Isolation of high molecular weight DNA from Hemophilus influenzae

J. Mol. Biol. (1965) 11, 476-490

Isolation of High Molecular Weight DNA from Hemophilus influenzae K. I.

BERNS AND

C. A. THOMAS,

JR.

Biophysics Department, Johns Hopkins University, Baltimore, Md., U.S.A. (Received 19 October 1964) The nuclear DNA complement of Hemophilus injluenzae has been determined to be 700 to 800 X 10 6 daltons, An isolation procedure has been developed which gives high yields of DNA with a maximum molecular weight of 400x 106 , or one-half of the nuclear DNA complement. Linkage of previously unlinked markers has been obtained in transformation assays. The isolation procedure has been extended to Escherichia coli and Serratia marcescens.

1. Introduction Work in recent years has shown that all the genes of Escherichia coli are found in a single DNA-containing structure called the bacterial chromosome [Taylor, Gierer & Adelberg, 1960; Jacob & Wollman, 1961; Nagata, 1962,1963; Bonhoeffer & Gierer, 1963). The work of Yoshikawa & Sueoka (1963a,b) indicates that this is also true in the case of Bacillus subtilis. The autoradiographs of Cairns (1963a,b) provide the most direct evidence on the replicating E. coli chromosome, which is revealed as a circular DNA duplex. In order to develop this subject further and to open the way for the study of structural irregularities along the length of the bacterial chromosome, we have attempted to isolate, in high yield, intact bacterial DNA molecules. The major difficulty in such an approach is the sensitivity to. shear displayed by DNA because of its extended configuration (Davison, 1959; Burgi & Hershey, 1961,1962). Unfortunately, the procedures involving minimal shear which have been successful in the extraction of intact bacteriophage DNA's are not applicable to bacteria. Second, existing methods for the extraction of bacterial DNA's involve intolerable levels of shear, as is evident from the fact that such preparations rarely contain DNA with a molecular weight of greater than 2 X107 (Marmur, 1961; Cavalieri, Deutsch & Rosenberg, 1961). Therefore it was necessary to develop a new method for the extraction of high yields of high molecular weight bacterial DNA. Hemophilus injluenzae was chosen as the experimental organism because it has a small DNA content which would enhance our chances of success. A well-defined transformation system exists for Hemophilus (Alexander & Leidy, 1953; Goodgal & Herriott, 1961). The extraction of DNA of much higher molecular weight than previously obtainable should result in the co-transformation of previously unlinked markers. 476

HIGH MOLECULAR WEIGHT BACTERIAL DNA

477

2. Materials and Methods (a) Bacterial growth

A Hemophilus Rd strain resistant to streptomycin, 2500 /Lg/mi., cathomycin, 2'5 /kg/mi., erythromycin, 8/kg/mi., and viomycin, 150/kg/ml. was obtained from Dr R. Herriott. The cells were grown in Difco brain-heart infusion medium supplemented with NAD, 2 /kg/mI., and hemin, 10 ILg/ml. (Goodgal & Herriott" 1961). E. coli and Serratia marcescens were grown overnight with aeration in broth (10 g Bactopeptone; 5 g NaCI; I g glucose; 100 mi. I M-tris, pH 7·5; 10 mi. 0·10 M.MgS0 4 ; 874 ml, water; Burgi, personal communication). Bacterial nucleic acids were labeled by adding to each mi. of growth medium 32p, 10 to 20 /kc, or [3H]thymidine, 20 /kc. 32p was obtained from E. R. Squibb & Sons, New York; and [3H]thymidine was obtained from Schwarz Bioresearch, Inc., Orangeburg, N.Y. Bacteria were counted in the Petroff-Hausser bacterial counting chamber. (b) DNA isolation procedure

One-tenth mi. of an overnight culture was added to 10 mi. of supplemented brain-heart infusion medium and the bacteria allowed to grow into the log phase (I to 2 X 109/mI.). The bacteria were centrifuged, resuspended in brain-heart infusion medium minus NAD and hemin and incubated under these starvation conditions at 37°C for 1·5 hr. The cells were then washed twice with SSC (0,15 M-NaCI-0'015 sr-sodium citrate) and finally resuspended in 27% sucrose in SSC. The bacteria were lysed with 0'2% SLSt, pronase (Calbiochem, Palo Alto, Calif.) added to a final concentration of I mgjml., and the lysate digested 7 hr at 37°C. The lysate was extracted twice with an equal vol. of water-saturated phenol (chromatography grade, Mallinokrodt Chemical Co., St. Louis, Mo.) by rolling at 60 rev.jmin (Frankel, 1963). Next the aqueous layer was extracted once gently with an equal vol. non-anhydrous ether and the aqueous layer was dialyzed overnight versus SSC. (c) DNA-RNA determinations

