Dynamics of replication foci in early S phase as visualized by cross-correlation function.

To monitor gradual changes in the replication foci distribution during early S phase, different segments of newly synthesized DNA were visualized by immunocytochemical mapping of two consecutively incorporated deoxythymidine analogs in pulse-chase-pulse experiments in HeLa cells. The resulting dual-labeled fluorescence images were evaluated using cross-correlation function (CCF) analysis. General changes of CCF shape due to image deterioration caused by blur, noise, and lateral sampling (pixel size) were also discussed. Using CCF analysis of model images simulating either random initiation of new replication foci, or the firing of new foci in close proximity to completed ones, we were able to ascribe the changes in the early S replication foci distribution to the latter mechanism. In contrast to the data published previously, we monitored the dynamics of all replication foci for up to 3 h. In addition, we showed that the replication foci dynamics is well described by random walk model, so that the average de-localization of individual foci is proportional to square root of the applied chase.

Released chromatin: linearized DNA for high resolution fluorescence in situ hybridization.

Free DNA was prepared from routinely harvested and fixed cells for high resolution FISH mapping using either a sodium hydroxide/ethanol mixture or 70% formamide. Hybridization signals from cosmid probes appeared as extended lines. The average length of signals on DNA prepared with sodium hydroxide was significantly greater than with formamide. A set of overlapping cosmids from the HLA class II region was used to determine how precisely the actual overlap or gap between probes can be calculated from the measured overlap or gap between their signals. Lengths of the probe signals and their known kilobase lengths were used as an internal ruler. The mean values calculated from the measured length from 30 or more signals for each probe pair showed remarkable conformity with the known kilobase lengths of their overlaps and gaps. Immediately adjacent probes could also be ordered on the released DNA. These simple procedures dramatically increase the speed with which relationships between probes can be determined during contig construction.

Epidermal growth factor receptors in Rous sarcoma virus-transformed cells of different hosts.

It was found that epidermal growth factor (EGF) binding on cells transformed by Rous sarcoma virus (RSV) differed in mice and hamsters. While EGF binding was considerably reduced in mouse tumours, the binding activity of hamster cells did not change after RSV transformation.

Two simple procedures for releasing chromatin from routinely fixed cells for fluorescence in situ hybridization.

We describe two methods for releasing chromatin from routinely harvested and fixed cells. Using fluorescence in situ hybridization with combinations of probes from the HLA class II region, we show that good signals can be obtained on free chromatin fibers enabling determination of relationships between closely adjacent or overlapping probes.

c-myc gene activation as a permanent trait of RSV-infected quail cells.

A high level of c-myc gene expression was found to be a constant feature of v-src transformation. The c-myc gene product was analyzed in quail embryo cells transformed by different mutants of Rous sarcoma virus (RSV) that were temperature-sensitive with respect to various parameters of v-src function. The high-level expression of c-myc proved not to be temperature-sensitive: at both permissive (35 degrees C) and non-permissive (41 degrees C) temperatures, the same high levels of c-myc gene product were found for all RSV mutants tested. This result, in agreement with earlier evidence for a v-src-induced proliferative stimulus which was unaffected by ts mutants at the non-permissive temperature, shows that certain v-src functions have not yet been fully characterized.

Immunofluorescence detection of the vimentin epitope in chromatin structures of cell nuclei and chromosomes.

We describe immunofluorescence detection of the vimentin epitope, recognized by monoclonal antibody VI-01, in chromatin structures of eukaryotic cell nuclei and chromosomes. The approach used is based on increased sensitivity of 5-bromodeoxyuridine (BrdU)-substituted DNA to UV irradiation-induced crosslinking of DNA with proteins in vivo, by which the proteins interacting with chromosomal DNA can be immunovisualized in situ.

Effect of poly(I). poly(C) on the expression of cellular genes.

The effect of poly(I) . poly(C) on DNA and RNA synthesis induced in chicken embryo fibroblasts stimulated by calf serum from a quiescent to a growing state has been investigated. The results show that 1. poly(I) . poly(C) inhibits the induction of DNA synthesis in CEF stimulated by serum. 2. This inhibitory effect is dependent on the time between administration of poly(I) . poly(C) and stimulation, and is manifested at two discrete time intervals, at quiescent (Go) and late presynthetic (Gp) phases. 3. The 4-h incubation of quiescent cells with poly(I) . poly(C) before stimulation with calf serum inhibits and delays the onset of synthetic (S) phase, as manifested by the inhibition and retardation of induced DNA and RNA synthesis and by differences in the sedimentation profiles of total RNA synthesized at middle presynthetic phase of the stimulated cells.