Changes in the sedimentation properties of HeLa cell DNA and nucleoprotein during replication

484

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96352

CHANGES IN T H E S E D I M E N T A T I O N P R O P E R T I E S OF H e L a CELL DNA AND N U C L E O P R O T E I N D U R I N G R E P L I C A T I O N J O E L F. H A B E N E R * , B A R B A R A S. B Y N U M A N D J O S E P H SHACK**

Laboratory of Physiology, National Cancer Institute, National Institutes o[ Health, Bethesda, Md., 2OOl4 (U.S.A.) (Received J u n e 3oth, 1969)

SUMMARY

I. Short pulses of [3H]thymidine were used to label the newly replicated DNA of H e L a suspension cultures which had been grown for a generation time in [uC]thymidine. DNA was extracted by several methods from whole cells, nuclei and dispersed nucleoprotein, and the relative sizes of the newly replicated and bulk DNA were compared by sedimentation in alkaline and neutral sucrose gradients. 2. The results show that DNA is first synthesized in small pieces (less than 12 S) which are joined within I rain into intermediate replicon-sized segments (26 S) and in 30-45 rain into the final DNA product ( > 39 S). 3. Dispersed nucleoprotein containing the newly replicated DNA sediments faster than the bulk nucleoprotein. However, when extracted from nucleoprotein by alkali or 2 M NaC1, the newly replicated DNA sediments more slowly than the bulk DNA. 4- Evidence is presented which suggests that DNA labeled with high specific activity E3H]thymidine can undergo significant radiolytic degradation within I week.

INTRODUCTION

Various lines of evidence indicate that mammalian DNA is synthesized in multiple segments termed replicons 1-3. Autoradiographs show DNA synthesis occurring simultaneously at numerous fork-like growing points which proceed in opposite directions from adjacent initiation sites and join after a replicating segment (replicon) of 30" lO6-12o • 106 daltons is completed 4,5. PAOLETTI et al. 6 have reported that newly synthesized DNA extracted from Erhlich ascites cells has a lower sedimentation rate in neutral or alkaline sucrose gradients than the bulk of the cellular DNA. OKAZAKI et al. 7,8 have found that DNA of bacteria is first made in very small pieces, on the order of 0. 5 • IO6 daltons and are rapidly joined during replication, presumably by the action of DNA ligases 9. While our work was in progress both SCHANDL AND TAYLOR 1° and PAINTER AND SCHAEFER11 have also reported finding similar very small replieon precursors during the synthesis of mammalian DNA. In this report • P r e s e n t address: Endocrine Unit, Massachusetts General Hospital, Boston, Mass. 02114, U.S.A. • * To w h o m r e p r i n t r e q u e s t s should be sent.

Biochim. Biophys. Acta, 195 (1969) 484-493

SEDIMENTATION PROPERTIES OF

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DNA

485

we present our observations on the sequence of events that occur during DNA synthesis in H e L a cells; from the synthesis of very small segments, through replicon-sized units, to the final very large product. Since DNA exists within the mammalian cell as a nucleoprotein we have obtained analogous data on dispersed nucleoprotein. Using double isotope labeling, in order to allow direct comparisons between the newly replicated and bulk DNA, we have examined the sedimentation properties of DNA extracted b y several methods from whole cells, nuclei and dispersed nucleoprotein on alkaline and neutral sucrose gradients. We have made estimates of the molecular size of the intermediates formed during synthesis and have correlated them with some of the current concepts concerning DNA synthesis in mammalian cells.

MATERIALS AND METHODS

Cell culture Suspension cultures of the H e L a $3 clone of PUCK et al. TM were grown at 37 ° in modified Eagle's minimum essential media 1~ supplemented with 5 ~/o horse serum and containing penicillin (IOO units/ml) and streptomycin (IOO/~g/ml). The generation time was between 18 and 24 h. The cell line was kept in continuous culture at concentrations of from I . lO 5 to I . IOs cells/ml b y adding fresh media every 2-3 days. Cells in logarithmic growth phase were used in all experiments.