Nucleic acids were precipitated with cold 0·6 N·PCA or 10% TCA. If only DNA was to be precipitated, the RNA was hydrolyzed in 0·5 N-NaOH at 37°C for 2 hr prior to acid precipitation. Precipitates were collected by filtration through a 1·2 JL Millipore filter. DNA phosphorus determinations were performed using a modification of the method of Fiske & SubbaRow (1925). DNA was hydrolyzed in I N-HCI at 100°C for 3 hr. The assay mixture contained 1·0 ml. hydrolyzed sample, 1·0 mi. molybdate-H2S0 4 (5,0 g ammonium molybdate + 200 mI. 5 N-H 2S0 4 ) , 0·25 mi. Elon-bisulfite (I g Kodak Elon + 3 g sodium bisulfite, 99 ml. distilled water), and 2·75 ml. water. The mixture was allowed to stand at room temperature 30 min before measuring optical density at 750 mu in the Zeiss M4 QUI spectrophotometer. (d) Protein determinations

1. Amino acid analyzer A DNA-RNA preparation was precipitated with cold 5% TCA and the pellet washed with absolute ethanol to remove the TCA. The pellet was dried overnight in vacuo and redissolved in 2·0 mi. 6·0 N-HCI. The solution was put into thick-walled glass tubing, heated to drive off air and sealed. The HCI solution was hydrolyzed (120°C) for 24 hr, the HOI exhausted, the residue resuspended in acetate buffer, pH 2, and assayed on the Spinco amino acid analyzer by Dr M. Young.

2. Colorimetric measurements A purified DNA-RNA preparation was concentrated using a Rinco evaporator (Rinoo Instrument Co., Greenfield, IlL) until it contained more than 50 JLg/mi. DNA. A 4-mi. sample was made 0·5 N in NaOH by adding 0·36 mi. 6 N-NaOH and digested for 2 hr at

t Abbreviations used: SLS, sodium lauryl sulfate; PCA, perehloric acid; TCA, tricWoroacetic acid; BSA, bovine serum albumin; H DNA, Hemophilus DNA; S, O. E and V, genetic markers for resistance to streptomycin, cathomycin, erythromycin and viomycin, respectively. 33

478

K. I. BERNS AND C. A. THOMAS, JR.

37°C. The tube was then chilled, 0·42 rnl, 12 N~PCA added, and the tube kept in ice for 15 min. The samples were centrifuged and the D 2 6 0 of the supematant solutions was measured to determine RNA. The pellet was resuspended in 0·60 ml, 0·2 N·NaOH and 0·10 ml. removed and diluted 20 times in SSC to measure D 2 6 0 for DNA. Control tubes containing known amounts of BSA and 312 p.gsalmon sperm DNA in 4·0 ml, were treated identically. The protein assay was performed according to the method of Lowry, Rosebrough, Farr & Randall (1951; Colowick & Kaplan (1957)), except that the amount of Folin reagent used was doubled. The resultant color was measured at 700 mp' in the Zeiss spectrophotometer. (e) Radioactivity assays Samples containing either 32p or 14Cwere counted in a Nuclear Chicago gas-flow counter. Samples containing two isotopes or 3H were either precipitated by acid, collected on Millipore filters, dried, and placed over nichrome wires in glase vials; or absorbed directly onto small squares of Whatman no. 1 fitter paper on the bottom of a plastic vial (Hershey, personal communication). Ten ml. fluor was added to each sample, which was then assayed in a Nuclear-Chicago scintillation counter. The fluor was prepared by adding 4·0 g 2,5 diphenyloxazole and 0'10 g 1,4 bis.2.(5.phenyloxazolyl).benzene (obtained from the Packard Instruments Corp., Lagrange, IlL) to 1'0 L toluene.

