A unified phylogeny-based nomenclature for histone variants

Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure

High resolution mapping of Xenopus laevis 5S and ribosomal RNA genes by EM in situ hybridization.

We have developed a modification of in situ hybridization at the electron microscope level that permits simultaneous detection of at least two sequences. Probes are labelled with either biotin or AAF and detected with two distinct sizes of colloidal gold. This protocol has been applied to map the positions of Xenopus laevis oocyte-type 5S genes relative to ribosomal precursor genes in several independently derived cell lines. The results for the line TRXO, which expresses some oocyte 5S RNA, indicate that this inappropriate expression is not due to translocation from telomeric sites into the nucleolus organizer, as previously hypothesized. In addition we found that four other Xenopus cell lines, none of which express these genes, also contain distinct 5S oocyte translocations. These results suggest that an alteration in chromosome position is insufficient to result in gene activation and that sequences which are telomeric-proximal are exceptionally prone to translocation

Nucleosome dissociation at physiological ionic strengths.

Monomer nucleosomes purified on isokinetic sucrose gradients are shown to dissociate into component DNA and histones at physiological ionic strength upon dilution to a DNA concentration below 20 microgram/ml. The starting material is 11S, contains 145-190 BP DNA, and equimolar amounts of the four core histones with slightly less H1. Dilution of monomers in the presence of 0.14 M NaCl results in the rapid conversion of 10-40% of the 3H thymidine labeled material from 11S to 5S (5S is coincident with the S value of monomer length DNA). The proportion of nucleosomes which dissociate increases with increasing NaCl concentration between 0.15 M and 0.35 M and decreases with increasing DNA concentration above 1 microgram/ml. Recycling 11S monomers, which remain after dissociation, through a second dilution in salt generates an equivalent proportion of 5S material as seen after the initial dilution. Thus, the dissociation does not result from special properties of a subset of nucleosomes. An equilibrium between intact monomer and free DNA and histones appears to be rapidly established under the conditions described and the dissociated DNA will reassociate with histones to form 11S monomers if conditions of high DNA concentration and low ionic strength are established

Mouse satellite DNA, centromere structure, and sister chromatid pairing.

The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes

Deoxyribonucleic acid sequence mapping on metaphase chromosomes by immunoelectron microscopy.

Nucleic acid sequences can be localized on chromosomes in the electron microscope after hybridization with a biotinylated DNA probe followed by detection with a primary antibiotin antibody and a secondary antibody coupled to colloidal gold. Hybridization probes can also be labelled with alternative ligands such as N-acetoxy-2-acetylaminofluorene (AAF), Dinitrophenyl-dUTP and Digoxigenin-dUTP. Multiple labelling is possible if these differently modified DNA probes are used in conjunction with colloidal gold preparations of varying particle sizes. A substantial signal amplification can be achieved by incubating preparations with successive cycles of primary antibiotin antibody followed by a biotinylated secondary antibody. Detection is with Streptavidin-gold, and in the case of highly and moderately repeated sequences, the signal is visible in the light microscope. Detailed protocols are given for EM in-situ hybridization to whole mount metaphase chromosomes and include instructions necessary to perform multiple sequence localization and signal amplification

Mouse satellite DNA, centromere structure, and sister chromatid pairing.

The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes