The proteins of polytene chromosomes of Drosophila hydei

It is reported that chromatin can be prepared from highly purified polytene nuclei from the salivary glands of third instar larvae of Drosophila hydei; such chromatin differs from that of diploid nuclei mainly by deficiencies in certain nonhistone chromosomal proteins. It is suggested that these proteins are important components of constitutive heterochromatin, which is severely underrepresented in polytene chromosomes. Chromosome morphology, including the pattern of induced puffs, is maintained throughout the mass isolation of glands and sucrose gradient purification of nuclei, as indicated by studies on temperature-shocked and control larvae. No significant alteration in the chromosomal proteins following puff induction by heat shock could be detected on analysis of the isolated protein fractions by disc gel electrophoresis. More sensitive techniques must be developed to study the apparent rearrangement or accumulation of protein at puff sites, and to elucidate the role of this protein in gene activation.     

Ultrastructural distribution of ribonucleoprotein complexes during mitosis. snRNP antigens are contained in mitotic granule clusters

The great majority of snRNP and hnRNP ribonucleoproteins have been shown to be confined to the nucleus except during periods of cell division. We have now determined the fine structure distribution of polypeptides associated with these RNP complexes during interphase and mitosis in mammalian tissue culture cells using immunoelectron microscopy. Many hnRNP antigens are found at the periphery of heterochromatin masses, known to be the sites of non-rRNP proteins initially surround areas of condensing chromatin and later become generally dispersed throughout the mitotic cell. The Sm protein antigens of snRNP complexes are found diffusely distributed in interphase nuclei as well as concentrated in fields of interchromatin granules (ICG). Proteins of snRNP complexes, unlike those of hnRNP, are associated with discernible cellular structures during mitosis. By prometaphase/metaphase, dense granular clusters are observed to contain a high concentration of snRNPs. These mitotic granule clusters (MGCs) are often in close proximity to chromosomal masses by late anaphase/telophase. The MGC structures are morphologically similar to interchromatin granule fields found in interphase nuclei. Furthermore, like interchromatin granules, they are sites of a high concentration of snRNP antigens and do not contain detectable hnRNP proteins or DNA.     

A protein released by DNAase I digestion of drosophila nuclei is preferentially associated with puffs.

Antisera have been produced against five molecular weight subfractions of the Drosophila proteins readily extracted from nuclei following limited DNAase I digestion. Immunofluorescence staining techniques were used to assess the distributions of these proteins in the polytene chromosomes of Drosophila. In three cases, the antigens were widely distributed; in one case, the antigens appeared to be slightly more concentrated at active loci; and in one case, the antigens were strongly concentrated at a defined set of loci, including puffs and most of the loci which are active (puffed) at some time during third instar larval and prepupal development. The latter distribution pattern differs from that of RNA polymerase. Nonhistone chromosomal proteins of this type may have a key role in establishing and/or maintaining the altered chromatin structure characteristic of the active state.     

The myc proteins are not associated with chromatin in mitotic cells.

The protein products of cellular and viral myc oncogenes are detected in nuclei by immunofluorescence. No myc fluorescence is found in nucleoli. In mitotic cells the myc antigens are not found associated with metaphase chromosomes, but are diffusely distributed throughout the cytoplasm. Cytoplasmic myc fluorescence is first observed when chromatin begins to condense in early prophase. Granular nuclear myc fluorescence is again discerned in telophase cells, when the nuclear envelope is formed and becomes more prominent upon cytokinesis; concomitantly the diffuse cytoplasmic myc staining is lost. These results suggest that myc proteins not only bind to DNA or chromatin, but are also associated with other structural systems in the nuclei.     

Specific interactions of chromatin with the nuclear envelope: positional determination within the nucleus in Drosophila melanogaster.

Specific interactions of chromatin with the nuclear envelope (NE) in early embryos of Drosophila melanogaster have been mapped and analyzed. Using fluorescence in situ hybridization, the three-dimensional positions of 42 DNA probes, primarily to chromosome 2L, have been mapped in nuclei of intact Drosophila embryos, revealing five euchromatic and two heterochromatic regions associated with the NE. These results predict that there are approximately 15 NE contacts per chromosome arm, which delimit large chromatin loops of approximately 1-2 Mb. These NE association sites do not strictly correlate with scaffold-attachment regions, heterochromatin, or binding sites of known chromatin proteins. Pairs of neighboring probes surrounding one NE association site were used to delimit the NE association site more precisely, suggesting that peripheral localization of a large stretch of chromatin is likely to result from NE association at a single discrete site. These NE interactions are not established until after telophase, by which time the nuclear envelope has reassembled around the chromosomes, and they are thus unlikely to be involved in binding of NE vesicles to chromosomes following mitosis. Analysis of positions of these probes also reveals that the interphase nucleus is strongly polarized in a Rabl configuration which, together with specific targeting to the NE or to the nuclear interior, results in each locus occupying a highly determined position within the nucleus.     

Kinetochore components recognized by human autoantibodies are present on mononucleosomes.

We have developed a competitive enzyme-linked immunosorbent assay for solubilized kinetochore components, using human CREST (calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia) scleroderma autoimmune antibodies specific for these kinetochore elements. Using this quantitative assay, we found interphase persistent or "pre-kinetochore" components in low- and moderately high-salt (375 mM salt) extracts of micrococcal nuclease-digested rat liver and chicken erythrocyte nuclei. The release of antigen activity from nuclei under these conditions has been correlated with loss of pre-kinetochore foci as determined by immunofluorescence microscopy. Combined biochemical and competition assay analysis of chicken erythrocyte nuclear extracts indicates that pre-kinetochore components are tightly bound to chromatin of mononucleosome size. The conclusions based on competition assay data are supported by a direct binding assay, which confirms that antigens recognized by CREST sera are present on chromatin. These results raise the possibility that the kinetochore-specific chromosomal antigen(s) we have detected substitutes for "standard" mononucleosome components, such as histone H1. Furthermore, they suggest approaches to the isolation of kinetochore-specific DNA sequences from higher eucaryotes.     