(f) Zone sedimentation in sucrose density-gradients Linear sucrose density-gradients from 5 to 20%, w/v, were produced using a replica of the mixing chambers of Britten & Roberts (1960). Sucrose solutions were made up in SSC. All runs were performed in the Spinco model L ultracentrifuge using the SW39 rotor at 20 to 25°C, 25,000 rev./min, 2i to 21 hr. If very fast sedimenting material was expected, the run was' of shorter duration. Following the run the tube was placed in a special holder with a small cannula at the bottom, through which a pin was pushed to puncture the tube. Pressure to regulate the flow was maintained by a manometer. Burgi & Hershey (1963) have presented evidence that the relative distances traveled by several DNA species in zone sedimentation through a sucrose density-gradient are equal to the relative 8 values ofthe DNA's: 81/82 = dl/d2. Further, they have defined an equation relating relative 8 values to relative molecular weights: 81/82 = (Ml/M2)0.36. Therefore, if one knows the molecular weight of a marker DNA, it should be possible to calculate the molecular weight of the other DNA present. We were able to reproduce the results of Burgi & Hershey for whole T2 and T5 DNA.

(g)

osa density.gradients

The procedure used was that of Thomas & Berns (1961). csm was obtained from the Harshaw Scientific Co., Cleveland, Ohio. (h) Transformation Assays were performed in the laboratory of Dr R. Herriott with the able assistance of Miss E. Yamashita, following the procedure of Alexander & Leidy (1953).

(i) Light micr08copy of bacteria with stadmed nuclei The general method of Smith (1950) was used in staining Hemophilu.s for light micro. scopy, One to 2 X 1010 bacteria were centrifuged onto agar, which was cut out of the tube and placed on glass beads over a solution of 2% OS04 for varying periods of time. Fixation for 20 min was most satisfactory. Following fixation, the cover slide was pressed down gently to make good contact and it was drawn across the agar. The cover slide was placed in 1 N·HCI for hydrolysis (25 to 35 min, 60°0), rinsed with distilled water, placed in 1% formaldehyde 2 min, rinsed, placed in basic fuchsin for 30 sec, rinsed and mounted in a drop of tap water. The coverslip was sealed on a slide with melted paraffin wax using a camel's-hair brush. Slides were observed using a Zeiss photomicroscope under

PLATE 1. Photomicrograph of bacteria with their nuclei stained. (See text for details.)

[facing p. 479

HIGH MOLECULAR WEIGHT BACTERIAL DNA

479

oil immersion (oil on the slide and on the condenser) at 2500 X with a green filter (Zeiss VG9). Photomicrographs were made using Panatomie X film (Eastman Kodak, Rochester, N.Y.).

3. Results (a) Nuclear DNA complement

1. Ohemical determination of the average amount of DNA per bacterium

The amount of DNA per cell was determined for different growth conditions. Assays were made on cells from overnight cultures (11 to 12 hours) and on cells from log-phase cultures which had been resuspended and incubated in unsupplemented (no NAD, no hemin) brain-heart infusion containing chloramphenicol (50 fLg/mI.) for 1·5 hours at 37°C.Mter being counted in the Petroff-Hauser chamber, the bacteria were digested for two hours at 37°C in 0·5 N-NaOH and the solution then made 0·6 N in PCA and chilled to precipitate the DNA. The amount of DNA phosphorus in the precipitate was determined after hydrolysis in boiling 0·6 N-PCA. Cells from overnight cultures contained the minimum amount of DNA, 2·1 to 2·7 X 10- 9 fLg/cell (1300 to 1600 X106 daltons), while the starved, chloramphenicol-treated, log-phase cells contained 8 to 10 X10- 9 fLg/ cell. 2. Average 7I:umber of nuclear areas per cell

Bacteria from overnight cultures with their nuclei stained were observed in the light microscope (Plate I). A large majority of the cells had two nuclear areas. This was corroborated by electron microscopic examination of the same cells stained with uranyl nitrate. Thus the nuclear DNA complement of bacteria in overnight cultures is about 725 X106 daltons, one-half the mean cell DNA content of 1450 X106 daltons. (b) Oomposition of DNA preparation

