DNA replication in SV40 infected cells

J. Mol. Bid. (1970) 50, 549-568

DNA Replication

in SV40 Infected Cells

I. Analysis of Replicating

SV40 DNA

ARNOLD J. LEVINE, HYEN S. KANG

Department of BiochemicaEL3cience-qPrinceton University, P&c&n, N.J., U.B.A. AND FOSTER

E. BILIJSEIMER-~

Department of Biological Sciencea, Doug&8 Car.qu8, Rutgers University, New Brunswick, N.J., U.&A. (Received 16 October1969, and in revked form 21 January 1970) Ftadioisotope labeling has been used to analyze the replication of SV40 DNA in primary African green monkey kidney cells. The incorporation of [3H]thymidine into viral-specif?c DNA begins at about 16 hours tufter infection and reaches a maximum rate at 30 hours. Sedimentation of viral DNA through neutral sucrose gradients indicates that little or no supercoiled viral DNA (20 to 21 8) is synthesized during the Crst five minutes of labeling with [3H]thymidine (at 30 hr after infection). Instead, a viral DNA form that sedimented at 24 to 26 s is observed. The 3H-labeled 26 s DNA can be chased into viral supercoiled DNA by the addition of unlabeled thymidine to the culture. This viral precursor DNA, unlike mature viral closed circular DNA, is completely den&tumble in alkali and bands & a lighter density than closed circular SV40 DNA in an ethidium bromide-ce&m chloride equilibrium gmdient. Benzoylated-naphthoylated DEAE-cellulose column chromatography wa8 used to fractionate mature (21 S) and replic&.ng (26 S) SV40 DNA. Viral supercoiled DNA elutea from such a column between 0.66 and O-6 M-N&I. Under fhese same conditions, replicating SV40 DNA remains bound to the column and can be eluted later with caffeine. Electron micrographs of viral DNA obtained from the caffeine-eluded fraction of the column show replic&iug SV40 molecules with two branch points, three branches and no visible ends (circles). About 76% of these molecules have completed 90 to 96% of their replication.

1. Introduction During the past several years, information has accumulated about the mechanism of DNA replication for several different bacteriophages. In particular 93x174 and X have a stage in their replication cycle where double-stranded circular DNA is duplicated (Young BE Sinsheimer, 1964; Sinsheimer, Starman, Nagler & Guthrie, 1962). An analogous process is found for the papova viruses which, however, replicate their DNA in the mammalian cell nucleus. In the interests of broadening our understanding of DNA replication, and obtaining information of a comparative biochemical nature, a study of the mechanisms involved in SV40 DNA replication has been undertaken. t Present U.S.A.

address: Department

of Biologioel

Soienoes, Californie 649

State College, Calif., Pa. 15419,

550

A. J. LEVINE,

H.

S. KANG

AND

F. E. BILLHEIMER

Hirt (1966,1969) demonstrated that polyoma DNA replicates semiconservatively and that the replicating DNA molecule forms a structure with two branch points and three branches much like that observed with A (Tomizawa & Ogawa, 1968) and $X174 DNA’s &nippers, Whalley, & Sinsheimer, 1969). The nature of viral or cellular components that may be required for DNA replication, and the events that occur at the replication point, remain to be elucidated. In addition, the physical properties of the polynucleotide strands in replicating molecules are not well understood. In this paper, sedimentation analysis and column chromatography are used to determine some of the properties of replicating SV40 DNA. A precursor of mature viral DNA sedimenting at 24 to 25 s was the first viral-specific product labeled with short pulses of [3H]thymidine. This replicating form of SV40 DNA was fully denaturable in alkali and banded at a lighter density than mature viral DNA during ethidium bromide-cesium chloride equilibrium centrifugation. Little or no mature viral closed circular or supercoiled DNA was detected during the first five minutes after exposure of infected cells to [3H]thymidine. Following this initial lag period, and as the labeling time was increased, mature SV40 DNA molecules represented an increasingly larger proportion of the total amount of isotope incorporated. Benzoylated-naphthoylated DEAE-cellulose column chromatography was used to separate the mature (21 S) and replicating (25 S) forms of SV40 DNA. Electron micrographs of replicating SV40 DNA obtained from these columns show circular molecules with two branch points and three branches. This same type of replicating molecule has been observed in polyoma virus-infected cells (Hirt, 1969).

2. Materials and Methods (a) The SV40 large-plaque supplied by V. Defendi.

mutant

virus

was used throughout (b) T&&e

these studies.

This virus was kindly

culture

Monolayer cultures of primary African green monkey kidney cells? (Flow Laboratories) were grown on 100 mm x 20 mm plastic Petri dishes in Dulbecco’s modified Eagle’s medium (Grand Island Biological Co.) supplemented with 10% calf serum. (c) Preparation

of virus

Monolayer cultures were infected with SV40 at an input multiplicity of 25 to 100 plaqueforming units per cell in 2 ml. of Dulbecco’s medium with 2% calf serum. After 2 hr adsorption at 36°C an additional 10 ml. of medium was added. After 6 to 8 days the cells were scraped from the Petri dish surface, centrifuged out of suspension and resuspended in 0.1 vol. of PO,-buffered saline (0.15 M-NaCl in 0.01 a6-sodium phosphate buffer at pH 7.5). The cells were sonicated for 5 min with a Branson sonifier at 4°C. These stocks were titrated and stored at -20°C. (d) Infectivity

assay

Infectious virus was titrated on monolayers of monkey procedure described by Gilead & Ginsberg (1965) as modifled (e) PurQkation The procedure that was followed Crawford & Crawford (1964), except employed. t These will be called monkey

of SV40

cells using the plaque assay by Levine & Ginsberg (1967).

virus

in the purification was first described by Black, that trypsin treatment of the pelleted virus was not

cells in this paper.

