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Biochimica et Biophysica Acta, 407 (1975) 120--124 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA Report

BBA 91418

A PROCEDURE FOR THE RAPID LYSIS OF MAMMALIAN CELLS PRIOR TO ALKALINE SUCROSE DENSITY GRADIENT CENTRIFUGATION

R.A. WALTERS and C.E. HILDEBRAND

Cellular and Molecular Biology Group, Los Alamos Scientific Laboratory, University of California, Los Alamos, N.M. 87544 (U.S.A.) (Received June 10th, 1975)

Summary

Relatively long exposures to alkaline conditions are required to effect lysis and DNA strand separation in some cell lines prior to alkaline sucrose gradient centrifugation. We describe here a simple technique utilizing sodium lauroyl sarcosine and heparin which permits a reduction in alkaline lytic storage time of from 5--6 h to 45 min for Chinese hamster cells layered onto a gradient.

The technique of DNA sedimentation in alkaline sucrose density gradients has proven very useful in the study of DNA metabolism as well as DNA damage (and subsequent repair) induced by external agents. Since DNA purification prior to centrifugation can result in the selective loss of newly synthesized, low molecular-weight "Okazaki particles" [1 ] and will produce sheared non- replicating bulk DNA, lysing cells directly on the gradient with a minimum of handling is the method of choice. However, under similar lyric conditions, different cell lines vary widely in the time required to effect alkaline lysis and DNA strand separation [2]. Since newly replicated DNA may be as small as 100 nucleotides [1 ], diffusion during long lytic storage times will complicate interpretation of the sedimentation profile. Ionizing radiation has been used to accelerate DNA denaturation by introducing strand breaks [3], but this method is not particularly useful for those to whom such facilities are unavail- able. We describe below a simple, rapid technique which permits a reduction of alkaline lytic storage time of Chinese hamster cells from 5--6 h to 45 min.

Exponentially growing Chinese hamster cells {line CHO) [4] were labeled for 36 h with 0.05 uCi/ml [Me-14C] thymidine (Schwarz-Mann, 55.3 Ci/mol), followed by a 2 h chase in nonradioactive medium. Cells were harvested by low-speed centrifugation, washed twice with phosphate-buffered saline (pH 7.0), and resuspended in 0.05 M cacodylic acid (pH 7.0). For all experiments

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described, cells (2- 10 s, ~ 1.3 ~g DNA) in 0.1 ml of 0.05 M cacodylic acid buffer were layered, under the conditions described below, onto linear 5--20% (w/v) sucrose gradients (0.4 M NaOH, 0.1 M sodium EDTA, 0.1% sodium lauroyl sarcosine (sarkosyl)). Gradients were centrifuged in a 8W 27 rotor in a Beckman L3-50 ultracentrifuge; the gradients were fractionated from the top. Fractions (1.2 ml) were collected in vials, 2 ml water added, and counted with 15 ml PCS* (Amersham/Searle) in a Packard Tri-Carb liquid scintillation spectrometer.

Gradients were calibrated using k (kindly supplied by Dr B.J. Barnhart) and T: (Miles Laboratories, Inc.) phage DNAs assuming single-strand molecular weights of 1 . 6 . 107 (k) and 6 . 5 . 1 0 7 (T2). The sedimentation coefficient and molecular weight of the peak of Chinese hamster DNA were calculated as described previously [5,6] .

The common procedure for obtaining cell lysis and DNA denaturation prior to centrifugation entails addition of cells to an alkaline lysing solution previously layered onto the gradient, followed by a storage period to allow lysis and DNA denaturation. When 0.1 ml of cells was added to 0.9 ml of an alkaline lysing solution [2] and stored at 23--25 °C for varying lengths of time prior to centrifugation, the results shown in Fig.IA-E were obtained. Only after a minimum of 5 h lytic storage (Fig. lA) could we obtain, on a routine basis, reasonably sharp DNA sedimentation profiles (with a peak at

