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.

No abstract

Temperature-sensitive (ts) A 1S9 mouse L cells continue to synthesize double-stranded covalently closed mitochondrial (mt) DNA at a temperature (38.5 degrees C) which is nonpermissive for chromosomal DNA replication. The amount of mt DNA made appears to be quantitatively linked to nuclear DNA synthesis. Nuclear DNA replication proceeds normally for 6-8 h after the cells are shifted to 38.5 degrees C, and then declines to reach a minimum at 20-24 h. The level of mt DNA synthesis remains high during this period and decreases once the ts lesion has been established.

Mouse adenovirus multiplies, apparently without impediment, in temperature-inactivated ts A1S9, ts C1 and ts 2 mouse fibroblasts. Thus, the DNA of mouse adenovirus can replicate in the absence of functional DNA topoisomerase II, a DNA-chain-elongation factor, and a protein required for traverse of the Gi/S interface, respectively, encoded in the ts A1S9, ts C1 and ts2 genetic loci. These results are compared with those obtained with polyoma virus.

Mouse L-cells treated with cytosine arabinoside, hydroxyurea, fluorodeoxyuridine, methotrexate, or mitomycin C rapidly cease DNA synthesis and stop dividing. Such inhibition of DNA replication is followed by interruption of formation of lysine- and arginine-containing proteins, including chromatin-bound histones, and by a major reorganization of the heterochromatin of the central nucleoplasm, manifest as disaggregation of large clumps of this condensed chromatin. Morphometric analysis revealed both cell and nuclear enlargement in cells treated with such antimetabolites of DNA replication. These observations are in contrast to those made with WT-4 cells starved of isoleucine or treated with cycloheximide. Isoleucine depletion was associated with inhibition of DNA synthesis and continued increase of cell and nuclear volume, but not with massive disaggregation of heterochromatin. Cycloheximide produced inhibition of DNA synthesis and protoplasmic growth, and also prevented structural reorganization of chromatin. A model is presented which suggests that initiation of chromatin replication is associated with a process, dependent upon de novo protein synthesis, which results in chromatin disaggregation. This can be revealed by inhibition of the correct replication of chromatin DNA and chromatin protein.