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Differential denaturation of Haemophilus influenzae DNA
BIOCHIMICAET BIOPHYSICAACTA
234 BBA 96790
DIFFERENTIAL
DENATURATION
OF
HAEMOPHILUS
INFLUENZAE
DNA ALAN D. COOPER AND P. C. HUANG Department o] Biochemistry, The Johns Hopkins University, School o/Hygiene and Public Health, Baltimore, Md. 212o5 (U.S.A.) (Received September I4th, I97o)
SUMMARY
The objective of this study is to determine whether differential denaturation of various genes can be observed. B y treatments with heat or b y titration with hydroxyl ions at subcritical levels, genetic markers from Haem@hilus influenzae have been inactivated in the order of streptomycin resistance > erythromycin resistance > novobiocin resistance. The kinetics and extent of inactivation depends on whether a thermal or alkaline method is used. Results obtained with an assay system which allows the expression of single-stranded DNA are complementary to those testing the transforming activity of double-stranded DNA.
INTRODUCTION The stability of DNA helices does not always follow the prediction of simple statistical mechanics, since DNA is a heterogeneous, aperiodic lattice in an equilibrium state of the order-disorder transition. In this context, it is not surprising to find t h a t both the base sequence and particularly the G • C content, determine to a large extent the thermal melting behavior of a given DNA molecule 1. In fact, it has been suggested that the A • T- and G • C-rich regions of DNA m a y be mapped b y controlled denaturation. For instance, FELSENFELD AND HIRSCHMAN~ reported a preferential denaturation of the A • T regions in DNA. This was based on the computation of hyperchromic shifts resulting from denaturation. The contributions of A • T and G . C base pairs to absorbance changes are quantitatively and qualitatively different. CROTItERS3 has presented a model with which one can calculate the partition function and entropy required in the denaturing process and can also locate genetic loci of specific base composition. Recent studies with electron microscopy have provided still other evidences for the preferential denaturation by mild alkali of specific regions in viral chromosomes 4. Biological approaches to examine the differential inactivation of genetic markers have also been taken. The assumption was that different genes are sufficiently dissimilal in their base sequences so that a preference in the melting of the A • T-rich segments will eliminate genes so enriched. Thus, using thermal denaturation, several genetic markers in DNA have been shown to be inactivated differentially in the tiansforming system of Diplococcus pneumoniae 5-7. These inactivations were attributed to Biochim. Biophys. Acta, 232 ~I97I) 234-245
DIFFERENTIAL DENATURATION OF
DNA
235
the collapse of selected areas in the DNA molecule following partial separation of the strands. These differential inactivations were attributed to localized differences in G - C content but not necessalily to the overall base composition. CHEVALLIER AND BERNARDI8 examined the thermal inactivation of three genes from Haemophilus in[luenzae but only minor differences were noted in the critical inactivation temperatures. RUDNER et al. 9 observed that in fully denatured samples of Bacillus subtilis DNA, whether inactivated b y dialysis, heat or alkali, the proportions of the residual activities with respect to three genetic markers, Adenine, leucine and methionine were approximately 2 : i : I. The similar behavior of the three markers suggested to them that regardless of the manner in which the loss of secondary structure is induced the stability of a marker must be inherent in its unique base-paired sequence. Alkaline denaturation of DNA has now been more closely examined t°-l~. From these studies it is clear that denaturation by alkali, like denaturation by heat involves the rupturing of hydrogen bonds which basically provide the difference in stability between A • T and G • C pairs. Consequently, a preferential inactivation of different genes by alkaline treatment of the DNA is to be expected. In this study, using the H. influenzae system, a comparison of the transforming activities of several genetic markers was made after both thermal and alkaline denaturation. The basic approach takes advantage of subcritical denaturation conditions; thus allowing minor differences to be expressed. The results suggest the existence of preferential denaturation of certain markers implying that some genes examined are indeed different in their base composition. MATERIALS AND METHODS
(z) DNA Transforming DNA was isolated from H. in[luenzae according to the method of GOODGALAND HERRIOTT13. DNA prepared b y this procedure has a molecular weight of about 2 • lO7-4.1o ~ as determined b y sucrose density gradient sedimentation. The general properties of the DNA* used in this study are as follows: The DNA had a hyperchromicity of about 26 % at 26o nm and a Tin, melting temperature, of 84.5 ° in o.15 M NaC1. ESN DNA, SN DNA and SNK DNA had absorbance ratios, A~n0n m / A ~ o am, greater than 2.2 and A~6o nm/A280 nm greater than 1. 9 . (2) Strain of bacteria A non-encapsulated (R) strain of H. in]luenzae type d was obtained originally from Hattie E. Alexander of Columbia University. (3) Trans/ormation The basic method of assay for transformation was that of GOODGAL AND HERRIOTTis. Cells were made competent after the procedure of HERRIOTT et al. 14 " The D~qA p r e p a r a t i o n s are designated b y s y m b o l s for t h e antibiotic resistance m a r k e r s t h e y carry. E = e r y t h r o m y c i n ; S = s t r e p t o m y c i n ; N = novobiocin (also k n o w n as cathomycin) a n d K = kanomycin. The subscript denotes t h e level of resistance; t h u s Ere m e a n s resistance to i o p g / m l of e r y t h r o m y c i n .
Biochim. Biophys. Acta, 232 ¢i97 I) 234-245
236
A . D . COOPER, P. C. HUANG
in competence-developing medium (MV), resuspended in a storage medium (MII) and stored in 15 % glycerol at --7 °0 until used. Three transformation assay procedures were used. In the first, the cells were thawed and diluted I : IO with MII medium. In the second and third assays the competent cells were thawed, washed with o.15 M NaC1, and resuspended in 0.3 % bactopeptone (Difco) and o.15 N[ NaC1 either at p H 7.0 for double-stranded DNA assay or at p H 4.8 in the presence of 3 × lO-4 M E D T A for single-stranded DNA assay 15-17. Following the addition of DNA the cells were incubated at 37 ° with slight shaking at 60 strokes/min. In the case of the single-stranded assay, the p H was changed after IO min from p H 4.8 to p H 7.0 b y the addition of o.1 M N a O H 15-17. The total time of incubation was 3 ° min. At the end of this time, the cells were diluted, plated, and incubated for 12o min and then overlaid with agar containing twice the desired concentration of the challenging antibiotic. Throughout this paper, the three methods for assaying transforming DNA will be referred to as the MII method, the p H 7.0 method, and the p H 4.8 method.
(4) Thermal denaturation DNA at a concentration of 5 × lO-2/~g/ml was incubated in a volume of i.o ml of o.ool 5 M Nacl--o.oooI5 M sodium citrate, p H 6.8, in a screw cap vial. Heat treatment was done in a thermograd. The thermograd consists of four parallel rows of 19 separate chambers. Temperature is controlled differentially by two thermostats at the extreme ends so that a linear temperature gradient of any desired range can be generated. Denaturation was terminated by chilling the vials in an ice-water-salt (--IO °) bath. Aliquots of DNA were used for transformation with the p H 7.0 and p H 4.8 methods. In all cases, the concentration of DNA used is within the linear dosage range for transformation, i.e. 2 × lO -3/~g/Io 8 cells.
(5) Alkaline denaturation Two buffer systems were used for the denaturation of DNA by alkali. One was the glycine-NaCI-NaOH buffer at an ionic strength of o.I is. The other was a series of buffers covering the range 7.o-13.o and were at an ionic stlength of 0.21° consisting of Tris-HC1-NaC1, NaHCO3-NaOH-NaC1, Na2HPO4-NaOH-NaC1 or KC1-NaOHNaCI. A constant ionic strength was maintained because the relaxation time of a double-stranded DNA molecule shows a very definite dependence on ionic strength z°. The p H ' s were measured at ambient temperature with a Beckman Research p H meter equipped with a KC1-AgC1 electrode (pH o-14.o ). The p H ' s were determined before and after the experiment. Occasional discrepancies greater or equal to 0.2 p H due to CO2 absorption were encountered. When this occurred, the experimental point was discarded. The protocol of alkaline denaturation and assay was usually as follows: To 9.9 ml of buffer contained in a sterile scintillation vial and stirred gently b y a magnetic stirrer at ambient temperature was added o.I ml of DNA usually at a concentration of 2.5/~g/ml. The concentration of DNA in the denaturing buffer was usually less than I/~g/ml, so that after dilution the DNA would be at a concentration within the linear dosage range for transformation. Furthermore, the concentration of DNA was kept constant during denaturation as DNA unwinding is partly hydrodynamically limited, hence there is a dependence of the rate on concentration 2°. At the appropriate Biochim. Biophys. Acta, 232 (1971) 234-245
DI FFERENT IAL DENATURATION OF
DNA
237
time, o.I ml was removed and added to 4.9 ml of competent cells in MII medium which terminates the alkaline action. Bactopeptone mediums (pH 7.0 and pH 4.8 methods) replaced MII medium whenever the transforming activity of single-stranded DNA was determined. MII at pH 4.8 is not a good medium to transform cells by singlestranded DNA (unpublished observation), although it is a good buffer. Addition of buffer with a high pH never changed the pH of the MII competence medium by more than o.I pH unit. Whenever bactopeptoue replaced MII medium neutralization of the denatured DNA with H3PO , followed by a I : IO dilution in bactopeptone was necessary before adding o.1-4. 9 ml of competent cells.
