Fractionation of human H1 subtypes and characterization of a subtype-specific antibody exhibiting non-uniform nuclear staining.

Four histone H1 subtypes and H1(0) were fractionated from human placental nuclei and purified to homogeneity by a combination of Bio-Rex 70 chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Polyclonal antibodies were generated in rabbits against one of these subtypes designated H1-3. Antibodies reacted only against this subtype in enzyme-linked immunosorbent assays and Western assays; subtype specificity was documented further by Western blotting of cell and nuclear extracts. They crossreacted with monkey H1, but not with H1 from other vertebrates tested. The epitope(s) recognized were mapped by immunoblotting against peptides prepared by cleavage with N-bromosuccinimide (NBS) and alpha-chymotrypsin; it includes the variant amino-terminal tail of the protein as well as a portion of the globular domain. The antibody stains mitotic chromosomes weakly but uniformly and, unlike antibodies that recognize total H1 which show uniform nuclear staining after indirect immunofluorescence localization, anti-H1-3 exhibits preferential labelling of the nuclear periphery. This non-uniform staining suggests compartmentalization of this subtype which may have functional significance with respect to differential chromatin condensation.

Ultrastructural localization of nucleic acid sequences in Saccharomyces cerevisiae nucleoli.

The putative nucleolus in Saccharomyces cerevisiae is visible in electron micrographs as a darkly stained, crescent-shaped structure associated with the nuclear envelope. The haploid yeast genome contains 100-200 tandem copies of a 9.1 kb ribosomal DNA (rDNA) repeat predicted to reside in this structure. We combined in situ hybridization of non-isotopically labeled probes to isolated S. cerevisiae nuclei with immunogold detection to localize rDNA and rDNA precursor sequences in nuclei at the electron microscope (EM) level. Gold particles are restricted to defined regions of nuclei which appear more electron dense than the bulk of the nucleus and which generally exhibit the crescent shape typical of the structure thought to be the nucleolus. In addition, snR17, the yeast homolog of mammalian U3, a nucleolar-restricted small nuclear RNA (snRNA), was localized to the same electron dense region of the nucleus. These data, in conjunction with published immunofluorescent localizations of nucleolar-associated antigens, provide definitive proof that the dense crescent is the nucleolus. Finally, the technique described is applicable to probing nuclear organization in a genetically manipulable system.