not containing hydrolytic breaks. The experiments of section 2(e), Table 3 are only a beginning in this direction; the appropriate criteria of purity and integrity are currently being worked out by others (Burgi & Hershey, 1961; Freifelder & Davison, 1962). Nevertheless, these experiments do provide evidence on the following point. It appears that in T2 DNA (at least), type I reversibility is not exclusively due to a single, uniquely stable nucleotide sequence. For instance, when preparation 7 (Table 3) is shear degraded to 0'3 = 26 molecules, the entire i-transition moves to lower temperatures; it does not split into two parts, and it is not broadened. Were a unique, GO-rich sequence responsible for the type I reversibility of the parent molecule, then a much more stable fragment should be found in the collection of predominantly quarter and eighth molecules comprising the 8 = 26 mixture (Rubinstein et al., 1961). The other experiments summarized in Table 3 give the same result. This suggests the following explanation for the origin of the size dependence of type I reversibility: we postulate that the temperature range over which irreversible denaturation is observed is that over which the small, most stable parts of the secondary structure of a double-stranded DNA molecule dissociate. A molecule retaining a minimum number (we do not at present know what that minimum is) of such residual ordered sections will re-form a double helix. In larger molecules, having a greater number of these, a smaller fraction of intact nuclei will provide type I reversibility, so that the i-transition will occur at a higher temperature. I am grateful to M. Roger, N. Davidson, W. Guild and R. Haselkorn for most helpful discussions, to R. Wahl, W. Dove, D. Freifelder, L. Kozloff and R. Mackal for gifts of phage, bacteria and DNA, and to S. Staves and A. Daniels for expert technical assistance. This research was supported by a grant from the United States Public Health Service (0-5007). 32

486

E. PETER GEIDUSCHEK

REFERENCES Beer, M. & Thomas, C. A. (1961). J. Mol. Biol. 3, 699. Belozherski, A. N. & Spirin, A. S. (1959). In The Nucleic Acids, ed. by E. Chargaff & J. N. Davidson, Vol. 3, Chapter 32. New York: Academic Press. Berns, K. 1. & Thomas, C. A. (1961). J. Mol. Biol. 3, 289. Borenfreund, E., Fitt, E. & Bendich, A. (1961). Nature, 191, 1375. Bunville, L. G. & Geiduschek, E. P. (1960). Biochem. Biophys. Res. Oomm. 2, 287. Burgi, E. & Hershey, A. D. (1961). J. Mol. Biol. 3, 458. Burton, K. (1960). Biochem, J. 77, 547. Cavalieri, L. (1957). J. Amer. ahem. Soc. 79, 5319. Cavalieri, L. & Rosenberg, B. H. (1957). J. Amer. ahem. Soc. 79, 5352. Chargaff, E. (1955). In The Nucleic Acids, ed. by E. Chargaff & J. N. Davidson, Vol. 1, Chapter 10. New York: Academic Press. Davison, P. F. (1959). Proc. Nat. Acad. Sci., Wash. 45, 1560. Davison, P. F., Freifelder, D., Hede, R. & Levinthal, C. (1961). Proc, Nat. Acad. Sci., Wash. 47, 1123. Doty, P. (1961). Harvey Lectures, 55, 103. Doty, P., McGill, B. B. & Rice, S. A. (1958). Proc. Nat. Acad. s«, Wash. 44, 432. Doty, P., Marmur, J., Eigner, J. & Schildkraut, C. (1960). Proc, Nat. Acad. Sci., Wash. 46,461. Doty, P., Marmur, J. & Sueoka, N. (1959). Brookhaven Symposia, 12, 1. Eigner, J. (1959). Thesis, Harvard University. Flory, P. J. (1961). J. Polymer Sci. 49, 105. Freifelder, D. & Davison, P. F. (1962). Biophys. J. in the press. Freifelder, D., Davison, P. F. & Geiduschek, E. P. (1961). Biophys. J. 1, 389. Geiduschek, E. P. (1958). J. Polymer Sci. 31, 68. Geiduschek, E. P. (1961a). Fed. Proc, 20, 353. Geiduschek, E. P. (1961b). Proc. Nat. Acad. Sci., Wash. 47, 950. Geiduschek, E. P. & Herskovits, T. T. (1961). Arch. Biochem. Biophys. 65, 99. Geiduschek, E. P. & Holtzer, A. (1958). Advanc. Biol. Med. Phys. 6, 432. Geiduschek, E. P., Moohr, J. W. & Weiss, S. B. (1962). Proc. Nat. Acad. Sci., Wash. 48, in the press. Ginoza, W. & Zimm, B. H. (1961). Proc. Nat. Acad. s«, Wash. 47, 639. Guild, W. (1961). Abstr. Biophys. Soc. p. FB9. Guild, W. (1962). Abstr. Biophys. Soc. p. WA12. Gulland, J. M., Jordan, D. O. & Taylor, H. E. W. (1947). J. ahem. Soc. 1131. Hamaguchi, K. & Geiduschek, E. P. (1962). J. Amer. ahem. Soc. in the press. Haselkorn, R. (1959). Thesis, Harvard University, Jones, A. S. (1953). Biochim. biophys. Acta, 10, 607. Josse, J., Kaiser, A. D. & Kornberg, A. (1961). J. Biol. ahem. 236, 864. Mandell, J. D. & Hershey, A. D. (1961). Anal. Biochem, 1, 66. Marmur, J. (1961). J. Mol. Biol. 3, 208. Marmur, J. & Doty, P. (1959). Nature, 188, 1427. Marmur, J. & Doty, P. (1961). J. Mol. Biol. 3, p85. Marmur, J. & Grossman, L. (1961). Proc. Nat. Acad. Sci., Wash. 47, 778. Marmur, J. & Lane, D. (1960). Proc. Nat. Acad. Sci., Wash. 46, 451. Nester, J. & Lederberg, J. (1961). Proc. Nat. Acad. Sci., Wash. 47, 52. Rice, S. A. (1955). Thesis, Harvard University. Rich, A. & Tinoco, I. (1960). J. Amer. ahem. Soc. 82, 6409. Roger, M. & Hotchkiss, R. (1961). Proc. Nat. Acad. s«, Wash. 47, 653. Rolfe, R. & Ephrussi-Taylor, H. (1961). Proc, Nat. Acad. s«; Wash. 47, 1450. Ross, P. & Sturtevant, J. M. (1961). Abstr. Amer. ahem. Soc. p. 57C. Chicago. Rubinstein, I., Thomas, C. A. & Hershey, A. D. (1961). Proc, Nat. Acad. s«, Wash. 47, 1113. Schneider, N. S. (1953). Thesis, Harvard University. Shapiro, H. S. & Chargaff, E. (1957). Biochim. biophys. Acta, 26, 608. Shapiro, H. S. & Chargaff, E. (1960). Biochim. biophys. Acta, 39, 68.

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Simmons, N. S. (1957). As quoted by V. L. Stephens & E. L. Duggan. J. Amer. ahem. Soc. 79, 5703. Sinsheirner, R. L. (1959). In The Nucleic Acids, ed. by E. Chargaff & J. N. Davidson, Vol. 3, Chapter 33. New York: Academic Press. Sturtevant, J. & Geiduschek, E. P. (1958). J. Amer. ahem. Soc. 80, 879. Sueoka, N., Marmur, J. & Doty, P. (1959). Nature, 188, 1429. Zimm, B. H. (1960). J. ahem. Phys. 33, 1349.