Radioactive pulse labeling The bulk DNA was in every case labeled b y growing cells for a generation in E14CJthymidine (New England Nuclear Corp.), 26 mC/mmole, 30 ~C/ml. Excess E14Clthymidine was removed b y centrifuging the cells, resuspending them in fresh media and incubating for i h at 37 °. Pulse labeling was then done with E3Hjthymidine (New England Nuclear, 15.3-19. 3 C/mmole at a level of 2 ~C/ml for 0.5-0.3 rain at 37 °. L3HlThymidine incorporation was stopped by pouring an aliquot of the cells with rapid stirring into 0.5 w l . of frozen media. The cells were collected immediately by centrifugation at 4 ° and resuspended in o.15 M NaCl-o.oI 5 M sodium citrate (pH 7.8) and 0.002 M E D T A (sodium salt) at a cell concentration of I.lO7-2 . l o 7 cells/ml. The pulse was chased by adding a 5o-fold excess of nonradioactive thymidine to the culture vessel at the end of the pulse. The incorporation of E3Hlthymidine under these conditions was reduced to about 2-3 % of the rate during a 3-rain pulse.

Preparation o/ nuclei and nucleoprotein Nuclei were prepared b y lysing washed cells at a concentration of approx. 2. lO s cells/ml in 0. 5 % (v/v) Nonidet P-4 o detergent (Shell Chemical Co., New Y o r k ) 0.075 M NaCl-o.o24 M EDTA (pH 8.0) for 15 rain at o °. Lysis was complete under these conditions and only occasional cytoplasmic fragments remained adherent to the nuclei. The nuclei were washed twice in a solution consisting of equal volumes of phosphate-buffered saline and o.075 M NaCl-o.o24 M E D T A (pH 7.6). Nucleoprotein was prepared b y taking up a well-drained pellet of washed nuclei from 2. lO s to 4"lOS cells in IO ml of 0.25 mM EDTA, rapidly transferring to a Waring blender containing 200 ml of 0.25 mM E D T A at o ° and stirring for 20 sec at 9000 rev./min (Variac Setting of 60 V). Biochim. Biophys. Acta, 195 (1969) 484-493

486

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al.

DNA extraction DNA was extracted by three methods: Method I; direct alkali extraction s. An aliquot of a suspension of either cells or washed nuclei in o.015 M sodium citrate-o. 9 M NaCl-o.oI M EDTA or of the dispersed nucleoprotein in 0. 9 M NaCl-o.oI M EDTA, was lysed in o.i M NaOH and incubated at 37 ° for 3 ° min with occasional gentle stirring. The lysate was centrifuged at 2000 x g for 15 rain to remove particulate matter. Recovery of radioactivity in the supernatant was greater than 9° % by this procedure. Method II; modiJication o[ the THOMAS procedure 14. The cell suspension at a concentration of i. lO7-2 • lO7 cells/ml in o.15 M NaCl-o.o5 M trisodium citrate-o.oo2 M EDTA, was lysed in I ~o sodium lauryl sulfate at room temperature, incubated in pronase (Calbiochem) at 2 mg/ml for 16-18 h at 37 ° and, extracted twice with equal volumes of water-saturated phenol at 25 °. The extraction mixture was chilled to o ° and centrifuged, the upper aqueous layer decanted, the interface precipitates extracted with water once and the combined aqueous layers dialyzed exhaustively either against O.Ol5 M NaCl-o.ooI5 M trisodium citrate-o.oo2 M EDTA or against o.oi M phosphate buffer (pH 6.8). Recovery of DNA by this method was 60-80 % and determinations of the radioactivity remaining behind in the interface precipitate and phenol layers did not show any enrichment of either of the two isotopes. MethodlII; modi[ication o[ the KIRBY procedure 15. Cells at a concentration of 2. lO6 cells/ml were lysed and extracted simultaneously by stirring directly in 1 % sodium lauryl sulfate, 6 °/o sodium p-aminosalicylate, 0.002 M EDTA for 30 min at 7 o°, shaken for 40 min at 25 ° with 88 ~o phenol, 12 °/o m-cresol and o.I °/o 8-hydroxyquinoline, chilled to o ° and centrifuged, and the aqueous layer was exhaustively dialyzed against o.oi M phosphate buffer (pH 6.8). Most extractions were done with unlabeled denatured carrier HeLa DNA added to insure against any selective loss of the newly formed single-stranded DNA.

Denaturation o[ DNA The DNA extracted in the native state by Methods I I and I I I was denatured by incubating in o.I M NaOH and o.ooi M EDTA at 25 ° for 20 min.

