Nucleosome assembly will studied in vitro using extracts of HeLa cell nuclei as a source of somatic cell nucleosome assembly factors. The appropriate DNA, histone modification state, incubation conditions will be determined for study of in vitro assembly. The formation of pre-nucleosomal histone-histone complexes, histone-DNA complexes, and factor-DNA complexes will be examined in order to establish the assembly pathway. Chromatin assembled in vitro will be tested for its ability to be transcribed by injection into Xenopus oocytes. Incubation of chromatin extracts derived from cells actively expressing sequences to be tested will be performed before, during, and after assembly in order to study the formation of transcriptionally active chromatin.
