Heat denaturation of DNA in situ, in unbroken cells, was studied in relation to the cell cycle. DNA in metaphase cells denatured at lower temperatures (8~176 lower) than DNA in interphase cells. Among interphase cells, small differences

Heat denaturation of DNA in situ, in unbroken cells, was studied in relation to the cell cycle. DNA in metaphase cells denatured at lower temperatures (8~176 lower) than DNA in interphase cells. Among interphase cells, small differences between G1, S, and G2 cells were observed at temperatures above 90~ The difference between metaphase and interphase cells increased after short pretreatment with formaldehyde, decreased when cells were heated in the presence of 1 mM MgCI2, and was abolished by cell pretreatment with 0.5 N HC1. The results suggest that acid-soluble constituents of chromatin confer local stability to D N A and that the degree of stabilization is lower in metaphase chromosomes than in interphase nuclei. These in situ results remain in contrast to the published data showing no difference in D N A denaturation in chromatin isolated from interphase and metaphase cells. It is likely that factors exist which influence the stability of D N A in situ are associated with the super-structural organization of chromatin in intact nuclei and which are lost during chromatin isolation and solubilization. Since D N A denaturation is assayed after cell cooling, there is also a possibility that the extent of denatured D N A may be influenced by some factors that control strand separation and D N A reassociation. The different stainability of interphase vs. metaphase cells, based on the difference in stability of DNA, offers a method for determining mitotic indices by flow cytofluorometry, and a possible new parameter for sorting cells in rectaphase. The cell transition from interphase to metaphase involves a sequence of dramatic changes in the gross morphology of nuclear chromatin. The dispersed chromatin of the interphase nucleus undergoes condensation and packing into chromosome entities of a very characteristic morphology. Although the gross morphologic changes are well characterized (see references 9, 26, 30), the changes in molecular structure of chromatin that accompany this transition are poorly understood. This may be because most studies of chromatin structure have been carried out on isolated chromatin in solutions, i.e., under conditions where factors responsible for maintenance of the characteristic gross morphology are lost. Chromatin extraction which involves homogenization of nuclei, removal of divalent cations, shearing, and solubilization, destroys any chromatin superstructure that may exist in situ (4, 6, 15, 23) and that in turn may modulate the interactions between DNA and the macromolecules of the nuclear milieu. The gross morphology of chromatin, DNA-nuclear envelope interactions, and the presence of divalent cations appear to be of a special importance during transi128 TeE JOURNAL or CELL BIOLOOV " VOLUME 73, 1977 9 pages 128-138 tion from interphase to metaphase (9, 26, 30). In previous papers (10-14, 35), we have presented a method for studying the thermal stability of DNA in situ in large populations of unbroken, individual cells. Since ionic interactions between D N A and neighbor ing macromolecules influence the stability of the double helix (3, 18-20 , 32, 33, 36), the pa t te rns of thermal dena tura t ion of D N A in situ provide informat ion regarding the molecular s t ructure of nuclear chromat in . This me thod was used to investigate chromat in changes during