REPLICATING

SV40 DNA

551

(f) Cen&i&g&on lechnipe-s was performed by aedimenting O-2 ml. of the sample through a 6 to 20% linear sucrose gradient (1 M-NaCl, 0.01 M-Tris buffer at pH 7*2,0.01 MEDTA) for 3 hr at 40,000 rev./min in an SW60.1 rotor. Samples were collected through a hole punctured in the bottom of the tube onto Wbatman 3MM filter pads (2.3 cm). The filter pads were dried, washed twice with cold 10% trichloroacetic acid and once with cold acetone. The dried filters were placed in scintillation vials, a toluene spectrafluor(Amersham Searle) mixture wss added, and the samples counted in a Beckman liquid-scintillation counter. (g) Measurements of viral DNA synthesis Cells were infected aa described for the preparation of virus stocks. All experiments were performed 30 hr after infection unless otherwise noted. The rate of DNA synthesis was measured by the addition of [3H]thymidine (14.2 to 17.9 c/m-mole) (New England Nuclear Corp.) to infected cells. All of the radioactivity incorporated into trichloroacetic acidprecipitable material remained precipitable after treatment with 0.3 M-KOH for 18 hr at 37°C. This same material became acid-soluble after treatment with DNase (Worthington) for 2 hr at 37°C. Viral and cellular DNA were fractionated by the procedure described by Hirt (1967). At the end of the labeling period the medium was discarded, and the monolayers were washed twice with 10 ml. of cold PO&-buffered saline. The cells were lysed by the addition of 1 ml. of 06% dodecyl SO4t in 0.01 M-Tris buffer, pH 6.6, and 0.01 Mdisodium EDTA. The samples were gently scraped from the plates into centrifuge tubes and NaCl added to a 6nal concentration of 1 rd. These samples were kept at 4°C for 6 to 18 hr. The large molecular weight (cellular) DNA precipitated and was centrifuged out of SOI supernatant suspension at 12,000 x g for 30 min. A portion of the 1 M-NaCldodecyl was used to analyze viral DNA replication. To demonstrate that the great majority of [cH]thymidine incorporated into trichloroacetic acid-precipitable material in the NaCl-dodecyl SOr soluble fraction was indeed viral DNA the following experiment was performed. One infected and one uninfected Petri dish of monkey cells (3 to 6 x lo6 cells) were labeled with [3H]thymidine for 1 hr at 30 hr after infection. O-2 ml. of the NaCl-dodecyl SO*-soluble fraction was layered onto a 6 to 20% linear sucrose gradient and centrifuged at 40,000 rev./min for 3 hr in an SW60.1 rotor. Samples of the gradient were collected and analyzed as described below. Figure 1 presents the data obtained for this experiment. Greater than 90% of the acid-precipitable radioactivity cosediments with a 3aP-labeled super-coiled viral DNA sedimentation marker (see arrow). A small proportion of the label sediments slightly faster than the viral DNA. The uninfected cells contain little or no radioactivity in the region of viral DNA. These same samples sedimented through alkaline gradients indicated that greater than 90% of the acid-precipitable radioactivity was found in rapidly sedimenting closed circular viral DNA. (h) Purified &al DNA Closed circular viral DNA was obtained by the following procedure. Infected cells were washed with cold P&-buffered saline snd lysed by the addition of 1 ml. of O*S”Jododecyl SO4 in 0.01 I+Tris buffer, pH 6.6 and 0.01 Br-disodium EDTA. The viral DNA was then fractionated from large molecular weight cellular DNA by the procedure described by Hirt (1967). The small molecular weight fraction (viral DNA) was extracted with redistilled phenol saturated with 1 M-NaCl, Tris buffer (0.01 Y, pH 7.2) and 0.01 M-EDTA. The phenol was removed by dialysis against 0.01 a6-NaCl, 0.01 M-Tris-HCl (pH 7.2) and 0.01 YEDTA. This DNA was then heated for 4 min at 100°C and rapidly chilled in an ice bath. The NaCl concentration of this solution was adjusted to 0.3 M and the solution put on a benzoylated naphthoylated DEAE-cellulose column. Closed circular (non-denaturable) DNA was eluted with 1 m-Nacl. The denatured DNA remained on the column. Velocity centrifugation through neutral or alkaline sucrose gradients demonstrated that the viral DNA obtained from the 1 a6-NaCl fraction of the column was identical in its sedimentation properties to closed circular DNA isolated from purified SV40 virions. This procedure is a modification of that first used to isolate #X174 RF1 (Kamano & Sinsheimer 1968).

Sucrosegradient centrifugation

t Abbreviation

used: dodecyl SO,, sodium dodecyl aulfata.

552

A. J. LEVINE,

H.

S. KANG

AND

F. E. BILLHEIMER I

I

(a)

+

0

%MW~~a.*.W~~.8..88...~ I I IO 20

I 30

1

Fraction no. Fro. 1. Velocity sedimentation through a sucrose gradient of the 1 r,r-NaCl-dodecyl SO, supernatant from en infected and an uninfected monkey cell culture. One infected and one uninfected cell monolayer culture were labeled with [3H]thymidine (10 PC/ml.) for 1 br at 30 hr after infection. A 0*2-ml. sample of the 1 ar-N&l-dodecyl SO, supernatant was sedimented through a 6 to 20% linear sucrose gradient for 3 hr at 40,000 rev./min in a SW50.1 rotor. Samples were collected and counted as described in Materials end Methods. The arrow indicstes the position of a 3aP-18beled viral supercoiled merker DNA. The curve shows eH cts/min (a) plus SV40; (b) minus SV40. (i) DNA-DNA

hybridization

DNA-DNA hybridization was performed with the modifications described by Aloni, Winocour & Sachs (1968) of the procedure first used by Denhardt (1966). It was used to demonstrate that the large majority of [3H]thymidine incorporated into the NaCldodecyl SO,-soluble fraction was viral specific. At 20 hr after infection a culture was labeled for 1 hr with [3H]thyrnidine and the viral and cellular DNA’s were fractionated into 1 M-NaCl-dodecyl SO,-soluble and precipitable material as described by Hirt (1967). These two fractions were phenol-extracted and sonicated as described by Aloni et al. (1968). The results of DNA-DNA hybridization employing either viral supercoiled DNA or host cellular DNA (monkey cell) on the l?lter are given in Table 1. The controls with purified viral supercoiled DNA hybridized with viral or host cell DNA indicate the extent of crossreaction between viral and host DNA that is to be expected under these conditions. The degree of hybridization between the NaCl-dodeoyl SO4 supernatant fraction and viral or host cell DNA indicates that a minimum of 90% of the NaCl-dodecyl SO,-soluble DNA is viral specific. Additional experiments utilizing DNA labeled for 5 or 10 min with [3H]thymidine (but not sonicated) indicate that labeled DNA obtained from the NaCldodecyl SO,-soluble fraction after a short pulse is at least 90% viral specific (Table 1). (j)

Viral

DNA

for column c?rrorna$ography

Viral DNA was obtained by extracting the NaCl-dodecyl SO4 supernatant with redistilled phenol saturated with 1 m-NaCl, 0.001 na-EDTA and Tris-HCl buffer (O-01 M. pH 7.2). The phenol was removed by dialysis against 0.3 M-NaCl, 0.001 M-EDTA and 0.01 M-Tris-HCl buffer at pH 7.2.