159 + 5 S, molecular weight 4.8 + 0.3 • 108) coupled with adequate recovery of DNA {>85%). With increasing storage times up to 18 h (Fig.IB-E), the sedimentation profile broadened and shifted to a lower apparent molecular weight, presumably as a result of alkali-induced DNA degradation [7] . With storage times ~ 5 h under these conditions, DNA recovery was low and variable, and the DNA that did remain in the gradient sedimented anomalously [8] (see below). In all cases, protein remained at the top of the gradient (Fig. lA) with ~ 80% of the protein in the top three fractions. The DNA at (or near) the b o t t o m of the tube (e.g. fraction 30) may be due to wall effects, as discussed by others [9] . No corrections have been made for this effect. The required lytic storage time and the size of the DNA at the peak agree well with that obtained by others for Chinese hamster cells under similar con- ditions [7] .

The effect of reduced temperature during lyric storage was also examined in an a t t empt to reduce alkali-induced DNA degradation. The protocol of cell lysis was the same as above except that the gradients were kept at 4 °C during lytic storage. The results are shown in Fig.lF. Although some of the DNA in the gradient sedimented at ~ 159 S after 18 h storage, DNA also remained near the top of the gradient. In addition, DNA recovery in the gradient was only 33% of that added, the remainder having pelleted. When the lysing solu- tion was made to 1% in Sarkosyl, DNA recovery in the gradient was increased from 33% to 83%, bu t ~26% of the DNA in the gradient was recovered in the top two fractions. Although 4 °C lytic storage has been used b y others, it is doubt fu l that the DNA is completely denatured [10] . McBurney et al. [10] also used Sarkosyl during 4 °C lysis and suggested that it appeared to accelerate DNA degradation. However, it does no t appear here that extended exposure to Sarkosyl induced extensive degradation of the 159 S DNA (see below and

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Fig.2B). The large amount of DNA recovered in the top two fractions of Sarkosyl-lysed cells is more likely a result of anomalous sedimentation due to incomplete denaturation than to DNA degradation.

It is clear from the data in Fig.IA-E that (a) relatively long lytic times are required to reproducibly obtain bulk DNA in a reasonably sharp sedimenta- tion profile and (b) the DNA distribution pattern in the gradient can change over a relatively short time. In an effort to shorten the required lytic storage time without sacrificing DNA recovery and resolution in the gradient, we tested additional lytic conditions. For the sake of brevity, we will present only those conditions which we found to produce the best results; the data are shown in Fig.2. The data in Fig.2A-C were obtained by adding cells in 0.1 ml to 0.4-ml solutions (previously layered onto the gradient) containing 1 mg/ml heparin plus 1% sarkosyl (Fig.2A), 1% sarkosyl (Fig.2B), and distilled water { Fig.2C) rather than adding cells directly onto an alkaline lysing solution.

123

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F i ~ 2 . Effec t o f di f fer ing ly t i c c o n d i t i o n s o n D N A re leased f r o m cells. Cells in A- -C w e r e added to 0.4 ml o f (A) 1 m g / m l heparin plus 1% sarkosy l , r ecovery 0 .86; (B) 1% sa rkosy l , recovery 0 .73 ; and (C) dist i l led water , r ecovery 0.70. Af te r 15 rain, 0 .5 m l 1 M NaOH, 0.2 M E D T A w e r e added and centr i fuga- t i o n begun a f ter 4 5 rain. Cells in I:)--F w e r e added to 0 . 9 m l 0.56 M NaOH, 0.11 M E D T A conta in ing (D) 0.9 rng/ntl heparin plus 0 .9% sarkosy l , r ecovery 0 .69 ; (E) 0 .9% sarkosy l , r ecovery 0 .70 ; and (F) base only , r ecovery 0.67. Centr i fuga t ion was begun af ter 1 h. For al l gradients , lys is wa s at 2 3 - - 2 5 °C w i t h c e n t r i f u g a t i o n at 15 °C, 22 000 rev . /min , for 5 h. The d irec t ion o f s e d i m e n t a t i o n is f r o m l e f t to right.