Zone sedimentation 5-2o % sucrose gradients were made either alkaline in o.I M NaOH-o. 9 M NaCl-o.oo2 M EDTA (pH 12.o) or neutral in o.15 M NaCl-o.oI 5 M trisodium citrate0.002 M EDTA (pH 7.2). 0.5 ml of alkaline denatured DNA extract containing about 50/~g of DNA (5000-60 ooo counts/rain) was layered on top of 4.8 ml of a preformed gradient and centrifuged in the SW-39 rotor of the Spinco L ultracentrifuge (Spinco Division, Beckman Instruments, Palo Alto, Calif.) or 1-2 ml were centrifuged through 28 ml of gradient in the SW-25.I rotor, 3 °, for 8-12 h at 22 500-25 ooo rev./min. 0.2 ml (SW-39) or 2.0 ml (SW-25.I) fractions were collected by upward displacement with 60 % sucrose, precipitated with cold 5 % trichloroacetic acid in the presence of added carrier consisting of o.I °/o calf thymus DNA and 1. 5 °/o bovine serum albumin, washed twice with cold 5 % trichloroacetic acid and hydrolyzed in 5 % trichloroacetic acid at 9°0 for 20 min. 0.2 ml of the hot trichloroacetic acid extract was assayed for radioactivity. Part of the radioactivity (from IO to 40 %) was recovered as a pellet at the bottom of the tube; this is shown in the figures by the interrupted line. Re-

Biochim. Biophys. Acta, 195 (1969) 484-493

487

SEDIMENTATION PROPERTIES OF H e L a CELL D N A

covery, including the pellet, was in general greater than 9 ° %. Sedimentation constants shown on the figures refer to temperature of 3 ° and were calculated assuming a constant rate of sedimentation through a 5-20 % sucrose gradient le,1~. Molecular weights were calculated using equations given by STUDIER18.

Radioactivity determinations 0.2 ml of the hot trichloroacetic acid extract was counted in IO ml of scintillalation fluid (60 % toluene-4o% ethylene glycol monomethyl ether-8.o % naphthalene0.4% PPO-o.oo5 % POPOP) in the Packard Tri Carb liquid scintillation spectrometer (Packard Instruments Co., La Grange, Ill.). Counting data was recorded on punch tape and double-label efficiency corrections were done with an IBM 162o computer (International Business Machines Corp., White Plains, N.Y.). Sedimentation graphs were then generated by a Model 2D2 X-Y recorder (F. L. Mosley Co., Pasadena, Calif.). RESULTS

The sedimentation behavior of DNA extracted from pulsed and pulse-chased cells by Method I is shown in Fig. I. Although the sedimentation zones are broad, indicative of a rather large heterogenity in sizes, a peak fraction can be identified;

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Fig. I. A l k a l i n e sucrose s e d i m e n t a t i o n of pulse-labeled H e L a cells e x t r a c t e d b y Method I. D N A w a s e x t r a c t e d f r o m cells w h i c h were: A, pulse-labeled for 3 rain w i t h [ S H ] t h y m i d i n c . A 5o-fold excess of n o n r a d i o a c t i v e t h y m i d i n e w a s added a n d D N A w a s e x t r a c t e d at: B, 15 min; C, 3 ° rain; D, 45 m i n . S e d i m e n t a t i o n w a s carried o u t in t h e SW-39 rotor, 22 5oo r e v . / m i n for 8 h at 3 °. T h e last fraction s h o w n a b o v e the i n t e r r u p t e d line represents t h e r a d i o a c t i v i t y in t h e pellet (see text). O - O , SH radioactivity; G - C ) , 1'C r a d i o a c t i v i t y .

Biochim.-Biophys. Acta, 195 (1969) 484-493

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J.F. HABENER et al.

the s values shown refer to these peak fractions. By 3 min, the shortest pulse time examined b y this method, most of the newly replicated DNA sediments at about 26 S, considerably more slowly than the bulk DNA (39 S). A small amount of the pulselabeled DNA is discernible just beyond the meniscus as very slowly migrating material (less than io S). The newly replicated DNA progressively increases in size and by 45 min sediments coincident with the bulk DNA. A. 0 WEEK

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Fig. 2. T i m e - d e p e n d e n t degradation of 3H-labeled DNA. D N A wa~ extracted b y Method I from ceils which were g r o w n for a generation time in E14C]thymidine, pulse labeled for 3 rain with [3Hlthymidine and chased w i t h a 5o-fold excess of nonradioactive thymidine. D N A was extracted in alkali: A, at the time of radioisotope labeling and after the labeled cells had stood for: B, I week; C, 2 weeks; or D, after the alkaline e x t r a c t had stood for i week. Sedimentation was done on alkaline sucrose gradients, SW-39 rotor, 22 500 rev./min, 8 h, 3 °. F r a c t i o n 24 is the radioactivity in th~ pellet and varied for io to 50 ~o of the total radioactivity added. O - Q , 3H radioactivity; (D-C), 14C radioactivity.