(6) Sucrose gradient centri]ugation Molecular weights were determined by sedimentation in a 5-20 % sucrose gradient, according to the plocedure of BURGI AND HERSHEY~1 for neutral gradients and according to STUDIER~2 for alkaline gradients.
RESULTS
(I) Thermal denaturation The relative decrease in double-stranded and increase in single-stranded transforming activities of the El0 markers are both functions of the temperature of denaturation (Fig. i). In Fig. i it is shown that the relative transforming activity, as assayed by the pH 7.0 method, is a function of temperature. Conversely, there is an increase in the relative transforming activity when assayed by the pH 4.8 method. The activity found in the pH 7.0 method is a reflection of the activity of doublestranded DNA. The pH 4.8 method assays mainly the activity of single-stranded DNA. The temperature at the midpoints of the two activity curves is near 7 o°. In order to magnify the relative extent of denaturation of various genetic markers a subcritical melting temperature was chosen. In this way, the rate of thermal denaturation is reduced. The kinetics of denaturation of the E, S, N and the linked marker SN at 69 ° are shown in Fig. 2 and 3. The result (Fig. 2) indicates that the SN marker is inactivated more rapidly and extensively than either of the two single markers. Within 0.5 min, the S marker is already inactivated to 88 % of the original activity (i.e. zero time) while activity in both the E and N markers are not inactivated until I rain. The differences in the extent of denaturation of the S, E, and N markers are small but consistently in the same relative order. Even after complete denaturation (IO min at 95 °) the relative transforming activities remain in the same order. The kinetics of relative increase in the transforming activity of single-stranded I)NA of the markers tested is shown in Fig. 3- The E and S markers respond more rapidly to denaturation but the N and SN markers show a time lag in response. However, the maximum levels of transforming activities of the several markers are in the order E > N >/ S > SN. After extensive denaturation, the E marker reaches a transforming activity IO times that of the E marker that is undenatured, whereas for N this value is 9 times, for S, 8.5 times and for SN about 6 times. For the SN linked marker the relative activity decreases further after extensive denaturation (95 °, IO min) and reaches a value 4.5 times that of the value for undenatured DNA.
Biochim. Biopkys. Acta, 232 (1971) 234-255
23 8
A. D. COOPER, P. C. HUANG
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Fig. I. Activation and inactivation of a genetic m a r k e r u p o n t h e r m a l denaturation. El0 $2000 Ns.~ D N A was d e n a t u r e d at a concentration of 5.4 " 1o-3/~g/ml in o.ool 5 1V~ N a C l - o . o o o i 5 NI s o d i u m citrate at various t e m p e r a t u r e s . The samples in vials at several different t e m p e r a t u r e s were heated in a t h e r m o g r a d for 15 rain and t h e n quickly chi]led in an ice-salt-water m i x t u r e at --lO% To each of 2. 5 ml of cells in either p H 7.0 bactopeptone, 237 • lO 5 cells/m], or p H 4.8 b a c t o p e p t o n e in t h e presence of 3 " 1°-4 M E D T A , 238 • io 5 cells/ml was added o.1 ml of t r e a t e d D N A to a final concentration of 2.I • lO -3/~g/ml. I n this figure the relative t r a n s f o r m i n g activity as assayed b y the p H 7.0 m e t h o d as well as the p H 4.8 m e t h o d is plotted against t e m p e r a t u r e . The IOO ~/o p o i n t for the Elo m a r k e r as assayed b y the p H 7.o m e t h o d is 9.9 • lO3 t r a n s f o r m a n t s / ml and the 16 % point for the El0 m a r k e r as assayed b y the p H 4.8 m e t h o d is 2.8 • lO 3 transform a n t s / m l . O , values determined b y the p H 7.0 m e t h o d ; O , values determined b y the p H 4.8 method. Fig. 2. The relative inactivation of genetic m a r k e r s u p o n t h e r m a l d e n a t u r a t i o n . Ex0 $2000 lq2.5 D N A was d e n a t u r e d at a concentration of 5.4" Io-2/~g/ml in o.oo15 M N a C l - o . o o o i 5 M, s o d i u m cit r a t e at 69 °. At various times aliquots were r e m o v e d a n d chilled in an i c e - s a l t - w a t e r m i x t u r e at --lO °. The zero-time p o i n t represents D N A t h a t had been k e p t at 25 °. I n addition an aliquot of ES1W D N A was completely denatured b y t r e a t i n g it at 95 ° for io min, and chilled. To each of 2. 5 ml of c o m p e t e n t cells at a concentration of 233 • lO5 cells/ml in p H 7.o b a c t o p e p t o n e was added o.I ml of t r e a t e d D N A to a final concentration of 2.1 - lO -8/~g/ml. The cells were t r a n s f o r m e d and counted. The relative t r a n s f o r m i n g activity as assayed in the p H 7.o s y s t e m is plotted a g a i n s t the e x t e n t of denaturation. The ioo ~o points for t h e m a r k e r s are E, 2.89 • lO4; S, 1.2 • zo4; iW, 3.4 " lO4; and SN, 3-7 " lOS t r a n s f o r m a n t s / m l . F o r this a n d s u b s e q u e n t figures, the m a r k e r s are represented b y the following symbols: O , Ns.~; O , Sso00; [], SN; × , Elo. The a r r o w indicates completely d e n a t u r e d DBIA (i.e. 95 ° for IO mill). The insert depicts the inactivation during the first 3 min of d e n a t u r a t i o n on an e x p a n d e d time scale.
Biochim. Biophys. Acta, 232 (i97 x) 234-245
DIFFERENTIAL DENATURATION OF D N A
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Fig. 3. The relative activation of genetic m a r k e r s u p o n t h e r m a l d e n a t u r a t i o n . El0 Sl000 Nt. 5 D N A was d e n a t u r e d a t a concentration of 5.4 " zo-3/~g/ml in o.ooI 5 M N a C l - o . o o o i 5 M sodium citrate a t 59 ° as described in Fig. 2. A t various times aliquots were r e m o v e d and chilled in an i c e - s a l t - w a t e r m i x t u r e at --lO% The zero-time p o i n t represents D N A t h a t h a d been kept a t 25 °. I n addition an aliquot of E S N D N A w a s completely d e n a t u r e d b y t r e a t i n g it a t 95 ° for IO min a n d chilled. To each of 2. 5 ml of c o m p e t e n t cells at a concentration of I 9 I • lO s cells]ml in p H 4.8 b a c t o p e p t o n e in t h e presence of 3 • I o - 4 M E D T A w a s added o.i ml of t r e a t e d D N A to a final c o n c e n t r a t i o n of 2.I • lO 3/,g/ml. The cells were t r a n s f o r m e d and counted. The relative percent t r a n s f o r m i n g activity as assayed b y t h e p H 4.8 m e t h o d is plotted against t h e e x t e n t of d e n a t u r a tion. The reference p o i n t (zero time) for t h e m a r k e r s are E, 2380; S, 127 • io3; N, 2.95 • IO8; a n d SN, 6.o 5 • i o 3 t r a n s f o r m a n t s ] m l . The insert depicts the f i r s t 3 m i n of d e n a t u r a t i o n on an e x p a n d e d t i m e scale.
These results indicate that (I) the genetic markers of H. in/luenzae can be differentially denatured by heat, depending upon the condition employed; (2) the genetic markers examined are differentially inactivated, as measured by their transforming activity by the pH 7.0 method in the following order SN > S > E > N. Biochim. Bi ophy s . Acta, 232 ( I 9 6 I ) 234-245
240
A . D . COOPER, P. C. HUANG
(3) The genetic markers examined are differentially activated if measured by their transforming activity with the pH 4.8 method, in the order of SN < N < E ~< S within the earlier stages of denaturation. However, maximal activity is achieved in the following order E > N i> S > SN.