The recovery of DNA in the final preparation was usually 60 to 80%. Some DNA (0 to 15%) was found in the phenol layers, but about 90% ofthis could be re-extracted from the phenol with SSC. The remainder of the DNA was probably in interfacial material although little, if any, of this was apparent. In the final preparations the 32p was distributed equally in DNA, RNA and acid. soluble fractions. The colorimetric protein assay (Lowry et al., 1951) (Table 1) showed that on a weight basis there was 9% as much protein as DNA present. The amino acid analysis (Table 2), on the other hand, showed a protein content of 2'5%. All the amino acids can be detected with the exceptions of histidine, arginine, and 1/2 cystine. Glycine is the most common, but it is considered a degradation product of the purines (Bendich, 1955). Aspartic acid and serine were the next most abundant. Coupled with the fact that very little lysine was observed, the absence of histidine and arginine indicates that the residual protein is not histone. Possibly even less protein would be observed if a more severe phenol extraction were employed, but the chance of mechanical damage to the DNA would be greater. An important feature of the extraction is the digestion with pronase. The omission of pronase, a proteolytic enzyme with general specificity (Nomoto, Narahashi & Murakami, 1960a,b), from the procedure results in a large interfacial precipitate during phenol extraction which contains 95% of the DNA.

K. 1. BERNS AND C. A. THOMAS, JR.

480

TABLE

1

Protein determination using method of Lowry et al., 1951

'rube

Protein added (Ilg)

Protein present (Ilg)

Control series 1 0 2 0 3 5 4 20 5 40

312 312 312 312 312

Hemophilus sample H-l H-2 10

450 490

0 - 0·008 0·094 0·186 0·443

2700 2700

0·500 0·600

0 0 5 20 40 40 ± 3 48 ± 3

t RNA is material which is not acid- precipitable after OH digestion. PCA supernatant fraction.

TABLE

2

Amino acid analysis

Ilmoles

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic aeid Proline Glycine Alanine Half cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Total amino acids§

t Trace

=

Trace'[ None None 0·026 0·016 0·022 0·016 None 0·288 0·012 None Tracet Trace] Trace] Traeef Tracej Trace]' 0·140

% amino

acids/ nucleotide'[

0·46

o o

1·50 0·93 1·27 0·93

o

16·70 0·70

o 0·46 0·46 0·46 0·46 0·46 0·46 g'55

less than 0·01 Ilmole but detectable, assumed to be 0'0081lmole for calculation of

(% amino acids/nucleotide).

t

600 Ilg DNA present, assume 350 to be average nucleotide mol. wt, 600 X 10- 6 g = 1'72llmoles nucleotides 3 5 0 g / moe 1

§ Excluding glycine.

HIGH MOLECULAR WEIGHT BACTERIAL DNA

481

The presence of a high concentration of sucrose during the lysis has been suggested by Cairns (1962) so that the cells will not burst explosively, subjecting the DNA to high shearing forces. Omission of sucrose during lysis results in DNA with a maximum molecular weight only about one-third of that obtained when sucrose is present during lysis. (c) Characterization of the DNA

1. Sucrose gradients

We used sedimentation through sucrose density-gradients to characterize the DNA preparations. Figure 1 is a typical sedimentation pattern obtained when the final extraction procedure is used. A large part of the H DNA preparation has a higher S value than the T2 DNA marker. The trailing off ofthe H DNA distribution indicates r--,

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FIG. 1. Sedimentation pattern of [3H)H DNA in the presence and absence of [HC]T2 DNA. Presence of T2 DNA does not alter the pattern. H DNA extracted using final extraction procedure plus chloramphenicol. (__) 3H; ( ) HC.

heterogeneity, but it appears that none of the H DNA has a lower S value than T2 DNA. The peak of the H DNA distribution was considered to be a conservative choice of the midpoint of the distribution of the largest class of molecules. The equation of Burgi & Hershey (1963) has been used to estimate the molecular weight from the relative S values observed in the sucrose gradients. The relative S values

482

K. I. BERNS AND C. A. THOMAS, JR.

have ranged from 1·43 to 1·48 that of T2 DNA. This would indicate a molecular weight approximately 3 times that of T2 DNA (133 X 106 ) or about 400 X 106 • Since the average nuclear DNA complement is 725 X 106 daltons, a DNA molecule of 400 X106 daltons would represent one-half the nuclear complement. The extraction of overnight cells yields a preparation in which the very large DNA is still present, but there is considerably more heterogeneity, the H DNA distribution extending back to the meniscus (Fig. 2). The extraction oflog-phase cells gives results which are nearly comparable to those obtained when the cells are starved (Fig. 3),