REPLICATING TABLE

SV40 DNA

553

1

DNA-DNA hybriokatim of viral and ceUdar DNA with the 1 M-NaCl do~?&ylSO, swpemzcltant and precipitate fraction.8 Source of DNA

Virus supercoiled 1 n-NeCl-dodecyl SO1 SU$X3l’U8t8Ut

Monkey cells 1 br-NaC%dodecyl SO, precipitetc

Totel radioeotivity (ots/min)

Bound to sltert( %) Viml

Monkey oell

No DNA#

4170

33.3

2.7

o-45

77,420 16,420

30.0 2.4

3.2

19.2

0.13 0.23

21,206

3.0

20.1

0.41

4660

22-o

1.9

0.14

10,460

16.6

l-4

0.09

Not eonicated 1 E6-NeCl-dodccyl SO4 supermbtant ; S-min pulse 1 r.rn-NaCl+lodccyl SO1 supernatant; IO-min pulse

t Viral and monkey cell DNA, 20 &llter. $ Backgrounds not subtracted from o/0bound velues. Details of the procedures 8rc given in the Materials end Methods section.

(k) Benzoylabd mphthoylded DEAE-celldose chromatogmphy This cellulose preparation was synthesized by the procedure of Gillsm et al. (1967) as modified by Sedat, Lyon t Sinsheimer (1969). Viral DNA was obtained from the NaCIdodecyl SO4 supernatant fraction and was extracted at least twice with redistilled phenol as described above. The purified viral DNA in O-3 rd-NaCl, 0.001 M-EDTA and 0.01 MTris-HCl buffer (pH 7.2) was allowed to adsorb onto the column (1 cm x 1 to 2 cm) at a flow rate of 0.6 to 1.0 ml./min at 23°C. The column was then washed with 10 vol. of the same buffer. A gradient of 0.3 to I.0 M-NaCI was passed through the column at a flow rate of about 0.5 to 1.0 ml./min. Samples of 2.0 or 2.4 ml. were collected. When the column had equilibrated with 1 M-NaCl a gradient of 0 to 2% caffeine in 1 m6-NaCl, 0.001 MEDTA and 0.01 rc-Tris-HCI buffer (pH 7.2) was passed through the column. This procedure ~8s followed by a stepwise elution with 8 ~-urea and 8 M-urea plus 1 y. dodecyl SO1 (in 0.001 M-EDTA and 0.01 M-Tris-HCI buffer at pH 7.2). 100 pg of bovine serum albumin were added as a carrier and all samples were precipitated with 10% trichloroaoetic acid. The precipitates were collected on Whatman glass filter pads (2.3 cm) and the radioactivity ~8s measured as previously desoribed. (1) Electron tiogrcvpha of DNA The miorographs were obtained using a modified procedure of Kleinschmidt t Zahn (1969). Purified DNA was diluted to 1 to 2 pg/ml. in 2 M-FUIMIO~~UJI acetate and 0.01% cytochrome c. This mixture was allowed to flow down a vertical glass rod onto a hypophase of ice-cold distilled water. Samples were obtained by touching a Parlodion-coated carbonstabilized too-mesh grid to the monolayer. These grids were dehydmted for 10 set in absolute ethanol and drained dry on lens paper. The grids were then rotary shadowed for 4 min with a platinum-iridium (80:20) alloy at an angle of 6’. Grids were scanned and photographed with an RCA EMU-3G electron microscope. Measurements of the lengths of DNA molecules were obtained from tracings of these molecules using a calibrated map measurer.

554

A. J. LEVINE,

H.

S. KANG

AND

F. E. BILLHEIMER

3. Results (a) Kinetics

of viral DNA

synthesis

In order to establish t’he optimum time period for studying viral DNA replication, an experiment was performed to determine the rate of incorporation of [3H]thymidine at various times after infection. Eight monolayer cultures of monkey cells were infected (time, 0 hour) as described in the previous section. At 3, 9, 15, 24, 30, 45, 55 and 72 hours after infection a one-hour exposure to [3H]thymidine was administered to one of the eight plates. The NaCl-dodecyl SO, supernatant fraction was centrifuged through a 5 to 20% linear sucrose gradient and the total number of counts per minute sedimenting in the 20 to 21 s region determined. The results of this experiment are shown in Figure 2. In a similar experiment the cells were harvested, sonicated, and assayed for infectious virus. These data are also presented in Figure 2. [3H]Thymidine

-8

r; -7 .eln 5 6 F -5 ‘iz & -4 8 f x-x 0

P 20

I 40

I 60

I 80

Time (hr) Fro. 2. The kinetics of synthesis of SV40 DNA and virus production in monkey cells. Eight monolayer cultures of cells were infected and pulse-labeled with [sH]thymidine (5 pc/ml.) for 1 hr at the times indicated. The 1 a4-NaCl-dodecyl SO1 supernatants from each time period were sedimented through sucrose gradients as described in the Materials and Methods section and the quantity of radioactive label present in the 20 to 21 8 region of the gradient plotted. In a separate but similar experiment, each monolayer culture was harvested and titrated for plaquc- X-X -, Viral DNA 3H cts/min; -O-c)-, forming units/ml. as described previously. plaque-forming units/ml.

incorporation into viral DNA first began at about 15 hours after infection and rose to a maximum rate of synthesis at 30 hours. Thereafter the rate of incorporation of isotope slowly declined. The production of infectious virus was first noted at 24 hours and continued to increase for at least 72 hours. An average yield of 300 to 500 plaqueforming units per cell was usually obtained. (b) Velocity sedimentation analysis of replicating 5840 DNA To examine the sedimentation behavior of replicating SV40 DNA the following experiment was performed. Four monolayer cultures of monkey cells were infected with SV40 virus and 30 hours later a culture was labeled with r3H]thymidine for 2.5, 5.0, 10.0, or 20 minutes. A sample of the NaCl-dodecyl SO, supernatant was sedimented through a linear 5 to 20% sucrose gradient for three hours at 40,009 rev./min. The results of this experiment are presented in Figure 3. For labelling periods of 2.5 or

REPLICATING

SV40

666

DNA

5 min

-

2 \E s r

IO min 30002000 IOOO0

l -’

20 min

8000 6000 -

Fraction no. FIQ. 3. Sedimentation of SV40 DNA synthesized after short pulses of [eH]thymidine. At 30 hr after infection monkey cell monolayer aultures were pulse-labeled with [8H]thymidine (100 at/ml.) for 2.6, 6.0, IO.0 and 20 min. 0.2-n& samples of the 1 r.r-NaCl-dodeoyl SO, eupernatant fraatione were sedimented through sucrose gradients and analyzed ae described in the text. The arrow indicates the peak position of a 31P-labeled SV40 DNA sedimentation marker. The ourve shows 3H ots/min.

