Toward understanding the controls affecting eucaryotic chromosome replication, we used a runoff replication assay to investigate whether the activity of a gene is related to its use of an upstream or downstream replication origin. When in vivo-initiated DNA polymerases are allowed to complete replication in vitro in the presence of bromodeoxyuridine triphosphate the density label is preferentially incorporated into origin-distal regions of DNA. Isopycnic centrifugation and blot hybridization analysis of the relative bromodeoxyuridine triphosphate incorporation into fragments spanning the chicken alpha-globin locus indicate that this region is replicated from an upstream origin both in chicken lymphocytes and in erythrocytes. Thus the replication polarity of these genes does not change as a function of transcriptional activity, consistent with earlier suggestions that DNA replication in the transcriptional direction may be a necessary but not sufficient condition for gene expression.The ordered nature of eucaryotic chromosome replication is evident in the replication of particular chromosome domains during discrete intervals of the S phase and in the synchronous initiation of replicon clusters whose size and number vary in a tissue-specific and developmentally regulated manner (reviewed in references 16, 21, and 47). These observations are consistent with recent data showing that DNA sequences complementary to probes for individual transcribed genes preferentially replicate early in the S phase and that chromosome position influences the timing of gene replication (6,11,17,20). In Saccharomyces cerevisiae, electron microscopic and biochemical data imply that DNA synthesis initiates at specific loci (5,7,18,46,50); the clearest evidence for the existence of discrete origins in higher eucaryotes comes from studies of the amplification of dihydrofolate reductase genes in CHO cells (22,23) and chorion genes in Drosophila follicle cells (36,44), where the extent -of amplification decreases bidirectionally from a central domain.The apparent nonrandom selection of sites for replication initiation and the asymmetry of chromatin replication at the level of DNA synthesis (16, 35) and protein deposition (28) have contributed to the proposal that replication origin selection may regulate gene activity (42, 49). As an initial step toward testing this hypothesis we have used a runoff replication assay to determine the direction of replication of the chicken alpha-globin genes in cells where these sequences are expressed (erythrocytes) or quiescent (lymphocytes). Following the logic used to locate the simian virus 40 origin of replication (12, 34), isolated nuclei were incubated in a replication cocktail containing bromodeoxyuridine (BrdU) triphosphate (BrdUTP) and allowed to complete the synthesis of DNA chains initiated in vivo. The direction of replication was deduced based on the relative incorporation of BrdU into DNA fragments encompassing the globin locus; those fragments farthest from the origin of replication incorpora...

To study the cell type specificity of the direction of replication of the human c-myc genes and the relationship of replication polarity to transcriptional activity, we analyzed the directions of replication of the c-myc genes in two Burkitt lymphoma cell lines, CA46 and ST486, and in HeLa cells. On the basis of in vitro runoff replication of forks initiated in intact cells, we found that transcribed c-myc genes in the germ line configuration in HeLa cells were replicated in the direction of transcription from origins in the 5'-flanking DNA, while the repressed, unrearranged c-myc genes of CA46 and ST486 cells were replicated in the antitranscriptional direction. In contrast, the transcribed c-myc genes of CA46 cells were replicated in the transcriptional direction, while the translocated, amplified c-myc genes of ST486 cells showed no preferred polarity of replication. The data also provided evidence for the existence of an endogenous barrier to DNA polymerases in the flanking DNA immediately 5' to the HeLa c-myc genes.The replication of eucaryotic chromosomes is controlled through the selection of sites for the initiation of DNA synthesis (2,6,7,10,12,14,19,21,24,32,39) and through the temporal programming of the activity at these sites (10,12,19,23). Combined with evidence for the existence of proteins which recognize the replication origins of viral minichromosomes (11,17,28,44), these results suggest that chromosome replication can be regulated in trans by DNA sequence-specific protein binding.That the activity of eucaryotic genes is related to the replication of chromatin is supported by several observations that transcribed genes are replicated early in the S phase of the cell cycle (8,9,16,18) and by the demonstration that the efficient transcription of certain transfected genes requires the replication of their chromatin template (13, 42). Moreover, Miller and Nasmyth (31) have demonstrated that passage through the S phase is necessary for repression of the silent mating type loci in Saccharomyces cerevisiae. Smithies (37) has proposed that the processes of replication and transcription are linked such that transcriptionally active genes are replicated from upstream origins, whereas inactive genes are replicated from either upstream or downstream origins. Consistent with this proposal, we have shown that the avian histone H5 genes are replicated in the transcriptional direction in embryonic erythrocytes, where they are expressed, but in the antitranscriptional direction in lymphoblastoid cells and chicken embryo fibroblasts, where they are quiescent (43). In comparison, the avian alpha-pi and alpha-D globin genes are replicated in the transcriptional direction both in cells in which they are active (erythrocytes) and in cells in which they are inactive (lymphoblastoid cells and fibroblasts) (26; Y. I. Lindstrom and M. Leffak, manuscript in preparation).To investigate further the relationship between replication polarity and transcriptional activity, we used the in vitro runoff replication (IVR) assay (2...

No abstract

No abstract

To study the cell type specificity of the direction of replication of the human c-myc genes and the relationship of replication polarity to transcriptional activity, we analyzed the directions of replication of the c-myc genes in two Burkitt lymphoma cell lines, CA46 and ST486, and in HeLa cells. On the basis of in vitro runoff replication of forks initiated in intact cells, we found that transcribed c-myc genes in the germ line configuration in HeLa cells were replicated in the direction of transcription from origins in the 5'-flanking DNA, while the repressed, unrearranged c-myc genes of CA46 and ST486 cells were replicated in the antitranscriptional direction. In contrast, the transcribed c-myc genes of CA46 cells were replicated in the transcriptional direction, while the translocated, amplified c-myc genes of ST486 cells showed no preferred polarity of replication. The data also provided evidence for the existence of an endogenous barrier to DNA polymerases in the flanking DNA immediately 5' to the HeLa c-myc genes.