(2) Alkaline denaturation Alkaline tzeatmeut of DNA was done near the midpoint of the pH inactivation range where partial denaturation takes place ~3. The kinetics of alkaline denaturation of several markers at pH 11.8 with the midpoint of inactivation is depicted in Fig. 4-
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F i g . 4- Kinetics of i n a c t i v a t i o n of t h e S=000, Ne.5, and K10 m a r k e r s u p o n alkaline d e n a t u r a t i o n To 9 . 9 m l of glycine buffer at p H 11.8 at r o o m t e m p e r a t u r e w a s added o . i m l of S N K D N A at a c o n c e n t r a t i o n of 2. 5 / ~ g / m l a n d s l o w l y stirred. A t t h e t i m e s indicated o . i m l w a s r e m o v e d a n d a d d e d to 4-9 m l of c o m p e t e n t cells at a c o n c e n t r a t i o n of 8 9 9 • lO s c e l l s / m l i n M I [ m e d i u m . T h e final c o n c e n t r a t i o n of D N A w a s 5 • io-4/~g/ml. The relative percent t r a n s f o r m i n g a c t i v i t y is p l o t t e d against t h e e x t e n t of d e n a t u r a t i o n at p H 11.8. The i o o % p o i n t s for t h e m a r k e r s are N , 4.2 • lO4; S, z . 5 7 " lO4; K , 1 . 7 . Io4; and SN, 6 . 0 4 • lO 8 t r a n s f o r m a n t s ] m l . I , Kz0. T h e z e r o - t i m e p o i n t consisted of o . I m l of D N A diluted i : i o o in o . 1 5 N[ N a C 1 plus o . i m l of p H 11.8 buffer. I t s h o u l d be n o t e d t h a t t h e Klo m a r k e r is leaky, For this reason b a c k g r o u n d w a s n o t zero and w a s s u b t r a c t e d to o b t a i n t h e v a l u e s u s e d in this figure.
All markers, K, S, and N including one linked marker SN treated at pH 11.8 exhibit similar kinetics of inactivation. In each instance, there are two steps involved with a rapid loss of activity to about 6 % of the original activity during the first 4 rain, followed by a gradual decrease to 1- 3 % after 12o rain depending on the marker. Little difference can be detected in the initial rate of inactivation. Differences can be recognized, however, during the second step. The difference in the residual activity, namely 3 % for K, 1. 4 °/o for N, 1 % for S, and 0. 3 % for SN can not be accounted Biochim. Biophys. Acta,
2 3 2 (1971) 2 3 4 - 2 4 5
DIFFERENTIAL DENATURATION OF
DNA
241
for by their innate differences in transforming efficiency, since each is compared to its original activity. Results not shown here indicate that the kinetics of inactivation by alkali of E are intermediate between S and N. Figs. 5 and 6 show results from experiments in which SN DNA (I #g/m]), partially denatured for various periods of time at pH 11.6, a subcritical pH, was compared with native SN DNA and with completely denatured SN DNA (pH 12.6 for IO min). The kinetics of denaturation of these markers, S, N and SN when assayed by either the pH 7.0 or pH 4.8 methods are diffelent from each other. Fig. 5 shows that DNA, denatured at pH 11.6, exhibits during the first 4 min a rapid decrease in doublestranded DNA transforming activity for all three markezs when measured by the pH 7.0 method followed by a gradual decrease later. However, the S marker is inacti-
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Fig. 5- Relative activation of genetic m a r k e r s u p o n alkaline denaturation. $2000 N,. 5 D N A was d e n a t u r e d at p H 11.6 a t r o o m t e m p e r a t u r e at a concentration of i #g/ml, for various times, neutralized w i t h chilled H3PO 4 and f u r t h e r chilled in an ice-water mixture. I n addition a sample of D N A was t r e a t e d at p H i2.6 for io min. The samples were diluted i : io and o.I ml added to 5.0 ml of c o m p e t e n t cells in p H 4.8 b a e t o p e p t o n e in the presence of 3 • 1o-4 M E D T A at a concentration of 54 8 • IOs cells/ml. The t r a n s f o r m e d cells were plated a n d counted. The N/S ([2], m) and SN/S (O, • ) ratios are plotted against t h e e x t e n t of d e n a t u r a t i o n to indicate the relative differences in t h e levels of activation of t h e N and S markers. The relative t r a n s f o r m i n g activity of the $200o, N~. 5 and $2000 b]2.s m a r k e r s as assayed b y the p H 4.8 m e t h o d is plotted against the e x t e n t of denaturation. The reference points (zero time) for the m a r k e r s are S, i 8 i t r a n s f o r m a n t s / ml; N, 482 t r a n s f o r m a n t s / m l ; a n d SN, 82 t r a n s f o r m a n t s / m l . Fig. 6. Relative inactivation of genetic m a r k e r s u p o n alkaline denaturation. $2000 N2. 5 D N A was d e n a t u r e d at r o o m t e m p e r a t u r e at p H 11.6 at a concentration of i / z g / m l for various times, neutralized with chilled H3PO 4 and f u r t h e r chilled in an i c e - w a t e r mixture. I n addition a sample of D N A was t r e a t e d a t p H 12.6 for i o min. The samples were diluted I : IO and o.I ml added to 5.o ml of c o m p e t e n t cells in p H 7.0 b a c t o p e p t o n e at a concentration of 688 • lO 5 cells/ml. The t r a n s f o r m e d cells were plated and counted. The N/S and SN/S ratios are plotted against the ext e n t of d e n a t u r a t i o n to indicate the relative differences in the levels of inactivation of the N and S markers. The relative t r a n s f o r m i n g activity of the Se000, N 2.5 and S20o0 N2. s m a r k e r s as assayed by the p H 7.0 m e t h o d is plotted against the e x t e n t of d e n a t u r a t i o n . T h e lOO% p o i n t s for the m a r k e r s are S, 6.6. io $ t r a n s f o r m a n t s / m l ; N, 1.6 5 . lO4 t r a n s f o r m a n t s / m l ; and SN, 2.26. io n transformants/ml.
Biochim. Biophys. Acta, 232 (197 I) 234-245
242
A . D . COOPER, P. C. tIUANG
vated more extensively than the N marker throughout the process, the former reaching a value of 56 % and the latter a value of 79 % at 4 ° rain (not shown in Fig. 5). The SN marker reaches a value of 53 % at 4 ° min. After complete denaturation the residual activities ~,.5 for S, N and SN are reduced to a residual level of 3.2 %. 4.3 % and 1.3 %, respectively. If denatured at p H II.6 and assayed by the p H 4.8 method, the N marker is found to increase 6-fold after 4 ° min (Fig. 6). The S marker increases more (7.7-fold) and the SN double marker less (5-fold) as compared to time zero. The values obtained with completely denatured DNA (pH 12.6 for IO min) give somewhat different results. Both the S and N markers have reached 12- and I7-fold, respectively, but cotransformation of the SN markers has fallen 1o 1.3-fold. The initial rate of denaturation can not be distinguished among the markers, although the levels of denaturation differ (S /> N > SN). The data just presented can also be expressed as the N/S ratio. As assayed by the pH 7.0 method, the N/S ratio increases from 2.5 to 3.5 after 40 min of denaturation at pH 11.6 (see also Fig. 5)- After complete denaturation the N/S ratio is 3.3- When assayed by the pH 4.8 method, however, the N/S ratio decreases from 2.5 to 2.1 after 4 ° min (see also Fig. 6), but the ratio increases to 3.8 after complete denaturation. When assayed by the pH 7.0 method the double-stranded molecules composing the partially denatured DNA have the same SN/S ratio (o.31) as the double-stranded molecules composing native (i.e. zero time) DNA (0.34) (Fig. 5)- After complete denaturation the S and N markers have a ratio of o.14. When assayed by the pH 4.8 method the single-stranded molecules of partially denatured DNA possess an SN/S ratio of 0.30 (Fig. 6). Upon complete denaturation the SN/S ratio falls 6-fold to 0.05. In these experiments, the molecular weights of the native and partially denatured (i.e. pH 11.6 for 5 min) DNA as determined by sucrose gradient density centrifugation were both 3° (4-1o) • lO 6, corresponding to a single-stranded molecular weight of 15(4-5)'1o 6. The single-stranded DNA of the completely denatured DNA preparation (pH 12.6, IO min) when sedimented in an alkaline sucrose gradient had a molecular weight of 8 ( ± 3 ) " lO6. Thus, only a 2-fold reduction in the single-stranded molecular weight of the DNA has occurred even after extensive denaturation. These results indicate that (I) the genetic markers of H. influenzae can be differentially denatured by alkali depending upon the conditions employed; (2) the genetic markers are differentially inactivated as measured by their transforming activity by the pH 7.0 method in the following order: SN > S > N; (3) the genetic markers examined are differentially activated as measured by their transforming activity by the pH 4.8 method within the earlier stages of denaturation in the older S > N > SN. However, the maximal activity is achieved in the following order N > S > SN. The difference is not due to molecular size of the DNA.
DISCUSSION Results presented in this study show that preferential inactivation of certain genetic markers may be obtained either b y thermal or alkaline denaturation. Thus the S marker is more labile to heat and to alkali than is either the E marker or the N marker.
