Regulation of replication at the R/G chromosomal band boundary and pericentromeric heterochromatin of mammalian cells

An epichromatin epitope: Persistence in the cell cycle and conservation in evolution

“…Sites of DNA synthesis occur in different regions of the interphase nucleus during progression through S phase. [57][58][59] The most peripheral chromatin of the interphase nucleus appears to replicate during late S, yielding immunofluorescent images of incorporated nucleotides very similar to those of epichromatin staining, and containing the "G band" gene-poor heterochromatin. However, in our studies epichromatin staining of metaphase chromosomes does not resemble the alternating patterns of R (gene-rich) and G bands, traditionally employed for chromosome karyotyping.…”

Functional characterization of a mammalian transcription factor, Elongin A

“…A fairly scattered pattern of distribution was observed. This is probably reflects the fact that the rate of replication fork movement is not constant during the S phase of the cell cycle [21]. Most notably, however, the proportion of the replication forks that had progressed only a short distance, i.e., less than 20 kb, within a given time was significantly larger in Elongin A -/-MEFs than in wild-type MEFs.…”

“…After incubation at 37˚C with 5% CO 2 for 10 min, the samples were washed with serum-free medium and cultured in normal medium for 20 min or 30 min to introduce modified nucleotides into nascent DNA. DNA fiber preparation was performed as described previously [20,21]. Replication labeled cells and 10-to 20-fold unlabeled cells were mixed before sample preparation because DNA fibers were extended straight and each replication-labeled DNA fiber was well separated.…”

Non-Denaturing Fluorescencein SituHybridization to Find Replication Origins in a Specific Genome Region on the DNA Fiber

“…Each genomic locus or gene has distinct replication timing which is determined by firing of flanking origins, and its regulation is involved in higherorder chromatin structure, transcriptional activity, and spatial localization in the nucleus. [4][5][6] Fluorescence in situ hybridization (FISH) is a useful method of delineating the replication timing of the specific genomic loci in mammalian nuclei. 7,8) DNA probes give singlet and doublet hybridization signals in each nucleus, indicating before replication and after replication respectively.…”

“…Recently, a method of identifying the replication fork progression and the replication origins has been developed based on in vivo replication labeling and the DNA fiber spread technique. 6,9,10) Combination with DNA fiber FISH allows one to analyze replicon structures in specific regions in the mammalian genome. [10][11][12][13] Most studies have used halogenated deoxyribonucleosides such as bromo-, iodo-, and chlorodeoxyuridine (BrdU, IdU, and CldU respectively) to label replicating DNA.…”