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but the results were not so reproducible and the starvation step was retained in the final extraction procedure. Originally, the bacteria were also subjected to chloramphenicol, 50 p.g!ml., during the period of starvation, but starvation alone proved to be as effective in yielding preparations with a high S value and minimal heterogeneity.

osa density-gradients H DNA was banded in CsCI to see if it had the same density as the same DNA purified by the severe Marmur procedure (1961). In the model L ultracentrifuge, 97% of the [3H]H DNA was found in the band, which itself was superimposed on the admixed [14C]T5 DNA. This is as would be expected in view of their nearly identical base compositions (Belozersky & Spirin, 1960). In the analytical ultracentrifuge H DNA bands at'a density of 1·693 g/cm 3 • Schildkraut, Marmur & Doty (1962) have quoted a density of 1·698 for H DNA purified by the Marmur procedure (1961). 2.

HIGH MOLECULAR WEIGHT BACTERIAL DNA

483

g/cm 3

However, the same authors quote a density for T2 DNA which is 0'006 higher than we obtain for similar preparations, so it would appear that the differences reported for the banding position of H DNA are due to a systematic difference in the measurement of absolute density. Thus there is no protein associated with the H DNA obtained by our extraction procedure which is detectable by banding in OsCI.

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3. Tran8formation The cells from which the DNA was extracted were resistant to four antibiotics: streptomycin, cathomycin, erythromycin and viomycin. The transforming activity of the four single markers was assayed for a preparation of H DNA, a portion of which had been sheared through a no. 25 needle. Figure 4(a) shows the sedimentation pattern of the original preparation, and Fig. 4(b) shows the sedimentation pattern of the sheared DNA. From the relative sedimentation rates one may calculate that the original DNA is about 25 times larger than the sheared material. The results (Table 3) show that the unsheared DNA had a specific transforming activity which was about 25 to 50% greater. In other sedimentation experiments done on labeled H DNA, it was found that the specific transforming activity of each marker was constant over the sedimentation zone.

484

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FIG. 4. Sedimentation patterns of unsheared [3H]H DNA (a) and sheared [3H]H DNA (b) used for transformation assays. [ 140]T2 DNA is the marker. (--) 3H; (----) 14 0 .

TABLE

3

Comparison of transforming activity of sheared and unsheared H DNA Marker

s

Colonies counted unsheared sheared

E

252 426 565

V

513

o

200 286

440 353

Ratio unsheared :sheared 1·26 1·49 1·28 1·45

The large size of the DNA extracted by this procedure might lead one to expect the appearance of linkage between markers previously thought to be unlinked. This has proved to be the case with Sand E, which show a 50-fold increase in double transformants over the number expected on a random basis (Table 4). In normal DNA preparations Sand C are linked about 60% of the time. Here about 60% of the double transformants for Sand E were also resistant to C. While the increase of Sand E double transformants is significant, the frequency of their occurrence is very low. The demonstration of linkage between markers which occupy very distant

HIGH MOLECULAR WEIGHT BACTERIAL DNA TABLE

4

Transformation: linkage of

s, e and

Dilution from TF

Colony ets

E

1/15 1/ 15 1/ 15

SE

1/15 10°

284 690 631 171 19 10

Marker

S

a

sa

1/ 2

485

E markers Titer inTF

4·26 X 1·04 x 9·46 x 2·56 x

1()3 1()3 1()3 1()3 20

SOlS = 60%. Random doubles for E and S

~=

0·40

=

(4·26 xI03 ) (9,46 X 103 ) II i-ox i 08 ces

= 0·40.

50 X the numbers of SE doubles expect ed on a random basis.

positions on the DNA molecule is not likely, because the continuity of the molecule must survive many hazards: it must remain unbroken in the diluted transformation mixture, it must be completely taken up by a single cell, and finally, to produce a stable clone of bacteria, both markers must be incorporated into the recipient genome by recombination. (d) Problems of shear and DNase activity during extraction 1. Size of DNA in SLS lysate

If there were an active DNase present during the pronase digestion or if the phenol extraction were breaking the DNA, then DNA in the SLS lysate should have had a higher relative S value than DNA in the final preparation. Therefore, [3H]thymidinelabeled cells were lysed in 27% sucrose, dialyzed against sse, and sedimented without further purification through a sucrose density-gradient (Fig. 5). It can be seen that the relative S value of the H DNA from the lysate is not noticeably greater than that of the final preparation.