Bioch,m. Biophys. Acta, 232 (1971) 234-245
DIFFERENTIAL DENATURATION OF D N A
243
The data of CHEVALLIERAND BERNARDI8 indicate that for H. influenzae DNA, the melting temperatures in o.15 M NaCl-o.oI M sodium phosphate buffer (pH 7.0) are 88 ° for the streptomycin marker and 88.6 ° for the erythromycin and novibiocin markers s. By selecting a subcritical temperature below the Tm (i.e. 7 °0 in o.oo15 M NaCl-o.oooI5 M sodium citrate) and examining the kinetics of denaturation our results indicate that the S marker is inactivated more rapidly than either the E or N marker. Furthering the earlier data 8, the present data indicate that the relative order of inactivation of genetic markers is SN > S >1 E > N. For D. pneumoniae DNA, the novobiocin, erythromycin, and streptomycin markers are inactivated during thermal denaturation in the order N > E > Se. The order of inactivation is therefore opposite to the order we have found for the similar markers of H. in/luenzae DNA. It is possible that: (I) The N, E, S markers in D. pneumococcus do not have the same base sequence as the corresponding markers in H. in/luenzae; or (2) the N, E, S markers in D. pneumococcus and H. in/luenzae m a y be similar but complete separation of the double strands of the DNA molecule does not occur until a region of unique composition is denatured; or (3) the N, E, S markers in D. pneumococcus and H. in/luenzae are similar in base sequence but the regions immediately adjacent to them differ in base composition. If the first conclusion is correct then one may infer that the genes between the Haemophilus and Diplococcus species are not conserved. Controlled denaturation therefore may be another useful method in the determination of genetic relatedness of various species of bacteria. The apparent loss of the marker activity of double-stranded DNA undei examination is not due to some irreversible damage to the marker region, such as an extensive reduction in size. The marker activity can be efficiently recovered when assayed b y the pH 4.8 method. However, it can not be determined whether each particular marker that is inactivated when assayed b y the pH 7.0 method is also activated when assayed b y the pH 4.8 method due to the relatively low efficiency of the latter. It has been noted'that there is a Ioo-fold declease in the relative transforming activity of the DNA when assayed by the pH 7.0 method whereas there is only a Io-fold increase in activity when assayed by the pH 4.8 method (Fig. I). This is probably due to the fact that double-stranded DNA if present in the pH 4.8 system will transform the cells about 15 times less efficiently than at pH 7.0 ]e,*8,*e. Parallel studies of ours *e indicate that upon alkaline denaturation of the transforming DNA, there is a loss of transforming activity as measured by the pH 7.0 method and an increase in transforming activity as measured b y the pH 4.8 method, both of which are related to the secondary structure of the molecule. The results indicate that the denaturation process is uot an all-or-none process. The knowledge of partial denaturation by alkali 11,12,26 has enabled us to study the incompletely denatured molecules. In contrast to the use of extreme pH which causes complete collapse of the DNA molecules 10, the present study employed a milder treatment and examined both stages of denaturation in the biphasic kinetics of denaturation. However, the initial reaction is still too fast to allow accurate comparison of the markers used. It has been detected nevertheless that there is a difference in the activity of different markers during the second phase of denaturation. The inactivation b y alkali of various genetic markers as determined kinetically was in the order of SN > S > E > N > K. Such differences in the kinetics of inactivation imply difBiochim. Biophys. Acta, 232 (1971) 234-245
244
A . D . COOPER, P. C. HUANG
ferences in the rates of denaturation within localized regions of the DNA molecules, and perhaps differences in the A. T content of these regions. These differences in the rates of denaturation m a y be due to differences in cross-linkage ~, 35 in these regions, differences in the A . T content of these regions or a combination of the two. Differences in the rates of denaturation within localized regions of the DNA molecules also indicate t h a t denaturation is not an all-or-none process. The extent of the activation of the various genetic markers during partial denaturation is in the order S > N which implies that the loss of the marker activity when assayed b y the p H 7.0 method is balanced b y the increase in marker activity assayed by the p H 4.8 method. Apparently, in the p H 4.8 method all t h a t is necessary for the marker activity to be recovered is that the DNA molecule be in a singlestranded configuration. The removal of protons b y alkali from the bases composing DNA is due to their neutralization b y hydroxyl ions. Indeed, AGENO et al. (1969) 11 have shown that a greater change in the absorbance at 23 ° n m occurs at lower p H ' s than changes in absorbance at 260 nm. They suggest t h a t these changes are due to deprotonation of the nucleic acid in the non-hydrogen bonding atoms of the bases composing the DNA molecule and m a y or m a y not affect the secondary structure of the molecule. As the A • T-rich regions are more easily deprotonated than the G • C-rich regions, these regions upon deprotonation lose their hydrogen bonding capacity more readily. Such a region is probably present in the S marker. The S marker is inactivated much more rapidly during alkaline denaturation and also during thermal denaturation both in the earlier and later stages of denaturation than is the presumably less A • T-rich N marker. This interpretation suggests t h a t denaturation could begin at either one or both ends of the molecule or within the molecule depending upon the extent of localization of the A • T-rich region. Denaturation continues from the more A • Trich regions to the less A • T-rich regions until the entire double-stranded molecule is separated into two single strands or until a hidden break in one strand of the duplex is reached. At this point or possibly during the neutralization process a portion of one strand of the duplex is broken off leaving a partially double-stranded molecule with extensive single-stranded regions. Those molecules containing the A" Trich S marker will be inactivated (in the early stages of denaturation) much more rapidly than those molecules containing only the N or both S and N markels (Figs. 5 and 6). The 6-fold fall or SN/S ratio, however, after complete denaturation m a y indicate an apparent reduction in the size of the single-stranded molecule b y a factor of six. Yet, the molecular weights of the native DNA preparation and the partially denatured DNA preparation (i.e. p H 11.6 for 5 inin), both remain 3o(4-1o)" lO 6, corresponding to a single-stranded molecular weight of 15(4-5)" 1°6. The singlestranded molecular weight of the completely denatuled DNA preparation when sedimented in an alkaline sucrose gradient was found to be 8(4-3)'1o s. Thus tile SN/S ratios and the molecular weight determination from the sucrose gtadient sedimentation both indicate that no more than 2.4 hidden breaks per double-stranded molecule were present in the original native DNA preparation. An alternative interpretation is t h a t these breaks were introduced during the process of denaturation. Such breaks could be introduced during the process of neutralization which terminates the denaturation process. The undue stress at the fork during the winding could create a torque sufficient to break one or both strands of the unraveling double helix. Under Biochim. Biophys. Acta, 232 (I97 I) 234-245
DIFFERENTIALDENATURATIONOF DNA
245
either interpretation, the difference in marker sensitivity to denaturation under controlled conditions may prove to be useful for genetic manipulations.
ACKNOWLEDGEMENTS
The work is supported in part by a National Institute of Health, U.S. Public Health Service Postdoctoral fellowship to A. D. C. (FRo5445), and Research Career Development Award to P. C. H. (I-K3GM31-238-oI) and by a grant from National Science Foundation (GB 8058). We are grateful to William M. Swartz and Charles Braslow who have contributed, respectively, in defining some of the experimental conditions for thermal and alkaline denaturation reported here. We thank Dr. Roger M. Herriott for his encouragement. REFERENCES I 2 3 4 5 6 7 8 9
J. MARMUR, R. ROWND AND C. L. SCHILDKRAUT, Progr. Nucleic Acid Res., I (1963) 232. G. FELSENFELD AND S. Z. HIRSCHMAN, J. Mol. Biol., 13 (1965) 407 . D. M. CROTHERS, Biopolymers, 6 (1968) 1391. R. ]3. INMAN AND M. SCHN6S, J. Mol. Biol., 49 (197 °) 93. IV[. ROGER AND R. D. HOTCHKISS, Proc. Natl. Acad. Sci. U.S., 47 (1961) 653" W. GINOZA AND B. H. ZIMM, Proc. Natl. Acad. Sci. U.S., 47 (1961) 639. J. M. PETERSON AND W. R. GUILD, J. Mol. Biol., 20 (1966) 497. 1~¢1.R. CHEVALLIER AND G. BERNARDI, J. Mol. Biol., 32 (1968) 437R. RUDNER, H. J. LIN, S. E. M. HOFFMA.NN AND E. CHARGAFF, Biochim. Biophys. Acta, 149
IO ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
(1967) 199. E. ]3. FREESE AND E. FREESE, Biochemistry, 2 (1963) 7o7 . M. AGENO, E. DORE AND C. FRONTALI, Biophys. J., 9 (1969) 1281. M. AGENO, E. DORE AND C. FRONTALI, Biopolymers, 9 (197 °) 116. S. H. GOODGAL AND R. M. HERRIOTT, J. Gen. Physiol., 44 (1961) 1229. R. M. HERRIOT~, E. MEYER AND M. VOGT, J. Bacteriol., IOI (197 o) 517. E. H. POSTEL AND S. H. GOODGAL, J. Mol. Biol., 16 (1966) 317 • S. H. GOODGAL AND E. H. POSTEL, J. Mol. Biol., 28 (1967) 261. E. H. POSTEL AND S. H. GOODGAL, J. Mol. Biol., 28 (1967) 247. G. L. MILLER AND R. H. GOLDER, Arch. Biochem. Biophys., 29 (195 o) 420. R. G. BATES, in H. A. SOBER, Handbook o[ Biochemistry, Chemical R u b b e r Co., 1968, p, JI9O. H. R. 1V[ASSIEAND B. H. ZIMM, Biopolymers, 7 (1969) 475. E. BURGI AND A. D. HERSHEY, Biophys. J . , 3 (1963) 309. F. W. STUDIER, J. Mol. Biol., I I (1965) 373. A. D. COOPER AND P. C. HUANG, J. Bacteriol., in the press. C. MULDER AND P. DOT~¢, J. Mol. Biol., 32 (1968) 423 . B. M. ALBERTS AND P. DOTY, J. Mol. Biol., 32 (1968) 379A. D. COOPER AND P. C. HUANG, Biochim. Biophys. Acta, (197 o) s u b m i t t e d .