The concentration of heparin (Sigma) was 100 times that required for maximal DNA deproteinization (Hildebrand, C.E., unpublished observation). The gra- dients were allowed to stand for 15 min, after which 0.5 ml of 1 M NaOH, 0.2 M EDTA were added for 45 min prior to centrifugation. The data in Fig.2D-E show the results obtained after 1 h lysis when cells were added directly into 0.9 ml alkaline lysing solution (0.56 M NaOH, 0.11 M EDTA) containing 0.9 mg/ml heparin plus 0.9% sarkosyl (Fig.2D), 0.9% sarkosyl (Fig.2E), and base alone (Fig.2F).

It appears that simply exposing the cells to hypotonic conditions alone for 15 min before adding alkali produces a more clearly resolved sedimentation pattern than obtained otherwise (compare Figs 2C and 2F) although, in each case, a large amount of DNA was found in the top fraction. The use of Sarkosyl or Sarkosyl plus heparin either added before (Figs 2A and 2B) or mixed with

1 2 4

alkali (Figs 2D and 2E) greatly improved the sedimentation profiles. However, the recovery of DNA was best when Sarkosyl plus heparin were used, followed by addition of alkali (Fig.2A), and the DNA recovery and sedimentation profiles were not significantly different from that seen after 5 h lysis in alkali alone (Fig.lA). Although the DNA recovery in Figs 2D and 2E was the same, use of heparin produced a sharper profile and eliminated DNA from the top fraction of the gradient.

Detergents like sodium lauryl sulfate have been used to enhance DNA deproteinization [11 ] ; however, precipitation of sodium lauryl sulfate under our conditions was so severe as to limit its usefulness. On the other hand, heparin, a sulfated polysaccharide of molecular weight 10 000--33 000, is very effective in deproteinizing DNA [12] and, when used in combination with sarkosyl, allows a significant reduction in alkaline lytic storage time without inducing DNA degradation. The DNA released sediments at a peak of 159 S, produces no anomalously sedimenting DNA characteristic of Chinese hamster DNA under short lyric storage times [8] and, based on the observa- tions of Cleaver [13] , is single-stranded. The technique described above should be very useful in studying DNA replication where it is important to prevent the selective loss of newly replicated DNA, to minimize diffusion of low molecular weight intermediates, and to achieve maximum resolution of newly replicated and bulk nonreplicating DNA.

This work was performed under the auspices of the U.S. Atomic Energy Commission.

References

1 Huberman, J.A. and Horwitz, H. (1973) Cold Spring Harbor Symp. Quant. Biol. 38, 233--238. 2 Cleaver, J.E. (1974) Radiat. Res. 57 ,207- -227 . 3 0 r m e r o d 0 M.G. and Lehman, A.R. (1970) Biochim. Biophys. Acta 204, 128--143. 4 Waiters, R.A. and Petersen, D.F. (1968) Biophys. J. 8, 1475--1486. 5 Abelson, J. and Thomas, C.A. (1966) J. Mol. Biol. 18, 262--279. 6 Studier, F.W. (1974) J. Mol. Biol. 11, 373--390. 7 Lett, J.T., Kiucis, E.S. and Sun, C. (1970) Biophys. J. 10, 277--292. 8 Elkind, M.M. and Kamper, C. (1970) Biophys. J. 10, 237--245. 9 Lett, J.T. and Sun, C. (1970) Radiat. Res. 44, 771--787.

10 McBurney, M.W., GrAhAm, F.L. and Whitmore, G.F. (1972) Biophys. J. 12, 369--383. 11 Palcic, B. and Skarsgaard, L. (1972) Int. J. Radiat. Biol. 21 ,417- -433 . 12 Arnold, E.A., Yawn, D.H., Brown, D.G., Wyllie, R.C. and Goffey, D.S. (1972) J. Cell Biol. 53, 737--757. 13 Cleaver, J.E. (1974) Biochem. Biophys. Res. Commun. 59, 92--99.