On the chromatin structure of the amplified, transcriptionally active gene for dihydrofolate reductase in mouse cells.
The method for two-dimensional hybridization mapping of nucleosomes (Levinger, L., Barsoum, J., and Varshavsky, A. (1981) J. Mol. Biol. 146, 287-304) was used to analyze chromatin structure of the gene for dihydrofolate reductase (DHF reductase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase (EC 1.5.1.3)) in L5178Y-R mouse cells. The DHF reductase gene in these cells is amplified about 350-fold as a result of selection for resistance to methotrexate. Dramatic overproduction of DHF reductase mRNA in L5178Y-R cells suggests that most of the DHF reductase genes in these cells are transcribed. We report that all major mononucleosomal species resolvable by two-dimensional fractionation are detected by both DHF reductase- and satellite DNA-specific hybridization probes. Although the DHF reductase and satellite hybridization patterns differ somewhat from each other and from the total mononucleosomal pattern, their overall similarity is very high. In particular, no large differences in the abundance of mononucleosomes containing high mobility group non-histone proteins (HMG) 14 and 17 are seen between the DHF reductase and satellite chromatin regions under a wide variety of conditions for chromatin isolation, digestion, and fractionation. Possible interpretations of the apparent lack of selectivity of HMG-chromatin interactions in this system are discussed. We also found that the amplified DHF reductase genes possess a wide range of nucleosomal repeat lengths close to that in the bulk chromatin. In contrast, the range of nucleosomal repeat lengths in the satellite chromatin is much narrower than in both DHF reductase and bulk chromatin.
D000818:Animals; D002478:Cells, Cultured; D002843:Chromatin / Q000032:analysis / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D005796:Genes; D007940:Leukemia L5178 / Q000201:enzymology*; D007942:Leukemia, Experimental / Q000201:enzymology*; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D009693:Nucleic Acid Hybridization; D009707:Nucleosomes / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014158:Transcription, Genetic
D002843:Chromatin; D009707:Nucleosomes; D012333:RNA, Messenger; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate