Sedimentation studies of giant DNA molecules present in unsheared whole‐cell lysates of D. melanogaster cells

We have used band sedimentation in shallow density gradients of CsCl in the preparative centrifuge to analyze the distribution of sedimentation coefficients present in tritium labelled DNA from D. melanogaster cells. The cells were lysed according to the method of Kavenoff and Zimm to preserve very high molecular weight DNA. Sedimentation measurements have been carried out as a function of speed of centrifugation. The resulting distribution functions have been interpreted with the aid of the Zimm‐Schumaker equation for the speed dependence of the sedimentation coefficient of very high molecular weight DNA. Low‐speed centrifugation (3000 rpm) indicates that DNA molecules from the lysate are evenly distributed over values of S20,w from 0 to 514S. This distribution is very sensitive to changes in speed of centrifugation and is transformed into a bimodal distribution at 12,080 rpm. Analysis of this transformation allows us to postulate that perhaps 55% of the DNA in the lysate may have molecular weights in excess of 40 × 109 g/mol. Some of these molecules may also possess a variety of configurations including partially replicated branched structures.

[1]  B. Zimm,et al.  Anomalies in sedimentation. V. Chains at high fields, practical consequences. , 1976 .

[2]  J. Hearst,et al.  The so20,w of unsheared DNA from whole cell lysates of Escherichia coli. , 1976, Biophysical chemistry.

[3]  B. Zimm.  Anomalies in sedimentation: IV. Decrease in sedimentation coefficients of chains at high fields , 1974 .

[4]  S. B. Leighton,et al.  The influence of rotor speed on the sedimentation behavior in sucrose gradients of high molecular weight DNA's. , 1974, Biophysical chemistry.

[5]  V. Schumaker,et al.  A rotor speed dependent crossover in sedimentation velocities of DNA's of different sizes. , 1974, Biochemical and biophysical research communications.

[6]  D. Levin,et al.  Neutral sucrose sedimentation of very large DNA from Bacillus subtilis. I. Effect of random double-strand breaks and centrifuge speed on sedimentation. , 1973, Journal of molecular biology.

[7]  R. Kavenoff.  Characterization of the Bacillus subtilis W23 genome by sedimentation. , 1972, Journal of molecular biology.

[8]  I. Schneider,et al.  Cell lines derived from late embryonic stages of Drosophila melanogaster. , 1972, Journal of embryology and experimental morphology.

[9]  D. Freifelder.  Molecular weights of coliphages and coliphage DNA. IV. Molecular weights of DNA from bacteriophages T4, T5 and T7 and the general problem of determination of M. , 1970, Journal of molecular biology.

[10]  J. Vinograd,et al.  The evaluation of standard sedimentation coefficients of sodium RNA and sodium DNA from sedimentation velocity data in concentrated NaCl and CsCl solutions. , 1965, Biochimica et biophysica acta.

[11]  D. Crothers,et al.  Viscosity and sedimentation of the DNA from bacteriophages T2 and T7 and the relation to molecular weight. , 1965, Journal of molecular biology.

[12]  J. H. Taylor,et al.  THE ORGANIZATION AND DUPLICATION OF CHROMOSOMES AS REVEALED BY AUTORADIOGRAPHIC STUDIES USING TRITIUM-LABELED THYMIDINEE. , 1957, Proceedings of the National Academy of Sciences of the United States of America.

[13]  S. Colowick,et al.  Methods in Enzymology , Vol , 1966 .