Methodology has been developed to allow purification of yeast plasmid chromatin to apparent biochemical homogeniety. Several types of experiments have shown that the purified material is representative of greater than 90% of the total plasmid population and that the material is traanscriptionally active. Minichromosomes containing TRP1ARS1 yeast sequences contain seven nucleosomes (detected by both electron microscopy and determination of linking number), in agreement with earlier nuclease mapping data. Histones are the major protein constituents of the minichromosomes. Preliminary results indicate that purification of the minichromosome by a method using protein-nucleic acid affinity is feasible; this procedure is simpler and quicker than the purification using conventional biochemical techniques. Experiments aimed at understanding chromatin structure of regulated genes continue. The chromosomal organization of the CUP1 gene in yeast coding for a metal-inducible metallothionein-like protein has been determined for both basal and induced states. We have previously described changes in the organization of sea urchin histone genes during developmentally regulated transcription. One of the hypersensitive 5'-flanking regions of these genes selectively binds to a 55000 dalton protein which is present only when the genes are active. A Type IV collagen gene exon from Strongylocentrotus purpuratus has been cloned and sequenced. This gene is developmentally regulated, turning on at blastula. We have determined the spatial distribution of expression of the gene; synthesis of the collagen mRNA is confined largely, if not exclusively to primary mesenchyme cells throughout their differentiation.
