STRUCTURE OF INTERPHASE AND METAPHASE CHROMOSOMES

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We shall continue our analysis of the chromatin assembly reaction catalyzed by the high speed supernatant of Xenopus laevis oocytes (S-150). The fate of the histones and non-histone proteins during the reaction will be monitored by immunoblots with specific histone antibodies and by T25I labelling of the DNA-bound proteins in the nascent chromatin. We shall conduct parallel studies on the composition of nascent chromatin in a kidney cell line of Xenopus laevis, to ascertain whether the in vitro process of assembly which we have characterized reflects the in vivo reaction that takes place during chromatin replication. We will characterize the dynamic (torsionally strained), transcriptionally active chromatin assembled with TFIIIA, the positive transcription factor of the 5S RNA gene. The composition of the dynamic minichromosomes will be examined by immunoblots with the histone antibodies and by 125I labeling. The dynamic, "open" nucleosomes will be characterized by electron microscopy and by MNase (micrococcal nuclease) and DNase I digestion. We will continue the purification of the factors required for the assembly of transcriptionally active chromatin. In particular, we shall purify and characterize the eucaryotic DNA gyrase, an enzyme which plays a key role in the production and maintenance of the torsionally strained supercoils in dynamic chromatin. These studies should illuminate the mechanisms which govern gene expression in eucaryotes.