Structure of interphase nuclei in relation to the cell cycle. Chromatin organization in mouse L cells temperature-sensitive for DNA replication

Abstract

Mutant lines of mouse L cells, TS A1S9, and TS C1, show temperature-sensitive (TS) DNA synthesis and cell division when shifted from 34 ~ to 38.5~ With TS A1S9 the decline in DNA synthesis begins after 6-8 h at 38.5~ and is most marked at about 24 h. Most cells in S, G~, or M at temperature upshift complete one mitosis and accumulate in the subsequent interphase at G~ or early S as a result of expression of a primary defect, failure of elongation of newly made small DNA fragments. Heat inactivation of TS C1 cells is more rapid; they fail to complete the interphase in progress at temperature upshift and accumulate at late S or G2. Inhibition of both cell types is reversible on return to 34~ Cell and nuclear growth continues during inhibition of replication.Expression of both TS mutations leads to a marked change in gross organization of chromatin as revealed by electron microscopy. Nuclei of wild-type cells at 34 ~ and 38.5~ and mutant cells at 34~ show a range of aggregation of condensed chromatin from small dispersed bodies to large discrete clumps, with the majority in an intermediate state. In TS cells at 38.5~ condensed chromatin bodies in the central nuclear region become disaggregated into small clumps dispersed through the nucleus. Morphometric estimation of volume of condensed chromatin indicates that this process is not due to complete decondensation of ehromatin fibrils, but rather involves dispersal of large condensed chromatin bodies into finer aggregates and loosening of fibrils within the aggregates. The dispersed condition is reversed in nuclei which resume DNA synthesis when TS cells are downshifted from 38.5 ~ to 34~ The morphological observations are consistent with the hypothesis that condensed chromatin normally undergoes an ordered cycle of transient, localized disaggregation and reaggregation associated with replication. In temperature-inactivated mutants, normal progressive disaggregation presumably occurs, but subsequent lack of chromatin replication prevents reaggregation. 246J, CELL BIOLOGY t~ The Rockefeller University Press.

Modulation of lymphocyte nuclear matrix organization in vivo by 5,6‐dichloro‐1‐β‐D‐ribofuranosyl benzimidazole: an autoradiographic and immunofluorescence study

“…It has long been acknowledged that changes in the metabolic status of the nucleus, whether a result of differentiation [11,24], activation [10,13,14,28,53,54] or pathogenesis induced by mutation [52], drugs [9,23,33,48], virus infection [12,18] or malignancy [25], have profound effects on nuclear organization. Alterations in nuclear structure have generally been defined by differences in nuclear size and shape, extent of nucleolar development, and the state of chromatin aggregation.…”

Histone H1 deposition and histone-DNA interactions in replicating chromatin.

“…Newly replicated chromatin is probably unfolded (12,(24)(25)(26). As histone Hi is present very early during chromatin replication, it seems likely that further chromatin folding during the maturation process involves additional interactions between the linker DNA and the core histones and interactions between histone molecules of neighboring nucleosomes.…”

“…The higher order structure of newly replicated chromatin is probably unfolded (12,(24)(25)(26). Maturation of newly replicated chromatin is suggested to be mediated by histone Hi, which is known to play an important role in organization of the chromatosome and of the higher order structures of chromatin (27).…”

ts A1S9 locus in mouse L cells may encode a novobiocin binding protein that is required for DNA topoisomerase II activity.

“…7; unpublished data), which revealed that the chromatin-bound DNA is rendered inactive as an in situ template for replication, as a result of heat denaturation of the ts A1S9 gene product. These findings, and others which indicated that expression of the ts AlS9 defect results in modification of chromatin structure (8) and supercoiling of the nuclear DNA (9), suggested that the ts AlS9 mutation may affect a protein which determines DNA conformation-i.e., a topoisomerase.…”