CHROMATIN ORGANIZATION IN THE HOMOGENEOUSLY STAINING REGIONS OF A METHOTREXATE-RESISTANT MOUSE-CELL LINE - INTERSPERSION OF INACTIVE AND ACTIVE CHROMATIN DOMAINS DISTINGUISHED BY ACETYLATION OF HISTONE H4

We have analyzed the organization of the homogeneously staining region s (HSRs) in chromosomes from a methotrexate-resistant mouse melanoma c ell line, Fluorescence in situ hybridization techniques were used to l ocalize satellite DNA sequences and the amplified copies of the dihydr ofolate reductase (DHFR) gene that confer drug-resistance, in combinat ion with immunofluorescence using antibody probes to differentiate chr omatin structure, We show that the major DNA species contained in the HSRs is mouse major satellite, confirming previous reports, and that t his is interspersed with DHFR DNA in an alternating tandem array that can be resolved at the cytological level. Mouse minor satellite DNA, w hich is normally located at centromeres, is also distributed along the HSRs, but does not appear to interfere with centromere function, The blocks of major satellite DNA are coincident with chromatin domains th at are labelled by an autoantibody that recognizes a mammalian homolog ue of Drosophila heterochromatin-associated protein 1, shown previousl y to be confined to centric heterochromatin in mouse, An antiserum tha t specifically recognizes acetylated histone H4, a marker for active c hromatin, fails to bind to the satellite DNA domains, but labels the i ntervening segments containing DHFR DNA. We can find no evidence for t he spreading of the inactive chromatin domains into adjacent active ch romatin, even after extended passaging of cells in the absence of meth otrexate selection.