New origins of DNA replication identified near the human IFNA2 gene

Cell type-specific regulation of a replication origin residing within an AT-rich/LINE-1-rich isochore

“…The two orders of magnitude difference in the activity of the same ori observed here in two cell lines suggests that this difference cannot be attributed to cell transformation/malignancy and is more likely due to different states of normal cells. The high activity of this ori characteristic of not only cells of T-lymphoblast (this study) but also of fibroblast origin 14 might be explained by a particular state of chromatin ready to express interferon alpha genes in both these cell types. 15 Thus, our data obtained on a model of established cell lines confirm the have found a metazoan chromosomal region that contained closely located ARS element(s) and elements important for episomal maintenance.…”

“…13,14 In this work, we have examined the GC content and LINE-1 density for the 430-kb type I interferon gene cluster including three oris identified earlier.…”

“…In the map of this segment, the IFNA13 3'-terminal base is the first one; cen, centromere; tel, telomere. In the case of the human epithelial cell line A-549, ~100 millions of cells from the adherent cell culture at ~50% confluence were grown as described for CH cells 14 and used for isolation of DNS. In the case of the human T-lymphoblastic cell line Jurkat, the non-adherent cell culture was grown according to recommendations at http://www.dsmz.de/human_and_animal_cell_lines/, and ~220 millions of cells collected from the exponential stage culture, containing ~500 thousands cells/ml, were used for isolation of DNS.…”

Transfecting DNA is frequently inserted near DNA replication origins

“…11 It is located close to the human interferon α 2 gene (IFNA2). As a first step toward functional dissection of this origin, we tested its replicator activity in the region of a major replication start site, ori-1, by transferring the overlapping origin fragments F2067, F1283 and F458 (2,067, 1,283 and 458 bp length, respectively), cloned in the pEGFP-C1.1 vector, to random ectopic sites of the human genome (Fig.…”

“…dNA replication initiation activities within ectopic chromosomal insertions of vectors (peGFP-C1.1 and pcdNA6.Frt) and plasmid constructs containing a fragment (F2067, F1283 or F458) from the dNA replication origin located within the Homo sapiens genomic iFNA2 locus. in both (A and B) , below the histogram are shown the genetic structure of the ectopic ori-1-containing insertions (with the location of the Q-PCr primer sets/sequence-tagged sites) and a scheme of the endogenous (on human chromosome 9) iFNA2 locus with the replication start sites identified 11 and the cloned origin fragments. Shading of histogram bars corresponds to that of the origin fragments' scheme.…”