5.0 minutes the [3H]thymidine was incorporated into material that sedimented between 24 to 25 s as measured by reference to a 3aP-labeled viral DNA sedimentation marker (see arrow). After 10 minutes, in addition to the 25 s peak a shoulder w&s observed co-sedimenting with vim1 supercoiled DNA. By 20 minutes of labeling, two distinct peaks were seen at 26 and 21 S. The 25 s peak is about twice as broad as the viral supercoiled DNA peak and frequently has a sharper leading edge than trailing edge. As the labeling time is increased (past 20 min) the percentage of 3H-labeled material sedimenting in the position of supercoiled DNA increases (see Fig. 1). The 25 s DNA could be sedimenting faster than supercoiled DNA because it has protein or RNA associated with it. To examine this possibility the NaCl-dodecyl SO, supernatant was treated with either pronase (1 mg/ml.), or trypsin (I :250) for 30

566

A. J. LEVINE,

H.

S. KANB

AND

F. E. BILLHEIMER

minutes at 37°C in 0.5% dodecyl SO, and then sedimented as usual. No change in either the sedimentation rate or shape of the 25 s peak was observed. Indeed, phenol extraction of the NaCl-dodecyl SO, supernatant did not change the properties of 25 s DNA. RNase (5 pg/ml. for 30 min at 37°C) treatment of the phenol-extracted 25 s material had no effect on its sedimentation behavior. (c) Conversion of 25 s DNA to viral swpermiled DNA The kinetic experiment presented in Figure 3 suggests that [3H]thymidine is first incorporated into 26 s DNA and then is found in mature viral DNA. To see if the 25 s DNA is really a precursor of the viral supercoiled DNA a pulse-chase experiment was performed. At 30 hours after infection two monolayer cultures of monkey cells were pulse labeled with [3H]thymidine for one minute. One culture was lysed with 0.6% dodecyl SO, to atop the pulse. The medium from the second culture was discarded and the cells were washed three times with warm medium containing unlabeled thymidine (0.2 mM). Warm medium plus unlabeled thymidine was added to the cells and the monolayer was incubated an additional hour at 37°C. At the end of this time, the chase was terminated by the addition of 0.6% dodecyl S04. The NaCl-dodecyl SO, I

I

I

I

I

I

I

I

200 -

(a)

IOO-

(b

Fraction

no.

FIG. 4. Velocity sedimentation of SV40 DNA synthesized after 8 1-min pulse and a I-min pulse plus a 1-hr chase. At 30 hr after infection 2 monolayer cultures of monkey cells were pulse-labeled with [3H]thymidine (100 &ml.) for 1 min. One culture was lysed with O*So/0dodecyl SO1 while the second culture was weshed and incubated with unlabeled thymidine (0.2 man). After 1 hr at 37°C the chase was terminated. Samples of the 1 M-NaCl-dodeoyl SO1 supernatent fraction were sedimented through a suoroee gradient aa desaribed in the text. The arrow indicates the peak position of a 3ap-18beled sedimentation merker of SV40 supercoiled DNA. The curve shows aH ote/min 8fter (8) 8 I-min p&e; (b) 8 I-min p&e followed by I-hr oh8se.

REPLICATING

SV40 DNA

667

supernatants were obtained as usual and O-2 ml. of each was centrifuged for three hours at 40,000 rev./min through a 5 to 20% linear sucrose gradient. The results of this experiment are presented in Figure 4. During the one-minute pulse with [“H]thymidine, the radioactive label was incorporated into the 25 s material. After a one-hour chase most of the label is found cosedimenting with viral supercoiled DNA. A leading shoulder is seen after the chase period. This may be due to some labeled viral DNA entering the replicating pool as a template for DNA synthesis. In addition it can be seen that the chase was not 109% effective in ss much as a small amount of [3H]thymidine continued to be incorporated after the chase. Despite these problems, it is clear that the great majority of 25 s DNA is converted to a form that sediments like mature viral DNA. (d) Alkdine

velocity sediment&on an&y&

The previous results demonstrated that [3H]thymidine is first incorporated into a 25 s DNA for a five-minute period and then is altered so as to sediment like supercoiled viral DNA. If 25 s material represents replicating SV40 DNA, it would replicate semi-conservatively (Hirt, 1966; Levine, unpublished data for SV40) and therefore should be irreversibly denstured in alkali. On the other hand the eH-label observed after a ten-minute pulse sedimenting in the supercoil region should be dosed circular DNA and therefore sediment very rapidly in alkali. To test these possibilities, three cultures of monkey cells which had been infected 30 hours previously were pulse-labeled with [“H]thymidine for 5-, lo- and 20-minute periods. The NaCl-dodecyl SOc supernatent was adjusted to 0.1 M-NaOH and incubated for ten minutes at 37°C. A 0.2-ml. sample was then sedimented through a 5 to 20% elkaline sucrose gradient for l-5 hours at 40,000 rev./mm The results of these experiments sre presented in Figure 5. After a five-minute pulse, little or no DNA sediments like the SV40 DNA marker (see arrow). By ten minutes of 3H-labeling a small peak can be observed at the position of closed circular vim1 DNA. The majority of viral DNA is still at the top of the gradient. Twenty minutes after exposure to [3H]thymidine both open and closed circular DNA can be observed. The kinetics of formation of supercoiled DNA observed in alkaline velooity centrifugation agrees well with the results presented in Figure 3. As an independent measure of the time-lag observed in the formation of closed circular DNA (mature viral DNA), the experiment described above was repeated but the NaCl-dodecyl SO4 supernatrtnt fraction was analyzed by ethidium bromide-cesium chloride equilibrium centrifugation. Viral DNA synthesized during & five-minute pulse-labeling period banded at a lighter density than an SV40 closed circular DNA centrifugation marker. After ten minutes of labeling, closed circular DNA was present in the sample and by 20 minutes exposure to the isotope the majority of viral DNA banded at the same position as the SaP-labeled SV40 DNA centrifugation marker. The kinetics of appearance of closed circular DNA were similar to those observed in Figurea 3 and 5. (e) Beiwviw of mature and pulse-labeled viral DNA on benzoylatechvp~~y~ed DEAE-celluhe columns This type of column chromatography has been used to separate resting and replicating double-stranded circular lambda DNA (Kiger & Sinsheimer, 1969). This same ohromatographic technique was therefore used to obtain larger quantities of

558

A. J.

LEVINE,

H.

