Chromatin assembly in Xenopus oocytes: in vivo studies.
We have followed the time course of chromatin assembly, DNA supercoiling, and transcription on a Xenopus 5S RNA gene clone injected into germinal vesicles of Xenopus oocytes. During the first 2 hr after DNA injection, there is a gradual enhancement in transcription that correlates with the increase in superhelical density of the DNA template; on the other hand, nucleosome assembly is already completed by 10-30 min after DNA injection. To probe further the DNA structure in the assembled minichromosomes, we injected enzymes and chemicals into the germinal vesicle. DNAase I and topoisomerase I injections reveal that the circular DNA has been assembled into two discrete and equally abundant types of chromatin: one type, which we call "dynamic" chromatin, is torsionally strained and is thus fully relaxed by those two enzymes. The other type, which we call "static" chromatin, still yields supercoiled DNA molecules after deproteinization. The dynamic chromatin is also relaxed by injection of novobiocin, and simultaneously, 5S RNA transcription is turned off. The results of our in vivo experiments suggest that the dynamic chromatin is the one that is transcriptionally active. We discuss the biological relevance of these findings.
D000818:Animals; D002843:Chromatin / Q000502:physiology*; D003001:Cloning, Molecular; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D003850:Deoxyribonuclease I; D004706:Endodeoxyribonucleases / Q000378:metabolism; D005260:Female; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000378:metabolism; D009865:Oocytes / Q000502:physiology*; D010957:Plasmids; D012313:RNA / Q000235:genetics; D013997:Time Factors; D014158:Transcription, Genetic; D014981:Xenopus
D002843:Chromatin; D004278:DNA, Superhelical; D012313:RNA; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease; D004264:DNA Topoisomerases, Type I