In a series of control experiments (Fig. 2) it was observed that average s values of 3H-labeled DNA gradually decreased both with pulsed and with pulsechased DNA while the 14C-labeled bulk DNA remained essentially contant. The results were similar whether whole cells stood before extraction (Figs. 2A-2C) or whether they stood in alkali after the extraction (Fig. 2D). No detectable degradative changes occurred within 28 h after pulse incorporation and, accordingly, all studies were carried out within that time. Likewise, no changes were observed with time in cells Biochim. Biophys. Acta, 195 (1969) 484-493

SEDIMENTATION PROPERTIES OF H e L a

489

CELL D N A

which had been pulsed with [aH]thymidine of lower specific activity (o.8 C/mmole) suggesting that ~H radiolysis was causing degradation of the pulse-labeled DNA. Both PERSON AND SCLAIRTM and Z A J I C E K AND GROSS~° have reported finding degradation of DNA labeled with high specific SH activity, an effect they have attributed to internal radiation.

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Fig. 3. Alkaline s u c r o s e s e d i m e n t a t i o n of D N A e x t r a c t e d b y Method I I (A-C) a n d Method I I I (D) a n d d e n a t u r e d in alkali as described in t h e t e x t . A 5o-fold excess of n o n r a d i o a c t i v e t h y m i d i n e w a s a d d e d to t h e c u l t u r e a f t e r 3 m i n of [ a H ] t h y m i d i n e i n c o r p o r a t i o n . D N A w a s e x t r a c t e d at: A, o. 5 m i n ; ]3, I rain; C, 3 m i n ; D, 3 rain. S e d i m e n t a t i o n w a s done in t h e S W - 3 9 rotor, 3 °, I2 h, 22 5oo r e v . / m i n (A, C); 25 ooo r e v . / m i n (B) a n d IO h a t 22 5oo r e v . / m i n (D). T h e final f r a c t i o n ( i n t e r r u p t e d line) c o n t a i n s t h e r a d i o a c t i v i t y in t h e pellet (15-45 %). O - O , 3H r a d i o a c t i v i t y ; G - C ) , 14C r a d i o a c t i v i t y .

Fig. 3 shows the alkaline sedimentation patterns obtained from DNA extracted by Method I I (Figs. 3A-3C) and Method I I I (Fig. 3D). The results are qualitatively similar to those shown in Fig. I for DNA extracted by Method I but the sedimentatation constants, particularly of the bulk DNA, are somewhat lower, undoubtedly due to greater shear during extraction. This lowering is greater for DNA extracted by Method I I (Figs. 3A-3C); the DNA prepared by Method I I I exhibits sedimentation Bioehim. Biophys. Acta, 195 (1969) 484-493

j . F . HABENER gt al.

49 °

behavior much closer to that of the DNA directly extracted by alkali (Method I). With pulses of 0.5 and I min, there is considerable 3H-labeled DNA seen sedimentating at 9-12 S or less; much of this has been converted into larger material by 3 min. However, it is evident that even at 0. 5 rain, the shortest pulse time used, most of the newly replicated DNA is present at 17 S or larger pieces. Sedimentation through neural sucrose gradients of two of the same samples (Method II) used for runs in alkali (Fig. 3) are shown in Fig. 4- As reported previously, 28 S A. 05

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Fig. 4. S e d i m e n t a t i o n in n e u t r a l sucrose. T h e D N A e x t r a c t e d b y Method I I (of. Fig. 3) was sedim e n t e d o n n e u t r a l s u c r o s e g r a d i e n t s as described in t h e t e x t . Conditions of s e d i m e n t a t i o n : SW-39 rotor, 22 5o0 r e v . / m i n , 3 °, for io h. A, o . 5 - m i n pulse; B, I - m i n pulse. 0 - 0 , aH r a d i o a c t i v i t y ; O - O , 1*C r a d i o a c t i v i t y .

most (about 95 %) of the DNA extracted by Method I I is double-stranded ~1. It is evident that a reduced fraction of the newly formed material sediments more slowly than the bulk compared with results in alkali. This suggests that a substantial portion of the newly formed DNA consists of small pieces which, before denaturation, are attached to longer template strands. I A. 3 M/N

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Fig. 5. S e d i m e n t a t i o n of dispersed nucleoprotein. 1Yucleoprotein p r e p a r e d f r o m cells: A, pulselabeled for 3 m i n ; B, c h a s e d for 60 m i n (see t e x t ) w a s s e d i m e n t e d t h r o u g h sucrose g r a d i e n t s m a d e o.25 m M in E D T A (pH 7.6). C e n t r i f u g a t i o n done in t h e SW-25 rotor, 22 5oo r e v . / m i n 3°; A, 9.7 h; ]3, 16 h. 0 - 0 , 8H r a d i o a c t i v i t y ; O - O , 14C r a d i o a c t i v i t y .