S. KANG

I

AND

I

F.

E.

BILLHEIMER

I

2000 -

5 min

t

20 min

I

IO

20

30 Tube no.

FIG. 5. Velocity sedimentation of SV40 DNA pulse labeled for 5, 10, or 20 min with [3H]thymidine through an alkaline SUC~OSCgradient. Samples of pulse-labeled SV40 DNA were obtained as in Fig. 3. A sample of the 1 Ma-NaCl-dodecyl SO4 supernatant was incubated in 0.1 M-NaOH for 10 min at 37°C. This sample was then sedimented through an alkaline sucrose gradient (0.1 m-NaOH, 0.9 ~-N&cl, 0.001 M-EDTA) for 1.5 hr at 39,000 rev./min in an SW39 rotor. The arrow indicates the peak position of a 3aP-labeled super-coiled SV40 DNA marker. The curve shows “H cts/min.

replicating SV40 DNA and to improve the separation between supercoiled viral DNA (21 s) and replicating DNA (25 s). When purified viral supercoiled DNA was placed on this column, 9.5% of the DNA remained bound to the column. Of this, 92% of the supercoiled DNA eluted at, a salt concentration between 0.55 and 0.60 M-NaCl. An additional 2 to 3% of the supercoiled DNA wi~8 eluted with a 2% caffeine solution. The remaining 5 to 6% of the DNA was removed from the resin with an 8 M-urea and 1% sodium dodecyl sulfate solution. Using these procedures, recoveries of supercoiled or replicating DNA were always close to 100%. To examine some of the properties of pulse-labeled SV40 replicating DNA by column chromatography, the following experiment WLLF~ performed. Four monolayer cultures of SV40-infected monkey cells were labeled with [3H]thymidine for 2.5, 5.0 10.0 and 20.0 minutes at 30 hours after infection. The 1 M-NaCl-dodecyl SO, supernatant fractions were extracted with phenol and dialyzed against 0.3 M-N&I, 0~001M-

REPLICATING

SV40

559

DNA

EDTA in 0.01 M:-Tris-HCl buffer at pH 7.2. The labeled DNA was placed on the column and eluted as described in the Materials and Methods section of this paper. The elution profiles of the trichloroacetic acid-preoipitable [“Hlthymidine obtained from these experiments are presented in Figures 6 and 7. For the 24% and &O-minute pulses (Fig. 6) the great majority of radioactivity eluted early in the caffeine gradient. By ten minutes (Fig. 7) a pronounced peak was also present which eluted like viral supercoiled DNA (at 0.57 nn-Nacl). After 20 minutes of labeling, this DNA species represented the majority of DNA in the sample. Material labeled with [3H]thymidine representing up to 5% of the total radioactivity incorporated was eluted with 8 Murea or 8 M-urea plus 1% dodecyl SOc. In addition, trichloroacetic acid-precipitable 3H-labeled isotope is frequently observed eluting between O-6 and 1.0 M-NaQ. It should be pointed out that these latter species represent radioactive components of I 0.3 to I.OwNaC

Un

) to 2% Caffeine

1000 800 600 400 -

2400 2000 1600 I200 800 400 Sk 80

0 1

IO

20

30

90

100

Fraction no. FIG. 6, Benzoylated-naphthoylated DEAE-oellulose cbrometogrsphy of viral DNA pulselabeled with [aH]thymidine for 2.6 min (a) and 6.0 min (b). At 30 hr after infection of two monolayer cultures of monkey cells, [gH]thymidine (10 pa/ml.) wan added to each oulture for 2.6 or 6.0 min. The pulse labeling wee stopped by the addition of 0.6% dodeoyl SO1. The 1 M-NeCl-dodecyl SOI supernatant freotion we8 phenol-extra&ad and ohromatographed on 8 column aa described in the text. The ourve shows SH cts/min/%-ml. fraotion.

A. J. LEVINE,

660

H.

5. KANG

AND

F. E. BILLHEIMER 1+Um

40

I

1

I

I

8 M-Urea + 1% dodecyl SO4 (a)

30

20

IO r 0 x 2 0 \ :: 100 Y

3

(b)

PO807060x

50403020IOou5>N 1

IO

20

30

Fraction no. FIG. 7. Benzoylated-naphthoylated cellulose chromatography of viral DNA [3H]thymidine for 10 min (a) and 20 min (b). The experimental details are &s given in Fig. 6. 3H cts/min/t-ml. fraction.

pulse-labeled

with

less than 10% of the total 3H incorporated and therefore one cannot be sure they are virus specific. To determine if the caffeine fraction contains a precursor of viral supercoiled DNA, a puke-chase experiment similar to the one described in Figure 4 was performed. After a ten-minute puke with [3H]thymidine, 66% of the 3H label was found in the caffeine fraction, while after a two-hour chase with cold thymidine only I@/$ of the 3H-labeled isotope eluted with caffeine. Concomitantly there was an increase in the radioactivity which could be eluted with 1-O M-Nacl (from 29 to 79%) after the twohour chase period. The quantity of radioactivity found in the 8 M-urea-l% dodecyl SO, fraction could be reduced from 5 to 3% with a two-hour exposure to unlabeled thymidine. These data indicate that the DNA eluting after the addition of caffeine is a precursor to the DNA which can be removed from this column with 1 M-NaCh

REPLICATINQ

SVCO DNA

561

(f) Rechromatography of viral DNA on benzoylated-nuphthoylated cellulose To determine the specificity and reproducibility of this fractionation procedure, the viral DNA from the 1-Ora-NaCl, 2% caffeine td ure+clodecyl SO, fractions were rechromatqraphed on a second similar column. The results of this experiment are presented in Table 2. About 90% of the viral DNA obtained from the 1-O M-NaCl TABLE 2

Rechromdography of viral DNA on benzoyladed-nuphthoylated cellulose column-s

DNA from first column

DNA in fraotion from seoond oolumn (% ) se1t Cf&ille Urea-dodeoyl so4

salt

87

Ceffeine Urea-dodecyl

19 35

SO4

13 81 66

0 0 9

Viral DNA obtained from a oolumn like that shown in Fig. 7 ww preoipitated with ethanol, dialyzed and chromatographed on a seaond, similar oolumn to determine the apeaifioity of this fractionation proaedure.

