Interaction with the nuclear matrix of a chimeric construct containing a replication origin and a transcription unit.
We have studied the interaction of a chimeric construct containing an origin of replication (from bovine papilloma virus) and a hormonally regulated transcription unit (long terminal repeat from the mouse mammary tumor virus, driving the v-Ha-ras gene) with the nuclear scaffold and matrix from mouse fibroblasts. We used two experimental approaches because the nuclear matrix protein composition depends largely on the isolation conditions, making its definition mostly operational. In situ studies and in vitro experiments performed in 1361.5 cells, a cell line in which multiple copies of the construct have been established, indicate that two interesting regions of the construct interact with the nuclear matrix. The first region is located in the v-Ha-ras gene 5'-flanking sequences. These sequences come from the Harvey virus and contain a piece of the virus like 30S (VL30) sequences in which the v-Ha-ras gene is embedded. This DNA fragment was coupled to the thymidine kinase (TK) promoter driving the reporter luciferase gene and assayed in transient transfection experiments. Its insertion, in the sense orientation, upstream of the TK promoter resulted in a moderate enhancement (2-3-fold) of the luciferase activity. The second region is the most interesting from a physiological point of view. It contains the plasmid maintenance sequence 1 (PMS-1) and the core origin of replication of the bovine papilloma virus. Differences in the results from in situ (nuclear scaffold) and in vitro (nuclear matrix) experiments suggest that the components involved in the interaction with PMS-1 and the viral origin of replication are different. This may be of importance in the context of the recently proposed view that PMS-1 could be part of a composite origin of replication and provide information at a distance.
Adom
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JN
Unité de Recherches sur les Communications Hormonales, INSERM U-33, Hôpital du Kremlin Bicêtre, France.
Gouilleux
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F
Richard-Foy
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H
D016475:3T3 Cells; D000818:Animals; D052688:Bovine papillomavirus 1 / Q000235:genetics; D002461:Cell Line, Transformed; D004261:DNA Replication; D011905:Genes, ras; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D051379:Mice; D015530:Nuclear Matrix / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D008156:Luciferases; D013937:Thymidine Kinase; D001616:beta-Galactosidase