Biochim. Biophys. Acta, 195 (1969) 484-493

SEDIMENTATION PROPERTIES OF

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DNA

491

The sedimentation pattern of dispersed nucleoprotein prepared from cells pulsed for 3 min with [3H]thymidine is shown in Fig. 5 A. Following a 6o-min chase with an excess of cold thymidine (Fig. 5B), the patterns of pulsed and bulk nucleoprotein superimpose. In contrast to the sedimentation pattern seen with the DNA extracts, the nucleoprotein particles containing the newly replicated 3H-labeled DNA sediment faster than the l~C-labeled material. However, when the nucleoprotein is deproteinized by exposure to 2 M NaC1 and sedimented through a sucrose gradient made 2 M in NaC1 (Fig. 6A) or when it is denatured and deproteinized by alkali / / C

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Fig. 6. S e d i m e n t a t i o n of 3 - m i n - p u l s e d n u c l e o p r o t e i n p r e p a r a t i o n of 0.2 5 m M E D T A (pH 7.4); B, o.I M N a O H - o . 9 M N a C I - 2 m M E D T A ison, nuclei f r o m w h i c h n u c l e o p r o t e i n w a s p r e p a r e d , w a s e x t r a c t e d b y in alkaline sucrose for IO h, 2o ooo r e v . / m i n , 3 °, SW-2 5 rotor. 0 - 0 , 14C r a d i o a c t i v i t y .

Fig. 5 in: A, 2 M N a C I (pH 12.o). C, for c o m p a r Method I a n d s e d i m e n t e d 8H r a d i o a c t i v i t y ; O - Q ,

and sedimented through an alkaline sucrose gradient (Fig. 6B), the newly replicated DNA in each case sediments more slowly (15 S) than the bulk 1*C-labeled material. For comparison Fig. 6C shows the sedimentation profiles in alkali of DNA directly extracted from the nuclei from which the particular preparation of nucleoprotein was made. The lower s values of both nucleoprotein particles and their DNA components undoubtedly result from even the minimum shearing required to produce dispersed nucleoprotein. DISCUSSION

The results of these studies lend further support to the concept of replicon synthesis in mammalian cells. We have shown that pulse-label is initially incorporated into smaller DNA segments which are then joined into more rapidly sedimenting larger segments during the course of replication. Other workers have made similar observations on the sedimentation behavior of newly replicated mammalian DNA ~,1°,n,32 Some general correlations can be made between our sedimentation results and the size estimated for replicons from autoradiography 5. The DNA labeled during 3 min of synthesis and extracted directly in alkali with minimum shear sediments in alkali with a peak of 26 S; 80 % of the radioactivity sediments rather broadly Biochim. Biophys. Acta, 195 (1969) 484-493

492

J.F. HABENER et al.