Biochim. Biophys. Acta, 232 (197 I) 234-245
234 BBA 96790
DIFFERENTIAL
DENATURATION
OF
HAEMOPHILUS
INFLUENZAE
DNA ALAN D. COOPER AND P. C. HUANG Department o] Biochemistry, The Johns Hopkins University, School o/Hygiene and Public Health, Baltimore, Md. 212o5 (U.S.A.) (Received September I4th, I97o)
SUMMARY
The objective of this study is to determine whether differential denaturation of various genes can be observed. B y treatments with heat or b y titration with hydroxyl ions at subcritical levels, genetic markers from Haem@hilus influenzae have been inactivated in the order of streptomycin resistance > erythromycin resistance > novobiocin resistance. The kinetics and extent of inactivation depends on whether a thermal or alkaline method is used. Results obtained with an assay system which allows the expression of single-stranded DNA are complementary to those testing the transforming activity of double-stranded DNA.
INTRODUCTION The stability of DNA helices does not always follow the prediction of simple statistical mechanics, since DNA is a heterogeneous, aperiodic lattice in an equilibrium state of the order-disorder transition. In this context, it is not surprising to find t h a t both the base sequence and particularly the G • C content, determine to a large extent the thermal melting behavior of a given DNA molecule 1. In fact, it has been suggested that the A • T- and G • C-rich regions of DNA m a y be mapped b y controlled denaturation. For instance, FELSENFELD AND HIRSCHMAN~ reported a preferential denaturation of the A • T regions in DNA. This was based on the computation of hyperchromic shifts resulting from denaturation. The contributions of A • T and G . C base pairs to absorbance changes are quantitatively and qualitatively different. CROTItERS3 has presented a model with which one can calculate the partition function and entropy required in the denaturing process and can also locate genetic loci of specific base composition. Recent studies with electron microscopy have provided still other evidences for the preferential denaturation by mild alkali of specific regions in viral chromosomes 4. Biological approaches to examine the differential inactivation of genetic markers have also been taken. The assumption was that different genes are sufficiently dissimilal in their base sequences so that a preference in the melting of the A • T-rich segments will eliminate genes so enriched. Thus, using thermal denaturation, several genetic markers in DNA have been shown to be inactivated differentially in the tiansforming system of Diplococcus pneumoniae 5-7. These inactivations were attributed to Biochim. Biophys. Acta, 232 ~I97I) 234-245
DIFFERENTIAL DENATURATION OF
DNA
235
the collapse of selected areas in the DNA molecule following partial separation of the strands. These differential inactivations were attributed to localized differences in G - C content but not necessalily to the overall base composition. CHEVALLIER AND BERNARDI8 examined the thermal inactivation of three genes from Haemophilus in[luenzae but only minor differences were noted in the critical inactivation temperatures. RUDNER et al. 9 observed that in fully denatured samples of Bacillus subtilis DNA, whether inactivated b y dialysis, heat or alkali, the proportions of the residual activities with respect to three genetic markers, Adenine, leucine and methionine were approximately 2 : i : I. The similar behavior of the three markers suggested to them that regardless of the manner in which the loss of secondary structure is induced the stability of a marker must be inherent in its unique base-paired sequence. Alkaline denaturation of DNA has now been more closely examined t°-l~. From these studies it is clear that denaturation by alkali, like denaturation by heat involves the rupturing of hydrogen bonds which basically provide the difference in stability between A • T and G • C pairs. Consequently, a preferential inactivation of different genes by alkaline treatment of the DNA is to be expected. In this study, using the H. influenzae system, a comparison of the transforming activities of several genetic markers was made after both thermal and alkaline denaturation. The basic approach takes advantage of subcritical denaturation conditions; thus allowing minor differences to be expressed. The results suggest the existence of preferential denaturation of certain markers implying that some genes examined are indeed different in their base composition. MATERIALS AND METHODS
(z) DNA Transforming DNA was isolated from H. in[luenzae according to the method of GOODGALAND HERRIOTT13. DNA prepared b y this procedure has a molecular weight of about 2 • lO7-4.1o ~ as determined b y sucrose density gradient sedimentation. The general properties of the DNA* used in this study are as follows: The DNA had a hyperchromicity of about 26 % at 26o nm and a Tin, melting temperature, of 84.5 ° in o.15 M NaC1. ESN DNA, SN DNA and SNK DNA had absorbance ratios, A~n0n m / A ~ o am, greater than 2.2 and A~6o nm/A280 nm greater than 1. 9 . (2) Strain of bacteria A non-encapsulated (R) strain of H. in]luenzae type d was obtained originally from Hattie E. Alexander of Columbia University. (3) Trans/ormation The basic method of assay for transformation was that of GOODGAL AND HERRIOTTis. Cells were made competent after the procedure of HERRIOTT et al. 14 " The D~qA p r e p a r a t i o n s are designated b y s y m b o l s for t h e antibiotic resistance m a r k e r s t h e y carry. E = e r y t h r o m y c i n ; S = s t r e p t o m y c i n ; N = novobiocin (also k n o w n as cathomycin) a n d K = kanomycin. The subscript denotes t h e level of resistance; t h u s Ere m e a n s resistance to i o p g / m l of e r y t h r o m y c i n .
Biochim. Biophys. Acta, 232 ¢i97 I) 234-245
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A . D . COOPER, P. C. HUANG
in competence-developing medium (MV), resuspended in a storage medium (MII) and stored in 15 % glycerol at --7 °0 until used. Three transformation assay procedures were used. In the first, the cells were thawed and diluted I : IO with MII medium. In the second and third assays the competent cells were thawed, washed with o.15 M NaC1, and resuspended in 0.3 % bactopeptone (Difco) and o.15 N[ NaC1 either at p H 7.0 for double-stranded DNA assay or at p H 4.8 in the presence of 3 × lO-4 M E D T A for single-stranded DNA assay 15-17. Following the addition of DNA the cells were incubated at 37 ° with slight shaking at 60 strokes/min. In the case of the single-stranded assay, the p H was changed after IO min from p H 4.8 to p H 7.0 b y the addition of o.1 M N a O H 15-17. The total time of incubation was 3 ° min. At the end of this time, the cells were diluted, plated, and incubated for 12o min and then overlaid with agar containing twice the desired concentration of the challenging antibiotic. Throughout this paper, the three methods for assaying transforming DNA will be referred to as the MII method, the p H 7.0 method, and the p H 4.8 method.
(4) Thermal denaturation DNA at a concentration of 5 × lO-2/~g/ml was incubated in a volume of i.o ml of o.ool 5 M Nacl--o.oooI5 M sodium citrate, p H 6.8, in a screw cap vial. Heat treatment was done in a thermograd. The thermograd consists of four parallel rows of 19 separate chambers. Temperature is controlled differentially by two thermostats at the extreme ends so that a linear temperature gradient of any desired range can be generated. Denaturation was terminated by chilling the vials in an ice-water-salt (--IO °) bath. Aliquots of DNA were used for transformation with the p H 7.0 and p H 4.8 methods. In all cases, the concentration of DNA used is within the linear dosage range for transformation, i.e. 2 × lO -3/~g/Io 8 cells.
(5) Alkaline denaturation Two buffer systems were used for the denaturation of DNA by alkali. One was the glycine-NaCI-NaOH buffer at an ionic strength of o.I is. The other was a series of buffers covering the range 7.o-13.o and were at an ionic stlength of 0.21° consisting of Tris-HC1-NaC1, NaHCO3-NaOH-NaC1, Na2HPO4-NaOH-NaC1 or KC1-NaOHNaCI. A constant ionic strength was maintained because the relaxation time of a double-stranded DNA molecule shows a very definite dependence on ionic strength z°. The p H ' s were measured at ambient temperature with a Beckman Research p H meter equipped with a KC1-AgC1 electrode (pH o-14.o ). The p H ' s were determined before and after the experiment. Occasional discrepancies greater or equal to 0.2 p H due to CO2 absorption were encountered. When this occurred, the experimental point was discarded. The protocol of alkaline denaturation and assay was usually as follows: To 9.9 ml of buffer contained in a sterile scintillation vial and stirred gently b y a magnetic stirrer at ambient temperature was added o.I ml of DNA usually at a concentration of 2.5/~g/ml. The concentration of DNA in the denaturing buffer was usually less than I/~g/ml, so that after dilution the DNA would be at a concentration within the linear dosage range for transformation. Furthermore, the concentration of DNA was kept constant during denaturation as DNA unwinding is partly hydrodynamically limited, hence there is a dependence of the rate on concentration 2°. At the appropriate Biochim. Biophys. Acta, 232 (1971) 234-245
DI FFERENT IAL DENATURATION OF
DNA
237
time, o.I ml was removed and added to 4.9 ml of competent cells in MII medium which terminates the alkaline action. Bactopeptone mediums (pH 7.0 and pH 4.8 methods) replaced MII medium whenever the transforming activity of single-stranded DNA was determined. MII at pH 4.8 is not a good medium to transform cells by singlestranded DNA (unpublished observation), although it is a good buffer. Addition of buffer with a high pH never changed the pH of the MII competence medium by more than o.I pH unit. Whenever bactopeptoue replaced MII medium neutralization of the denatured DNA with H3PO , followed by a I : IO dilution in bactopeptone was necessary before adding o.1-4. 9 ml of competent cells.