fraction of the 6rst co1um.nrechromatographecl in an identical manner. About 80% of the DNA elutd with 2% caffeine also eluded in the ceEeine fraction of the second column. In contrast, the DNA obtained with the urea-dodecyl SO4 eluted procedure did not rechromatograph in the urea-dodecyl SO, fraction, but instead was eluted with 1 M-Nacl (one-third) and 2% oaffeine (two-thirds). (g) Sedimeti~ion analysis of DNA from the salt, caffeine and urea-&&y1 fradions of the benzoylided-nxqMq&td cellulose column

SO,

Sedimentation velocity analysis of replicating SV40 DNA indicated that viral DNA labeled for short periods sedimented at 24 to 25 s ad that this component w&8 a precursor to mature supercoiled viral DNA. To determine the relationship of the caffeine DNA to 25 s DNA, the salt, oaffeine and uretdodecyl SO4 fractions from this column were sedimented through a linear 6 to 20% sucrose gradient (neutral pH) for three hours at 40,000 rev&in. The sedimentation proties of the 3H-labeled viral DNA are presented in Figure 8. The great majority of DNA obtained from the 1-O M-salt fraction of the column co-sedimented with a 3aP-labeled supercoiled, viral marker DNA (see arrow). The DNA which elutd with 2% caffeine sediment& ahead of the supercoiled DNA marker with a peak at about 25 8. The viral DNA that was eluted with urea-dodecyl SO4 sedimented aa a mixture of the two components observed in the salt and caffeine fractions. The sedimentation profiles of the caffeine and salt fractions from the column sedimented in an alkaline sucrose velocity gradient are presented in Figure 9. Most of the 3H-label that eluted in 1-Om-salt co-sedimented rapidly with a 3aP-labeled supercoiled viral DNA marker. The viral DNA obtained from the 2% caffeine fraction of this column sedimented through the alkaline sucrose gradient w a broad peak of about 16 to 20 8.

562

A. J. LEVINE,

H.

S. KANG

AND

Fraction

F. E. BILLHEIMER

no

FIG. 8. Velocity sedimentation through neutral sucrose grrtdients of vir81 DNA eluted from 8 benzoylated-nephthoyleted cellulose column with 1 x-NaCl (8); 2% csffeine (b) ; or 8 Ilr-uretl-1 y0 dodeoyl SOa (c). SV40 DNA obtained from 8 oolumn similer to that presented in Fig. 7 was sedimented through 8 linear 5 to 20% neutral sucrose gradient for 3 hr 8t 40,000 rev./min in 8n SW50.1 rotor. The 8rrow mctrker. The curve indicetes the position of 8 3aP-18beled SV40 superooiled DNA sedimentation shows 3H cts/min in vir81 DNA obtained from the column.

(h) Electron micrographs of viral DNA

prepared from the benzoykzted-naphthoylated DEAE-cellulose columns

The results of the previous experiments indicate that mature viral supercoiled DNA is separated from replicating viral DNA by chromatography on this type of column. In order to visualize the replicating SV40 molecules, a viral DNA sample from the caffeine fraction of one of the columns was prepared for electron microscopy by the techniques described in the Materials and Methods section. To prepare enough DNA for electron microscopy, the procedures used previously were scaled up tenfold (5 x lo7 infected cells). It should be noted that when larger quantities of viral DNA were chromatographed, about 10 to 20% of the supercoiled DNA, which is usually found in the 1.0 M-Nacl fraction, contaminated the DNA that elutes with 2% caffeine. The reason for this is not clear. Selected electron micrographs of viral DNA molecules obtained from the caffeine fractions of this column are presented in Plate I. The replicating viral DNA molecules from the caffeine fraction are circular and contain two branch points. DNA molecules replicating in this same fashion have been observed in autoradiographs of Escherichia

PLATE

fraction

1. Electron micrographs of a benzoylated-nephthoylaterl

of replicating SV40 DEAE-cellulose

DNA

molecules obtained column. Magnification

from the cefftinv ix 44,000 times.

REPLICATING

SV40

DNA

I 300-

!

663 I

I

r (a)

’

I

-

I

I (b)

I

Fraction

no.

FIG. 9. Velocity sedimentation through 8n alkaline rmcrose gradient of vi& DNA eluted from 8 benzoyleted-naphthoylated cellulose column with 1 M-N&~ (b) or 2% caffeine (a). SV40 DNA obtained from a column similar to that presented in Fig. 7 ~8s sedimented through an 8lk8line 6 to 20% sucrose gmdient (0.1 M-NaOH end 0.9 M-N&~) for 3 hr 8t 39,000 rev./min in an SW60.1 rotor. The 8rrow indicates the peak position of a 3aP-18beled SV40 DNA sedimentation marker. The curve shows 3H cts/min.

coli DNA (Cairns, 1963), and in electron micrographs of mitochondrial DNA (Kirsohner, Wolstenholme & Gross, 1968), lambda DNA (Tomizawe t Ogawa, 1908), polyoma DNA (Hirt 1969) and +X174 DNA (Knippers et al., 1969). When viral supercoiled DNA molecules were stored at 0 to 4°C for one to two weeks before being placed on the grid for electron microscopy, a variable percentage of supercoiled molecules were converted to open circular molecules. 37 of these molecules were photographed and measured to obtain a distribution of lengths of viral DNA molecules. This length distribution is presented in Figure 10(a). Most of these molecules had a length of l-7 ~1but there was a distinct skewing of the distribution to the smaller side. The mean length of SV40 DNA from the large-plaque mutsnt was 1.66 /.L Measurements of the replicating SV40 DNA molecules indicated that the length of the presumably replicated arms of the molecules (on either side of the branch points) were equal to one another and that one replicated arm plus the remainder of the molecule (presumably not yet replicated) was equal to the size of a non-replicating circular DNA molecule (within a 10% error). The mean length of SV40 circles (one replicated arm plus the unreplicated portion) measured from replicating molecules was 1.67 CL. A distribution of the total lengths of replicating molecules (Fig. 10(b) ) demonstrates that the majority of DNA molecules observed on many grid preparations had completed 90% of their replication. A completely duplicated DNA molecule would be expected to have a total length of 3.32 to 3.34 CL.Of the replicating molecules observed, 75% had a total length of 2.9 to 3.2 CL.