between 15 and 4o S. This corresponds to a molecular weight for single-stranded DNA in alkali of 15" lO 6 daltons (ref. I7) or twice that, 3o" lO 6 daltons, for a comparable length of double-stranded DNA. The range of sizes is 8. lO6-8o • Io 6 daltons for doublestranded DNA. These m a y be taken as minimum values since some shearing is expected during cellular lysis and sedimentation in alkali. These molecular sizes determined b y sedimentation are in agreement with those reported by HUBERMAN AND RIGGSs for replicating segments. The very slowly sedimenting DNA (less than 12 S) replicated during the first o.5-1 min of pulse incorporation is equivalent to a molecular weight of about i-lO 62. io~ daltons and corresponds to the early intermediate previously reported s,l°,n. Our results differ somewhat from those of SCHANDL AND TAYLOR10 who found a pronounced accumulation of the very small segments (4.2 S) during the first 30-60 sec of pulse incorporation followed within the next 60 sec by almost of total conversion into larger (I8.7 S) segments. Our studies show that the small segments are joined immediately upon completion of synthesis; assuming the m a x i m u m rate of synthesis for a single DNA strand is 2/,. rain -1 or 2" lO 6 daltons.min -1, I5 sec or less would be required to complete a segment of 5" lO5 daltons. Since most of the DNA we see after 30 sec oi synthesis is greater than 2. lO 6 daltons and is already present in repliconsized segments, we m a y conclude that no discernible delay occurs between completion of synthesis of the very small segments and their joining into intermediate replicon-sized segments. These disparate observations on the rapidity of joining m a y be due to inherent differences in the cell types studied or, more likely, due to the quite dissimilar conditions under which the cells were grown and pulse-labeled; perhaps the treatment of the cells with fluorodeoxyuridine, used b y SCHANDL AND TAYLOR1° just prior to pulse-labeling, causes a lag in either the production or action of DNA ligases during the first minute or two. It is not presently known why pulsed nucleoprotein sediments faster than the bulk nucleoprotein, even though the individual DNA molecules extracted from it are smaller. It is possible that the pulsed nucleoprotein m a y contain a number of segments of double-stranded DNA, forked or cross-linked in some manner. Another possibility is that the pulsed nucleoprotein contains a higher percentage of protein than the bulk and that part of the excess consists of types which are specifically associated with replication, and in some way, change the shape or density of the nucleoprotein particle. Pertinent to this is a suggestion, based on partition between phases during extraction with chloroform-isoamyl alcohol, indicating that proteins are bound more firmly to that DNA which has just undergone synthesis 23. ANKNOWLEDGMENTS

We thank Dr. F. K. Millar for aiding us in the use of the computer and Dr. F. Defillipes for the sample of Non-Idet P-4 o detergent and for m a n y helpful suggestions regarding its use. REFERENCES

I J. H. TAYLOR, J. Mol, Biol., 31 (1968) 5792 R. B. PAINTER, D. A. JERMANY AND R. E. RASMUSSEN, J. Mol. Biol., 17 (1966) 47" 3 S. OKADA, Biophys. J,, 8 (1968) 65 o.

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4 J. CAIRNS, J. Mol. Biol., 15 (1966) 372. 5 J. A. HUBERMAN AND A. D. RIGGS, J. Mol. Biol., 32 {i968 ) 327 . 6 C. PAOLETTI, N. DUTHEILLET-LAMONTHI~ZIE, PH. JEANTEUR AND A. OBRENOVITCH, Biochim. Biophys. Acta, 149 (1967) 435. 7 K. SAKABE AND R. OKAZAKI, Biochim. Biophys. Acta, 129 (1966) 651. 8 R. OKAZAKI, T. OKAZAKI, K. SAKABE, K. SUGIMOTO AND A. SUGINO, Proc. Natl. Acad. Sci. U.S., 59 (1968) 598. 9 K. SUGIMOTO,Z. OKAZAKI AND R. OKAZAKI,Proc. Natl. Aead. Sci. U.S., 60 (1968) 1356. IO E. K. SCHANDL AND J. H. TAYLOR, Biochem. Biophys. Res. Commun., 34 (1969) 291. i i R. t3. PAINTER AND A. SCHAEFER,Nature, 221 (1969) 1215. 12 T. T. PUCK, P. I. MARCUS AND S. CIECIURA, J. Exptl. Med., lO 3 (1956) 273. 13 H. EAGLE, Science, 13o (1959) 432. 14 K. I. BERNS AND C. A. THOMAS, J. Mol. Biol., i i (1965) 476. 15 K. S. KIRBY, Progr. Nucleic Acid Res. Mol. Biol., 3 (1964) I. 16 R. J. BRITTEN AND R. B. ROBERTS, Science, 131 (196o) 32. 17 E. H. McCoNKEY, in L. GROSS AND K. MOLDAVE, Methods in Enzymology, Vol. 12, P a r t A, Academic Press, N e w York, 1967, p. 620. 18 V. W. STUDIER, J. Mol. Biol., i i (1965) 373. 19 S. PERSON AND ~V[. H. SCLAIR, Radiation Res., 33 (1968) 66. 20 G. ZAIICEK AND J. GROSS, Exptl. Cell Res., 34 (1964) 138. 21 J. F. HABENER, B. S. BYNUM AND J. SHACK, Biochim. Biophys. Acta, 186 (1969) 412. 22 D. L. FRIEDMAN AND G. C. MUELLER, Biochim. Biophys. Acta, 174 (1969) 253. 23 A. G. LEvis, V. KRSMANOVIC, A. MILLER-FAURES AND 1V[. ERRERA, European J. Biochem. 3 (1967) 57.

Bioehim. Biophys. Acta, 195 (1969) 484-493