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Evidence of existence and compactness of dna superstructure units in mammalian cells a micro dosimetric approach to radiation induced dna release assayed by neutral sucrose gradient sedimentation

, : Evidence of existence and compactness of dna superstructure units in mammalian cells a micro dosimetric approach to radiation induced dna release assayed by neutral sucrose gradient sedimentation. Radiation & Environmental Biophysics 21(3): 175-204

Experimental results obtained with the neutral sucrose gradient sedimentation techniques of analyzing mammalian DNA after irradiation in vivo (Chinese hamster cells V79) are evaluated theoretically in order to verify and extend a model by Ormerod and Lehmann that describes the gradual release of free DNA from the DNA-membrane complex. The model is based on the idea of chromatin organization in the form of membrane attached superstructure units (MASSU) defined by consecutive attachment points in intervals of M0 (DNA MW of a MASSU). DNA sedimentation after cell lysis with sarkosyl as detergent allows good separtion of the released free DNA from that remaining in the complex. The dose-dependence of both the percentage of DNA released and pertinent MW parameters as measured with X-rays and derived from the model confirms it and yields the MASSU size M0 = 2.4 .times. 109 g/mol. The model is evaluated with respect to high-LET radiations by a mathematical theory based directly on microdosimetry. This approach, which could be of general interest as a contribution to theoretical microdosimetry, reduces the measurable quantities explicitly to the single event distribution of lineal energy for the gross volume, V, occupied by a MASSU in the interphase nucleus. Experimental results obtained with .alpha.-particles agree with this extended version of the model, yielding 0.2-0.3 .mu.m for the diameter of V. The entire chromatin, at least the greater part of it, must be organized in regular superstructure units with no detectable variance of their lengths M0, which is suggested to be a general principle whose functional implications remain to elucidated. The value of V implies that these units are rather compact formations corresponding to densely packed chromatin. Due to the detergent-stable attachment points, the commonly used neutral lysis and sucrose gradient assay after irradiation yields free DNA in the form of excised fragments smaller than M0 only, so that determination of double-strand breaks at relatively low doses requires the special methods of evaluation presented, too.

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