(6) Sucrose gradient centri]ugation Molecular weights were determined by sedimentation in a 5-20 % sucrose gradient, according to the plocedure of BURGI AND HERSHEY~1 for neutral gradients and according to STUDIER~2 for alkaline gradients.
RESULTS
(I) Thermal denaturation The relative decrease in double-stranded and increase in single-stranded transforming activities of the El0 markers are both functions of the temperature of denaturation (Fig. i). In Fig. i it is shown that the relative transforming activity, as assayed by the pH 7.0 method, is a function of temperature. Conversely, there is an increase in the relative transforming activity when assayed by the pH 4.8 method. The activity found in the pH 7.0 method is a reflection of the activity of doublestranded DNA. The pH 4.8 method assays mainly the activity of single-stranded DNA. The temperature at the midpoints of the two activity curves is near 7 o°. In order to magnify the relative extent of denaturation of various genetic markers a subcritical melting temperature was chosen. In this way, the rate of thermal denaturation is reduced. The kinetics of denaturation of the E, S, N and the linked marker SN at 69 ° are shown in Fig. 2 and 3. The result (Fig. 2) indicates that the SN marker is inactivated more rapidly and extensively than either of the two single markers. Within 0.5 min, the S marker is already inactivated to 88 % of the original activity (i.e. zero time) while activity in both the E and N markers are not inactivated until I rain. The differences in the extent of denaturation of the S, E, and N markers are small but consistently in the same relative order. Even after complete denaturation (IO min at 95 °) the relative transforming activities remain in the same order. The kinetics of relative increase in the transforming activity of single-stranded I)NA of the markers tested is shown in Fig. 3- The E and S markers respond more rapidly to denaturation but the N and SN markers show a time lag in response. However, the maximum levels of transforming activities of the several markers are in the order E > N >/ S > SN. After extensive denaturation, the E marker reaches a transforming activity IO times that of the E marker that is undenatured, whereas for N this value is 9 times, for S, 8.5 times and for SN about 6 times. For the SN linked marker the relative activity decreases further after extensive denaturation (95 °, IO min) and reaches a value 4.5 times that of the value for undenatured DNA.
Biochim. Biopkys. Acta, 232 (1971) 234-255
23 8
A. D. COOPER, P. C. HUANG
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Fig. I. Activation and inactivation of a genetic m a r k e r u p o n t h e r m a l denaturation. El0 $2000 Ns.~ D N A was d e n a t u r e d at a concentration of 5.4 " 1o-3/~g/ml in o.ool 5 1V~ N a C l - o . o o o i 5 NI s o d i u m citrate at various t e m p e r a t u r e s . The samples in vials at several different t e m p e r a t u r e s were heated in a t h e r m o g r a d for 15 rain and t h e n quickly chi]led in an ice-salt-water m i x t u r e at --lO% To each of 2. 5 ml of cells in either p H 7.0 bactopeptone, 237 • lO 5 cells/m], or p H 4.8 b a c t o p e p t o n e in t h e presence of 3 " 1°-4 M E D T A , 238 • io 5 cells/ml was added o.1 ml of t r e a t e d D N A to a final concentration of 2.I • lO -3/~g/ml. I n this figure the relative t r a n s f o r m i n g activity as assayed b y the p H 7.0 m e t h o d as well as the p H 4.8 m e t h o d is plotted against t e m p e r a t u r e . The IOO ~/o p o i n t for the Elo m a r k e r as assayed b y the p H 7.o m e t h o d is 9.9 • lO3 t r a n s f o r m a n t s / ml and the 16 % point for the El0 m a r k e r as assayed b y the p H 4.8 m e t h o d is 2.8 • lO 3 transform a n t s / m l . O , values determined b y the p H 7.0 m e t h o d ; O , values determined b y the p H 4.8 method. Fig. 2. The relative inactivation of genetic m a r k e r s u p o n t h e r m a l d e n a t u r a t i o n . Ex0 $2000 lq2.5 D N A was d e n a t u r e d at a concentration of 5.4" Io-2/~g/ml in o.oo15 M N a C l - o . o o o i 5 M, s o d i u m cit r a t e at 69 °. At various times aliquots were r e m o v e d a n d chilled in an i c e - s a l t - w a t e r m i x t u r e at --lO °. The zero-time p o i n t represents D N A t h a t had been k e p t at 25 °. I n addition an aliquot of ES1W D N A was completely denatured b y t r e a t i n g it at 95 ° for io min, and chilled. To each of 2. 5 ml of c o m p e t e n t cells at a concentration of 233 • lO5 cells/ml in p H 7.o b a c t o p e p t o n e was added o.I ml of t r e a t e d D N A to a final concentration of 2.1 - lO -8/~g/ml. The cells were t r a n s f o r m e d and counted. The relative t r a n s f o r m i n g activity as assayed in the p H 7.o s y s t e m is plotted a g a i n s t the e x t e n t of denaturation. The ioo ~o points for t h e m a r k e r s are E, 2.89 • lO4; S, 1.2 • zo4; iW, 3.4 " lO4; and SN, 3-7 " lOS t r a n s f o r m a n t s / m l . F o r this a n d s u b s e q u e n t figures, the m a r k e r s are represented b y the following symbols: O , Ns.~; O , Sso00; [], SN; × , Elo. The a r r o w indicates completely d e n a t u r e d DBIA (i.e. 95 ° for IO mill). The insert depicts the inactivation during the first 3 min of d e n a t u r a t i o n on an e x p a n d e d time scale.
Biochim. Biophys. Acta, 232 (i97 x) 234-245
DIFFERENTIAL DENATURATION OF D N A
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Fig. 3. The relative activation of genetic m a r k e r s u p o n t h e r m a l d e n a t u r a t i o n . El0 Sl000 Nt. 5 D N A was d e n a t u r e d a t a concentration of 5.4 " zo-3/~g/ml in o.ooI 5 M N a C l - o . o o o i 5 M sodium citrate a t 59 ° as described in Fig. 2. A t various times aliquots were r e m o v e d and chilled in an i c e - s a l t - w a t e r m i x t u r e at --lO% The zero-time p o i n t represents D N A t h a t h a d been kept a t 25 °. I n addition an aliquot of E S N D N A w a s completely d e n a t u r e d b y t r e a t i n g it a t 95 ° for IO min a n d chilled. To each of 2. 5 ml of c o m p e t e n t cells at a concentration of I 9 I • lO s cells]ml in p H 4.8 b a c t o p e p t o n e in t h e presence of 3 • I o - 4 M E D T A w a s added o.i ml of t r e a t e d D N A to a final c o n c e n t r a t i o n of 2.I • lO 3/,g/ml. The cells were t r a n s f o r m e d and counted. The relative percent t r a n s f o r m i n g activity as assayed b y t h e p H 4.8 m e t h o d is plotted against t h e e x t e n t of d e n a t u r a tion. The reference p o i n t (zero time) for t h e m a r k e r s are E, 2380; S, 127 • io3; N, 2.95 • IO8; a n d SN, 6.o 5 • i o 3 t r a n s f o r m a n t s ] m l . The insert depicts the f i r s t 3 m i n of d e n a t u r a t i o n on an e x p a n d e d t i m e scale.
These results indicate that (I) the genetic markers of H. in/luenzae can be differentially denatured by heat, depending upon the condition employed; (2) the genetic markers examined are differentially inactivated, as measured by their transforming activity by the pH 7.0 method in the following order SN > S > E > N. Biochim. Bi ophy s . Acta, 232 ( I 9 6 I ) 234-245
240
A . D . COOPER, P. C. HUANG
(3) The genetic markers examined are differentially activated if measured by their transforming activity with the pH 4.8 method, in the order of SN < N < E ~< S within the earlier stages of denaturation. However, maximal activity is achieved in the following order E > N i> S > SN.