564

A. J. LEVINE,

H. S. KANG

I.7

2.3

AND

2.9

F. E. BILLHEIMER

3.5

Totalkicgthp ho. 10. The distribution of tote1 lengths for (a) resting and (b) replioeting molecules. Measuremerits of the lengths of DNA molecules were obteined from treoings of the origins1 molecules photographed in the electron microsoope using E calibrated msp measurer.

4. Discussion At 30 hours after infection, little or no mature SV40 supercoiled DNA is formed in the tirst five minutes of exposure to E3H]thymidine. Figure 11 presents a compilation of sedimentation velocity experiments that illustrates this point. Instead, a precursor of viral DNA is observed which sediments at 24 to 25 S. This fast sedimentation rate is not due to the association of proteins or RNA with the precursor DNA since pronwe, trypsin, phenol extraction or RNase treatment doea not alter its sedimentation behavior. The five-minute lag in the appearance of closed circular mature viral DNA was also observed by alkaline sucrose velocity sedimentation and ethidium bromidecesium chloride equilibrium centrifugation. These results are in agreement with those of Hirt (1966), who found in polyoma-infected cells that no radioactivity sedimented in the region of 16 to 20 s after a 2*5-minute pulse with r3H]thymidine. The five-minute delay in the appearance of supercoiled DNA is surprising, since all t’he viral DNA molecules are most likely replicating in an asynchronous fashion (some molecules are finishing and some starting at the same time) and one would therefore

expect to see some supercoiled DNA labeled immediately after a pulse. This lag could be explained if newly synthesized mature viral DNA possessed some unusual properties so that it might be found in the 1 M-NaCldodecyl SO, precipitate. To determine if this ww indeed the CW, the 1 M-NaCl-dodecyl SO, precipitate fraction was isolated (by dissolving the dodecyl SO, at room temperature) after a five-minute pulse with [3H]thymidine, and anelysed by neutral and alkaline velocity sedimentation

REPLICATING I

1

SV40

DNA

I

566 a

-j BO3” 3 60s “0 w 40.4 iJ 202

’ 0/ . &.--

0

0‘7:

y/O

/

5 Time after

IO

1 15

addition

I 20 of isotope

I 25

I 30

(min)

FIG. 11. The percentage of radioaotivity sedimenting in the supercoil region as a function of the length of exposure to [3H]thymidine. The experimental procedures are given in Fig. 3. The dats are presented as the percentage of radioactivity found in the 20 to 21 s region of a sucrose gradient after different lengths of pulselabeling times with [3H]thymidine. (A), (0) and (0) are from 3 experiments done et different times.

and by ethidium bromide-cesium chloride equilibrium centrifugation. Little or no labeled DNA with the properties of closed circular or supercoiled DNA was observed. These data demonstrate that no 3H-labeled material with the properties of mature SV40 DNA could be detected in any cell fraction after a five-minute pulse with [3H]thymidine. The second surprising fact that emerges from the sedimentation analysis is that pulsed-labeled (1 to 5 min) viral DNA sediments faster than might have been expected for replicating double-stranded circular DNA. The replicating DNA isolated from lambda-infected cells sediments (in 2 M-NaCl) as a heterogeneous collection of DNA molecules extending over a range of 1.2 to 1.8 times faster than lambda linear DNA (Young & Sinsheimer, 1968). The 25 s SV40 DNA precursor observed here sediments as a peak about 1.8 times faster than linear SV40 DNA (14 s). Chromatography on benzoylated-naphthoylated cellulose DEAE-columns separates mature and replicating SV40 DNA. Mature viral DNA elutes from the column between 0.55 and 0.6 M-NaCl, while replicating DNA remains on the column. The replicating viral DNA can be eluted with caffeine. In theory, the benzoyl and naphthoyl groups on the cellulose resin interact with the non-hydrogen-bonded pyrimidine and purine rings (denatured regions of DNA) through a base-stacking mechanism. If this is the case, replicating SV40 DNA should contain some denatured regions in its DNA. In the electron micrographs obtained from the caffeine fractions, replicating SV40 DNA consisted of molecules with two branch points, three branches and no visible ends (circles). Two of these branches (replicated branches) were always equal in length and one replicated branch plus the remaining unreplicated portion were equal in length to relaxed circular SV40 DNA (1.66 p for circular SV40 DNA and 1.67 p for replicating DNA). This type of replicating molecule has been observed with a variety of other circular DNA’s (Cairns, 1963 ; Kirschner et al., 1968 ; Tomizawa & Ogawa, 1963; Hi& 1969). The unusual feature found here was that about 75% of the replicating molecules had completed 90 to 95% of their replication. It should be noted however, that it is not clear whether these molecules are almost completely replicated 36

566

A. J. LEVINE,

H.

S. KANG

AND

F. E. BILLHEIMER

or are fully replicated. In the latter case the region assumed to be unreplicated would be four-stranded (i.e. two pairs of strands together). Whether the cytochrome c would penetrate a four-stranded structure to yield a double-width replica of the DNA is not known. In either case the great majority of DNA molecules are found in a late stage of replication. This fact could be explained by one of two possibilities: (1) the procedures employed to fractionate DNA4 molecules somehow select for DNA that is in a late stage of replication; or (2) there is a slow or rate-limiting step late in the replication process and as long as the rate of DNA synthesis is fast compared to this, most of the molecules observed will be blocked at the slow step. Several experimental facts suggest (but do not prove) that the first alternative is not correct. After a short pulse with [3H]thymidine (up to 5 min), 80% of the DNA found in the 1 nr-NaCl-dodecyl SO, supernatant is eluted from the column with caffeine. If the 1 M-NaCl-dodecyl SO1 supernatant contains most or all of the viral replicating DNA, then the caffeine fraction should be representabive of the replicating molecules found in vivo. Attempts to find replicating viral DNA in the phenol-extracted 1 M-NaCl-dodecyl SO, precipitate by benzoylated-naphthoylated cellulose chromatography and velocity sedimentation have failed. The second alternat.ive, that there is a rate-limiting step late in the replication process, helps explain several observations. Kinetic data from velocity sedimentation analysis and column chromatography indicate that little or no supercoiled DNA is made for the first five minutes of labeling with [3H]thymidine. The rate-limiting hypothesis would predict this lag in the appearance of 3H-labeled supercoiled DNA. If SV40 DNA replicated at the same rate as mammalian cell DNA (about 2 p/min; Huberman & Riggs, 1968), then it would take about 50 seconds to replicate a DNA molecule of 1.66 p (SV40 DNA). The experimental value of a five-minute doubling time for mature SV40 DNA would indicate that at any one time about 80% of the replicating DNA should be blocked at the rate-limiting step in replicat’ion. This estimate agrees well with the length distribution of replicating SV40 DNA molecules (about 75% are almost completely replicated, as observed in the electron microscope). In addition, if a slow step or block exists late in the replication of XV40 DNA, one would expect a viral DNA molecule a,bout twice the tot’al length of SV40 DNA to accumulate. If the bonds between the two replicat’ed SV40 circles are mechanically ridged, one can calculate that this molecule should sediment at about 1.75 t,imes the velocity of the linear SV40 molecule (Bloomfield, 1966; Fukatsu & Kurata, 1966; Kirkwood & Riseman, 1948). This prediction agrees with the experimental observation that t’he 25 s precursor DNA sediments about 1.8 times faster than 14 s linea,r XV40 DNA. What factors might cause a slow down in the rate of SV40 DNA replication near the end of the molecule ‘2 Several possibilities exist’. The synthesis of +X174 singlestranded DNA from a replicative form template will not occur in the absence of a functional coat protein (Lindqvist & Sinsheimer, 1967). Similarly h double-stranded linear DNA requires coat protein for its maturation (MacKinlay & Kaiser, 1968). It is possible that ST740 DNB also requires a coat protein to finish its replication. This may be a useful means of ensuring the packaging of viral DNA into its coat. A second alternative is that the last 10% of the SV40 DNA that is to be replicated is different in some way which makes it replicate more slowly than the rest of the molecule. In this connection Aloni, VVinocour, Sachs & Torten (1969) have reported that about