2. Passback: of T 2 and H DNA

To examine further the question of shear and DNase, labeled T2 DNA and H DNA of known size were added to the lysate in separate experiments during the extraction of cold bacteria. I n Fig. 6 it can be seen that T2 DNA still has a relative S compared to thcS ofT5 DNA of 1·20. Thus T2 DNA molecules are not broken during the extraction. The small amount of trailing material seen in this experiment was present in the original preparation. H DNA which had undergone two extractions (Fig. 7(b» presented nearly the same sedimentation pattern as the original preparation. (e) Extraction of DNA from E. coli and S. marcescens Extraction of DNA from E. coli and S . marWicens proceeded without any apparent difficulty. The bacteria were grown into the log phase (about 2 X108/m l.) and incubated

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FIG. 5. Sedimentation pattern of [3H]H DNA from an SLS lysate (27% sucrose present during Iysis.) [14C]T2 DNA is the marker. Run only 1·5 hr so that any rapidly sedimenting material could be observed. The H DNA has moved about 1·4 times as far as the T2 DNA. (--) 3H; ( ) 14C.

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Fraction no. FIG. 6. Sedimentation pattern of [32P]T2 DNA which was present during the extraction of cold Hemophilus, [14C]T5 DNA is the marker. dT2/dT5 = 1·20. Trailing material was present in the originalmaterial, (-_) 32p; ( ) 14C.

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1·5 hours in the presence of chloramphenicol, 50 J.LgJml., at 37°0. The DNA was t hen extracted using the same method as that developed for H emophilus, except that the cells were lysed by exposure to 1 % SLS at 60°0 for 10 minutes. The DNA yield from E. coli was 60 to 70% and from S . marcescens 75 to 87%. Again final prep arations labeled with 32p contained about equal amounts of DNA, RNA and acid-soluble material. The E. coli preparation cont ained 9% protein while t he preparation from S. marcescens contained 2 to 3%. Two prepara tions of DNA extra cted from E . coli were exami ned and found to have sedimentation distributions superimposed on t he T2 mark er DNA (Fig. 8(a)). The DNA from S . marcescens moved faster than T2 DNA and had an estimated molecular weight of 250 X 106 • The failure to achieve the higher molecular weights obtained for H DNA cannot be explained ; but in any event , the molecular weight of the DNA extracted by this procedure is considerably higher than that achieved by older methods.

488

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8

10 Fraction no.

FIG. 8. Sedimentation patterns of [3H]E. coli DNA (a) and [3H]Serratia DNA (b). [ 140]T2 DNA is the marker. (--) 3H; (----) 14 0 .

4. Discussion We have succeeded in extracting in high yield, from H. injluenzae, DNA which has a maximum molecular weight of about 400 X 106 , corresponding to half of the nuclear DNA complement. It seems likely from work with other bacteria that the genetic information of Hemophilus is contained on one chromosome. Our results make it unlikely that there are more than two chromosomes. The strength of our conclusions depends on the accuracy of the determination of the amount of DNA per nuclear complement and the validity of the extrapolation of the equation of Burgi & Hershey (1963) relating relative S value in a sucrose gradient to relative molecular weight. The chemical determination of the average amount of DNA per cell is in agreement with similar results from the laboratory of Dr R. Herriott (personal communication) and with results obtained with the electron microscope by measuring the contour length of DNA released from disrupted spheroblasts derived from overnight cells (MacHattie, Berns & Thomas, 1965). The number of nuclear areas per cell has been obtained from measurements in both the light