(2) Alkaline denaturation Alkaline tzeatmeut of DNA was done near the midpoint of the pH inactivation range where partial denaturation takes place ~3. The kinetics of alkaline denaturation of several markers at pH 11.8 with the midpoint of inactivation is depicted in Fig. 4-
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o
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' iO0
' 120
F i g . 4- Kinetics of i n a c t i v a t i o n of t h e S=000, Ne.5, and K10 m a r k e r s u p o n alkaline d e n a t u r a t i o n To 9 . 9 m l of glycine buffer at p H 11.8 at r o o m t e m p e r a t u r e w a s added o . i m l of S N K D N A at a c o n c e n t r a t i o n of 2. 5 / ~ g / m l a n d s l o w l y stirred. A t t h e t i m e s indicated o . i m l w a s r e m o v e d a n d a d d e d to 4-9 m l of c o m p e t e n t cells at a c o n c e n t r a t i o n of 8 9 9 • lO s c e l l s / m l i n M I [ m e d i u m . T h e final c o n c e n t r a t i o n of D N A w a s 5 • io-4/~g/ml. The relative percent t r a n s f o r m i n g a c t i v i t y is p l o t t e d against t h e e x t e n t of d e n a t u r a t i o n at p H 11.8. The i o o % p o i n t s for t h e m a r k e r s are N , 4.2 • lO4; S, z . 5 7 " lO4; K , 1 . 7 . Io4; and SN, 6 . 0 4 • lO 8 t r a n s f o r m a n t s ] m l . I , Kz0. T h e z e r o - t i m e p o i n t consisted of o . I m l of D N A diluted i : i o o in o . 1 5 N[ N a C 1 plus o . i m l of p H 11.8 buffer. I t s h o u l d be n o t e d t h a t t h e Klo m a r k e r is leaky, For this reason b a c k g r o u n d w a s n o t zero and w a s s u b t r a c t e d to o b t a i n t h e v a l u e s u s e d in this figure.
All markers, K, S, and N including one linked marker SN treated at pH 11.8 exhibit similar kinetics of inactivation. In each instance, there are two steps involved with a rapid loss of activity to about 6 % of the original activity during the first 4 rain, followed by a gradual decrease to 1- 3 % after 12o rain depending on the marker. Little difference can be detected in the initial rate of inactivation. Differences can be recognized, however, during the second step. The difference in the residual activity, namely 3 % for K, 1. 4 °/o for N, 1 % for S, and 0. 3 % for SN can not be accounted Biochim. Biophys. Acta,
2 3 2 (1971) 2 3 4 - 2 4 5
DIFFERENTIAL DENATURATION OF
DNA
241
for by their innate differences in transforming efficiency, since each is compared to its original activity. Results not shown here indicate that the kinetics of inactivation by alkali of E are intermediate between S and N. Figs. 5 and 6 show results from experiments in which SN DNA (I #g/m]), partially denatured for various periods of time at pH 11.6, a subcritical pH, was compared with native SN DNA and with completely denatured SN DNA (pH 12.6 for IO min). The kinetics of denaturation of these markers, S, N and SN when assayed by either the pH 7.0 or pH 4.8 methods are diffelent from each other. Fig. 5 shows that DNA, denatured at pH 11.6, exhibits during the first 4 min a rapid decrease in doublestranded DNA transforming activity for all three markezs when measured by the pH 7.0 method followed by a gradual decrease later. However, the S marker is inacti-
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Fig. 5- Relative activation of genetic m a r k e r s u p o n alkaline denaturation. $2000 N,. 5 D N A was d e n a t u r e d at p H 11.6 a t r o o m t e m p e r a t u r e at a concentration of i #g/ml, for various times, neutralized w i t h chilled H3PO 4 and f u r t h e r chilled in an ice-water mixture. I n addition a sample of D N A was t r e a t e d at p H i2.6 for io min. The samples were diluted i : io and o.I ml added to 5.0 ml of c o m p e t e n t cells in p H 4.8 b a e t o p e p t o n e in the presence of 3 • 1o-4 M E D T A at a concentration of 54 8 • IOs cells/ml. The t r a n s f o r m e d cells were plated a n d counted. The N/S ([2], m) and SN/S (O, • ) ratios are plotted against t h e e x t e n t of d e n a t u r a t i o n to indicate the relative differences in t h e levels of activation of t h e N and S markers. The relative t r a n s f o r m i n g activity of the $200o, N~. 5 and $2000 b]2.s m a r k e r s as assayed b y the p H 4.8 m e t h o d is plotted against the e x t e n t of denaturation. The reference points (zero time) for the m a r k e r s are S, i 8 i t r a n s f o r m a n t s / ml; N, 482 t r a n s f o r m a n t s / m l ; a n d SN, 82 t r a n s f o r m a n t s / m l . Fig. 6. Relative inactivation of genetic m a r k e r s u p o n alkaline denaturation. $2000 N2. 5 D N A was d e n a t u r e d at r o o m t e m p e r a t u r e at p H 11.6 at a concentration of i / z g / m l for various times, neutralized with chilled H3PO 4 and f u r t h e r chilled in an i c e - w a t e r mixture. I n addition a sample of D N A was t r e a t e d a t p H 12.6 for i o min. The samples were diluted I : IO and o.I ml added to 5.o ml of c o m p e t e n t cells in p H 7.0 b a c t o p e p t o n e at a concentration of 688 • lO 5 cells/ml. The t r a n s f o r m e d cells were plated and counted. The N/S and SN/S ratios are plotted against the ext e n t of d e n a t u r a t i o n to indicate the relative differences in the levels of inactivation of the N and S markers. The relative t r a n s f o r m i n g activity of the Se000, N 2.5 and S20o0 N2. s m a r k e r s as assayed by the p H 7.0 m e t h o d is plotted against the e x t e n t of d e n a t u r a t i o n . T h e lOO% p o i n t s for the m a r k e r s are S, 6.6. io $ t r a n s f o r m a n t s / m l ; N, 1.6 5 . lO4 t r a n s f o r m a n t s / m l ; and SN, 2.26. io n transformants/ml.
Biochim. Biophys. Acta, 232 (197 I) 234-245
242
A . D . COOPER, P. C. tIUANG
vated more extensively than the N marker throughout the process, the former reaching a value of 56 % and the latter a value of 79 % at 4 ° rain (not shown in Fig. 5). The SN marker reaches a value of 53 % at 4 ° min. After complete denaturation the residual activities ~,.5 for S, N and SN are reduced to a residual level of 3.2 %. 4.3 % and 1.3 %, respectively. If denatured at p H II.6 and assayed by the p H 4.8 method, the N marker is found to increase 6-fold after 4 ° min (Fig. 6). The S marker increases more (7.7-fold) and the SN double marker less (5-fold) as compared to time zero. The values obtained with completely denatured DNA (pH 12.6 for IO min) give somewhat different results. Both the S and N markers have reached 12- and I7-fold, respectively, but cotransformation of the SN markers has fallen 1o 1.3-fold. The initial rate of denaturation can not be distinguished among the markers, although the levels of denaturation differ (S /> N > SN). The data just presented can also be expressed as the N/S ratio. As assayed by the pH 7.0 method, the N/S ratio increases from 2.5 to 3.5 after 40 min of denaturation at pH 11.6 (see also Fig. 5)- After complete denaturation the N/S ratio is 3.3- When assayed by the pH 4.8 method, however, the N/S ratio decreases from 2.5 to 2.1 after 4 ° min (see also Fig. 6), but the ratio increases to 3.8 after complete denaturation. When assayed by the pH 7.0 method the double-stranded molecules composing the partially denatured DNA have the same SN/S ratio (o.31) as the double-stranded molecules composing native (i.e. zero time) DNA (0.34) (Fig. 5)- After complete denaturation the S and N markers have a ratio of o.14. When assayed by the pH 4.8 method the single-stranded molecules of partially denatured DNA possess an SN/S ratio of 0.30 (Fig. 6). Upon complete denaturation the SN/S ratio falls 6-fold to 0.05. In these experiments, the molecular weights of the native and partially denatured (i.e. pH 11.6 for 5 min) DNA as determined by sucrose gradient density centrifugation were both 3° (4-1o) • lO 6, corresponding to a single-stranded molecular weight of 15(4-5)'1o 6. The single-stranded DNA of the completely denatured DNA preparation (pH 12.6, IO min) when sedimented in an alkaline sucrose gradient had a molecular weight of 8 ( ± 3 ) " lO6. Thus, only a 2-fold reduction in the single-stranded molecular weight of the DNA has occurred even after extensive denaturation. These results indicate that (I) the genetic markers of H. influenzae can be differentially denatured by alkali depending upon the conditions employed; (2) the genetic markers are differentially inactivated as measured by their transforming activity by the pH 7.0 method in the following order: SN > S > N; (3) the genetic markers examined are differentially activated as measured by their transforming activity by the pH 4.8 method within the earlier stages of denaturation in the older S > N > SN. However, the maximal activity is achieved in the following order N > S > SN. The difference is not due to molecular size of the DNA.
DISCUSSION Results presented in this study show that preferential inactivation of certain genetic markers may be obtained either b y thermal or alkaline denaturation. Thus the S marker is more labile to heat and to alkali than is either the E marker or the N marker.