REPLICATING

SV40

DNA

567

10% of the SV40 DNA molecule contains sequences which hybridize with host cell DNA. Yet a third alternative is suggested from the fact that replicating doublestranded circular lambda DNA contains longer than unit length polynucleotide strands (Kiger & Sinsheimer, 1969). If th e almost completed replicating SV40 molecules contained a double-length linear strand of DNA, then an endonuclease and ligase step would be required to produce (by recombination) two unit-length circles. Thus viral or more likely the host cell recombination enzymes may be required to finish the replication of SV40 DNA. One might expect that the normal levels of recombination or repair enzymes in stationary phase cells would be too low to meet the needs of hundreds of replicating SV40 DNA molecules. This could then represent the slow step late in replication of the SV40 DNA. The excellent technical assistance of Mrs Angelika K. Teresky is gratefully This research was supported by grant CA 11049-01 of the Public Health part by the Stuart Frankel Foundation.

acknowledged. Service and in

After this paper was submitted for publication, Bourgaux, Bourgaux-Ramoisy & Dulbecco (1969) described a procedure for the isolation and characterization of replicating polyoma DNA. These authors use 0.25% deoxycholate for cell lysis and a high-speed centrifugation to separate viral and cellular DNA. On the basis of velocity sedimentation experiments they have apparently isolated polyoma replicating DNA molecules in all stages of replication. We have repeated the experiment described in Figure 3 of this paper using the Bourgaux et al. lysis and fractionation procedure. The results of these experiments are identical to those presented in Figure 3 of this paper. Thus, employing a cell lysis and DNA fractionation procedure known to yield a heterogeneous collection of replicating polyoma DNA molecules results in an apparently more homogeneous, rapidly sedimenting, collection of SV40 DNA molecules. REFERENCES Aloni, Y., Winocour, E. & Sachs, L. (1968). J. Mol. BioZ. 31, 415. Aloni, Y., Winocour, E., Sachs, L. & Torten, J. (1969). J. Mol. Biol. 44, 333. Black, P. H., Crawford, E. M. & Crawford, L. V. (1964). Virology, 24, 381. Bloomfield, V. A. (1966). Proc. Nat. Acud. Sci., Wash. 55, 717. Bourgaux, P., Bourgaux-Ramoisy, D. & Dulbecco, R. (1969). Proc. Nat. Acad. Sk., Wash. 64, 701. Cairns, J. (1963). J. Mol. BioZ. 6, 208. Denhardt, D. T. (1966). Biochem, Biophy8. Res. Comm., 23, 641. Fukatsu, M. & Kurata, M. (1966). J. Chem. Phys. 44, 4539. Gilead, Z. & Ginsberg, H. S. (1965). J. Bmt. 90, 120. Gillam, I., Millward, S., Blew, D., von Tigerstromm, M., Wimmer, E. & Tener, G. M. (1967). Biochemistry, 6, 3043. Hirt, B. (1966). Proc. Nat. Acad. Sci., Wash. 55, 997. Hirt, B. (1967). J. Mol. BioZ. 26, 365. Hirt, B., (1969). J. Mol. BioZ. 40, 141. Huberman, J. A. & Riggs, A. D. (1968). J. Mol. BioZ. 32, 327. Kamano, T. & Sinsheimer, R. L. (1968). Biochim. biophys. Acta, 155, 295. Kiger, J. & Sinsheimer, R. L. (1969). J. Mol. BioZ. 40, 467. Kirkwood, J. G. & Riseman, I. (1948). J. Chem. Phys. 16, 565. Kirschner, R. H., Wolstenholme, D. R. & Gross, W. J. (1968). Proc. Nat. Acad. Sci., Wash. 60, 1466. Kleinschmidt, A. & Z&n, R. K. (1959). Naturf. lab, 770. Knippers, R., Whalley, M., & Sinsheimer, R. L. (1969). Proc. Nat. Acad. Sci., Wash. 64, 275. Levine, A. J. & Ginsberg, H. S. (1967). J. ViTology, 1, 747. Lindqvist, B. H. & Sinsheimer, R. L. (1967). J. Mol. BioZ. 30, 69.

568 MacKinley, Sedat, J., Sinsheimer, Tomizawa, Young, E. Young, E.

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S. KANG

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F. E. BILLHEIMER

A. G. & Kaiser, A. D. (1968). J. Mol. BioE. 39, 679. Lyon, A. & Sinsheimer, R. L. (1969). J. Mol. Biol. 44, 415. R. L., Starman, B., Nagler, C. & Guthrie, S. (1962). J. Mol. Biol. 4, 142. J. & Ogawa, T. (1968). Cold Sp. Hat-b. Symp. Qua&. Biol. 33, 533. T. & Sinsheimer, R. L. (1964). J. Mol. Biol. 10, 662. T. & Sinsheimer, R. L. (1968). J. Mol. Biol. 33, 49.