HIGH MOLECULAR WEIGHT BACTERIAL DNA

489

microscope and the electron microscope. We have assumed that the stained regions were indeed independent nuclear areas. We think that the higher S values ofH DNA are the result of the fact that they are longer, not more compact, structures. This is shown most directly from the electron microscopy of the DNA molecules taken from a purified preparation (MacHattie et al., 1965). The observed lengths fit the molecular weight values calculated from sedimentation very well. Further, the shear-fragility of these DNA molecules is in accord with an extended linear structure. Our goal was to preserve the integrity of as large a DNA molecule as possible, therefore the phenol extraction was performed very gently. Some protein was present in the final preparation and it is possible that a more vigorous phenol extraction would have removed it. The final preparation contained equal amounts of DNA, RNA and acid-soluble material. The DNA can be separated from the other two components by sedimentation through sucrose gradients or by adsorption to hydroxyapatite. Unfortunately, both procedures result in one to three breaks in the molecule. We believe this to result from increased fragility of the DNA when it is diluted the five- to ten-fold necessary to perform the above procedures. We thank Dr E. Moudrianakis, who observed Hemophilus stained with uranyl nitrate in the electron microscope. We are also indebted to Mr .T. Abelson for supplying us with labeled bacteriophage DNA and to Drs S. R. Suskind and P. Hartman for helpful discussions. Part of this work was supported by the National Science Foundation (GB.708). One of us (K. 1. B.) was aided by a Shell Oil Fellowship. REFERENCES Alexander, H. E. & Leidy, G. (1953). J. Exp. Moo. 97, 17. Belozersky, A. N. & Spirin, A. S. (1960). In The Nucleic Acid8, ed, by E. Chargaff & .T. N. Davidson, vol. 3, p. 307. New York: Academic Press. Bendich, A. (1955). In The Nucleic Acid8, ed. by E. Chargaff & .T. N. Davidson, vol. 1, p. 117. New York: Academic Press. Bonhoeffer, F. & Gierer, A. (1963). J. Mol. Biol. 7, 534. Britten, R . .T. & Roberts, R. B. (1960). Science, 131, 32. Burgi, E. & Hershey, A. D. (1961). J. Mol. Biol. 3, 458. Burgi, E. & Hershey, A. D.(1962). J. Mol. Biol. 4, 313. Burgi, E. & Hershey, A. D. (1963). Biophy8. J. 3,309. Cairns, .T. (1962). J. Mol. Biol. 4, 407. Cairns, .T. (1963a). J. Mol. Biol. 6, 208. Cairns, .T. (1963b). Oold Spr. Harb. Symp. Quant. Biol. 28, 43. Cavalieri, L. F., Deutsch, .T. & Rosenberg, B. H. (1961). Biophys. J. 1, 301. Colowick, S. P. & Kaplan, N. O. (1957). In Method« in Enzymology, vol. 3, p. 448. New York: Academic Press. Davison, P. F. (1959). Proc. Nat. Acad. Sci., Wa8h.45, 1560. Fiske, C. H. & SubbaRow, Y. (1925). J. Biol. Chem, 66,375. Frankel, F. R. (1963). Proc, Nat. Acad. Sci., Wa8h. 49,366. Goodgal, S. A. & Herriott, R. M. (1961). J. Gen. PhY8iol. 44, 1201. Jacob, F. & Wollman, E. L. (1961). Sexuality and Genetics of Bacteria. New York: Academic Press. Lowry, O. H., Rosebrough, N . .T., Farr, A. L. & Randall, R. .T. (1951). J. Biol. Chem, 193, 265. MacHattie, L., Berns, K. 1. & Thomas, C. A., Jr. (1965). J. Mol. Biol. 11, 648. Marmur, .T. (1961). J. Mol. Biol. 3, 208. Nagata, T. (1962). Biophqs. Biochem, Re8. Comm. 8, 348. Nagata, T. (1963). Proc. Nat. Acad. s«; Wash. 49, 551. Nomoto, M., Narahashi, Y. & Murakami, M. (1960a). J. Biochem., Japan, 48, 593.

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Nomoto, M., Narahashi, Y. & Murakami, M. (1960b). J. Biochem., Japan, 48, 900. Schildkraut, C. L., Marmur, J. & Doty, P. (1962). J. Mol. Biol. 4,430. Smith, A. G. (1950). J. Bact. 59, 575. Taylor, A. L., Gierer, A. & Adelberg, E. A. (1960). Genetics, 5, 1233. Thomas, C. A. & Berns, K. 1. (1961). J. Mol. Biol. 3, 277. Yoshikawa, H. & Sueoka, N. (1963a). Proc. Nat. Acad. s«, Wash. 49, 559. Yoshikawa, H. & Sueoka, N. (1963b). Proc. Nat. Acad. sa; Wash. 49, 806.