Bioch,m. Biophys. Acta, 232 (1971) 234-245
DIFFERENTIAL DENATURATION OF D N A
243
The data of CHEVALLIERAND BERNARDI8 indicate that for H. influenzae DNA, the melting temperatures in o.15 M NaCl-o.oI M sodium phosphate buffer (pH 7.0) are 88 ° for the streptomycin marker and 88.6 ° for the erythromycin and novibiocin markers s. By selecting a subcritical temperature below the Tm (i.e. 7 °0 in o.oo15 M NaCl-o.oooI5 M sodium citrate) and examining the kinetics of denaturation our results indicate that the S marker is inactivated more rapidly than either the E or N marker. Furthering the earlier data 8, the present data indicate that the relative order of inactivation of genetic markers is SN > S >1 E > N. For D. pneumoniae DNA, the novobiocin, erythromycin, and streptomycin markers are inactivated during thermal denaturation in the order N > E > Se. The order of inactivation is therefore opposite to the order we have found for the similar markers of H. in/luenzae DNA. It is possible that: (I) The N, E, S markers in D. pneumococcus do not have the same base sequence as the corresponding markers in H. in/luenzae; or (2) the N, E, S markers in D. pneumococcus and H. in/luenzae m a y be similar but complete separation of the double strands of the DNA molecule does not occur until a region of unique composition is denatured; or (3) the N, E, S markers in D. pneumococcus and H. in/luenzae are similar in base sequence but the regions immediately adjacent to them differ in base composition. If the first conclusion is correct then one may infer that the genes between the Haemophilus and Diplococcus species are not conserved. Controlled denaturation therefore may be another useful method in the determination of genetic relatedness of various species of bacteria. The apparent loss of the marker activity of double-stranded DNA undei examination is not due to some irreversible damage to the marker region, such as an extensive reduction in size. The marker activity can be efficiently recovered when assayed b y the pH 4.8 method. However, it can not be determined whether each particular marker that is inactivated when assayed b y the pH 7.0 method is also activated when assayed b y the pH 4.8 method due to the relatively low efficiency of the latter. It has been noted'that there is a Ioo-fold declease in the relative transforming activity of the DNA when assayed by the pH 7.0 method whereas there is only a Io-fold increase in activity when assayed by the pH 4.8 method (Fig. I). This is probably due to the fact that double-stranded DNA if present in the pH 4.8 system will transform the cells about 15 times less efficiently than at pH 7.0 ]e,*8,*e. Parallel studies of ours *e indicate that upon alkaline denaturation of the transforming DNA, there is a loss of transforming activity as measured by the pH 7.0 method and an increase in transforming activity as measured b y the pH 4.8 method, both of which are related to the secondary structure of the molecule. The results indicate that the denaturation process is uot an all-or-none process. The knowledge of partial denaturation by alkali 11,12,26 has enabled us to study the incompletely denatured molecules. In contrast to the use of extreme pH which causes complete collapse of the DNA molecules 10, the present study employed a milder treatment and examined both stages of denaturation in the biphasic kinetics of denaturation. However, the initial reaction is still too fast to allow accurate comparison of the markers used. It has been detected nevertheless that there is a difference in the activity of different markers during the second phase of denaturation. The inactivation b y alkali of various genetic markers as determined kinetically was in the order of SN > S > E > N > K. Such differences in the kinetics of inactivation imply difBiochim. Biophys. Acta, 232 (1971) 234-245
244
A . D . COOPER, P. C. HUANG
ferences in the rates of denaturation within localized regions of the DNA molecules, and perhaps differences in the A. T content of these regions. These differences in the rates of denaturation m a y be due to differences in cross-linkage ~, 35 in these regions, differences in the A . T content of these regions or a combination of the two. Differences in the rates of denaturation within localized regions of the DNA molecules also indicate t h a t denaturation is not an all-or-none process. The extent of the activation of the various genetic markers during partial denaturation is in the order S > N which implies that the loss of the marker activity when assayed b y the p H 7.0 method is balanced b y the increase in marker activity assayed by the p H 4.8 method. Apparently, in the p H 4.8 method all t h a t is necessary for the marker activity to be recovered is that the DNA molecule be in a singlestranded configuration. The removal of protons b y alkali from the bases composing DNA is due to their neutralization b y hydroxyl ions. Indeed, AGENO et al. (1969) 11 have shown that a greater change in the absorbance at 23 ° n m occurs at lower p H ' s than changes in absorbance at 260 nm. They suggest t h a t these changes are due to deprotonation of the nucleic acid in the non-hydrogen bonding atoms of the bases composing the DNA molecule and m a y or m a y not affect the secondary structure of the molecule. As the A • T-rich regions are more easily deprotonated than the G • C-rich regions, these regions upon deprotonation lose their hydrogen bonding capacity more readily. Such a region is probably present in the S marker. The S marker is inactivated much more rapidly during alkaline denaturation and also during thermal denaturation both in the earlier and later stages of denaturation than is the presumably less A • T-rich N marker. This interpretation suggests t h a t denaturation could begin at either one or both ends of the molecule or within the molecule depending upon the extent of localization of the A • T-rich region. Denaturation continues from the more A • Trich regions to the less A • T-rich regions until the entire double-stranded molecule is separated into two single strands or until a hidden break in one strand of the duplex is reached. At this point or possibly during the neutralization process a portion of one strand of the duplex is broken off leaving a partially double-stranded molecule with extensive single-stranded regions. Those molecules containing the A" Trich S marker will be inactivated (in the early stages of denaturation) much more rapidly than those molecules containing only the N or both S and N markels (Figs. 5 and 6). The 6-fold fall or SN/S ratio, however, after complete denaturation m a y indicate an apparent reduction in the size of the single-stranded molecule b y a factor of six. Yet, the molecular weights of the native DNA preparation and the partially denatured DNA preparation (i.e. p H 11.6 for 5 inin), both remain 3o(4-1o)" lO 6, corresponding to a single-stranded molecular weight of 15(4-5)" 1°6. The singlestranded molecular weight of the completely denatuled DNA preparation when sedimented in an alkaline sucrose gradient was found to be 8(4-3)'1o s. Thus tile SN/S ratios and the molecular weight determination from the sucrose gtadient sedimentation both indicate that no more than 2.4 hidden breaks per double-stranded molecule were present in the original native DNA preparation. An alternative interpretation is t h a t these breaks were introduced during the process of denaturation. Such breaks could be introduced during the process of neutralization which terminates the denaturation process. The undue stress at the fork during the winding could create a torque sufficient to break one or both strands of the unraveling double helix. Under Biochim. Biophys. Acta, 232 (I97 I) 234-245
DIFFERENTIALDENATURATIONOF DNA
245
either interpretation, the difference in marker sensitivity to denaturation under controlled conditions may prove to be useful for genetic manipulations.
ACKNOWLEDGEMENTS
The work is supported in part by a National Institute of Health, U.S. Public Health Service Postdoctoral fellowship to A. D. C. (FRo5445), and Research Career Development Award to P. C. H. (I-K3GM31-238-oI) and by a grant from National Science Foundation (GB 8058). We are grateful to William M. Swartz and Charles Braslow who have contributed, respectively, in defining some of the experimental conditions for thermal and alkaline denaturation reported here. We thank Dr. Roger M. Herriott for his encouragement. REFERENCES I 2 3 4 5 6 7 8 9
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(1967) 199. E. ]3. FREESE AND E. FREESE, Biochemistry, 2 (1963) 7o7 . M. AGENO, E. DORE AND C. FRONTALI, Biophys. J., 9 (1969) 1281. M. AGENO, E. DORE AND C. FRONTALI, Biopolymers, 9 (197 °) 116. S. H. GOODGAL AND R. M. HERRIOTT, J. Gen. Physiol., 44 (1961) 1229. R. M. HERRIOT~, E. MEYER AND M. VOGT, J. Bacteriol., IOI (197 o) 517. E. H. POSTEL AND S. H. GOODGAL, J. Mol. Biol., 16 (1966) 317 • S. H. GOODGAL AND E. H. POSTEL, J. Mol. Biol., 28 (1967) 261. E. H. POSTEL AND S. H. GOODGAL, J. Mol. Biol., 28 (1967) 247. G. L. MILLER AND R. H. GOLDER, Arch. Biochem. Biophys., 29 (195 o) 420. R. G. BATES, in H. A. SOBER, Handbook o[ Biochemistry, Chemical R u b b e r Co., 1968, p, JI9O. H. R. 1V[ASSIEAND B. H. ZIMM, Biopolymers, 7 (1969) 475. E. BURGI AND A. D. HERSHEY, Biophys. J . , 3 (1963) 309. F. W. STUDIER, J. Mol. Biol., I I (1965) 373. A. D. COOPER AND P. C. HUANG, J. Bacteriol., in the press. C. MULDER AND P. DOT~¢, J. Mol. Biol., 32 (1968) 423 . B. M. ALBERTS AND P. DOTY, J. Mol. Biol., 32 (1968) 379A. D. COOPER AND P. C. HUANG, Biochim. Biophys. Acta, (197 o) s u b m i t t e d .
Biochim. Biophys. Acta, 232 (197 I) 234-245