Nonlamin components of the lamina: a paucity of proteins.

Current models of nuclear organization propose that nuclear functions are modulated in part by reversible tethering of chromatin loops to structural elements of the nucleoplasm and the nuclear envelope. Lamins are the best-characterized proteins of the lamina portion of the nuclear envelope and are involved in binding chromatin to the inner nuclear membrane. However, they are not a universal feature of eukaryotic nuclei and do not account fully for the putative functions of the lamina in all organisms. It is possible that nonlamin components of the lamina may substitute for lamins in organisms from which they are absent and modify the properties of lamins during development and the cell cycle. We review the properties of the relatively small number of such components that have been reported, including the young arrest (fs(1)Ya) protein of Drosophila, statin, circumferin, and the MAN antigens. The experimental evidence indicates they are a diverse group of proteins, and that at least some have the potential to modulate the interactions of chromatin, lamins, and the nuclear membranes.     

The photoaddition of trimethylpsoralen to Drosophila melanogaster nuclei: a probe for chromatin substructure.

Derivatives of the furocoumarin, psoralen, can penetrate intact cells or nuclei and cross-link opposite strands of the chromosomal DNA under the influence of long wave-length ultraviolet light. The potential of trioxsalen (4,5',8-trimethylpsoralen) as a probe for chromatin structure has been investigated. The DNA in both embryo nuclei and tissue culture cells from Drosophila melanogaster was found to be about 90% protected from trioxsalen binding relative to purified DNA. Digestion of trioxsalen-treated nuclei by micrococcal nuclease and gel electrophoresis of the resulting DNA gave the same type of band pattern that is characteristic of native, untreated nuclei are digestion. Nuclease digestion was therefore used to examine the distribution of bound trioxsalen in the DNA. The resulting DNA fragments were analyzed both by radioactivity measurements and quantitative electron microscopy. The nuclease cleaved intact photoreacted nuclei in such a way that preferential excision of trioxsalen containing regions of the DNA occurred, but, when acting upon purified DNA that contained bount trioxsalen, it attacked the trioxsalen-free regions preferentially. It was thus concluded that trixosalen binds at the sites corresponding to the regular nuclease-sensitive regions of the chromatin in nuclei.     

The human origin recognition complex protein 1 dissociates from chromatin during S phase in HeLa cells

We investigated the association of human origin recognition complex (ORC) proteins hOrc1p and hOrc2p with chromatin in HeLa cells. Independent procedures including limited nuclease digestion and differential salt extraction of isolated nuclei showed that a complex containing hOrc1p and hOrc2p occurs in a nuclease-resistant compartment of chromatin and can be eluted with moderate high salt concentrations. A second fraction of hOrc2p that dissociates in vitro at low salt conditions was found to occur in a chromatin compartment characterized by its high accessibility to micrococcal nuclease. Functional differences between these two sites become apparent in HeLa cells that synchronously enter the S phase after a release from a double-thymidine block. The hOrc1p/hOrc2p-containing complexes dissociate from their chromatin sites during S phase and reassociate at the end of mitosis. In contrast, the fraction of hOrc2p in nuclease-accessible, more open chromatin remains bound during all phases of the cell cycle. We propose that the hOrc1p/hOrc2p-containing complexes are components of the human origin recognition complex. Thus, the observed cell cycle-dependent release of the hOrc1p/hOrc2p-containing complexes is in line with previous studies with Xenopus and Drosophila systems, which indicated that a change in ORC stability occurs after prereplication complex formation. This could be a powerful mechanism that prevents the rereplication of already replicated chromatin in the metazoan cell cycle.     

Chromatin fine structure of the histone gene complex of Drosophila melanogaster.

We have used salt extractions of nuclei and long agarose gels to dissect the chromatin fine structure of the histone gene repeat of Drosophila melanogaster. Extraction of nuclei with 0.35 M KCl removes many non-histone chromosomal proteins but does not significantly disturb the overall nucleosome arrangement of the repeat unit. After extraction of nuclei with 0.55 M KCl, which also removes histone Hl, the basic arrangement of nucleosome core particles in the repeat unit is not greatly disturbed and the exposed DNA segments near the 5' ends of the histone genes are also retained. Extraction of nuclei with 0.75 M or higher KCl concentrations causes extensive nucleosome sliding and rearrangement with accompanying changes in the nucleoprotein organization of the histone gene complex and loss of the 5' hypersensitive sites. Our results indicate that the histone gene repeat displays a highly organized chromatin structure in vivo.     

Nuclear envelope assembly around sperm chromatin in cell-free preparations from Drosophila embryos

Chicken sperm chromatin initiated an assembly of interphase-like nuclei in a cell-free cytoplasmic preparation from 1-6 h old Drosophila melanogaster embryos. The formation of these interphase-like nuclei from the condensed sperm chromatin happened in a series of distinct steps. Anti-Drosophila lamin monoclonal antibody stained the assembled nuclei in a pattern indistinguishable from normal Drosophila nuclei. This assembly process required an ATP regenerating system and could be blocked by the addition of novobiocin into the cell-free extract.