Ultrastructural, autoradiographic and electrophoretic examinations of Chara tomentosa spermiogenesis

Ultrastructure of a spermatid nucleus changes many times during spermiogenesis. Condensed chromatin forms irregular clusters during phases I-II, a continuous ring adjacent to a nuclear envelope during phases III-V and a network occupying the whole nucleus during phase VI. In advanced spermiogenesis dense chromatin disappears and short randomly positioned fibrils arise, then long parallel ones are found (phase VIII) which during phase IX form a lamellar structure. In mature spermatozoids (phase X) chromatin becomes extremely condensed. 3H-arginine and 3H-lysine incorporation into spermatids during 2-min incubation is intensive during phases IN, decreases during phases VI, VII and becomes very low during phases VIII-IX. Capillary electrophoresis has shown that during Chara tomentosa spermiogenesis replacement of histones with basic proteins whose mobility is comparable to that of salmon protamines takes place. At the beginning of spermiogenesis core and linker histones are found in spermatids. During early spermiogenesis protamine-like proteins appear and their amount increases in late spermiogenesis when core histones are still present. In mature spermatozoids only protamine-like proteins represented by 3 fractions: 9.1 kDa, 9.6 kDa, 11.2 kDa are found. Disappearance of linker histones following their modification precedes disappearance of core histones. The results indicate that dynamic rearrangement of chromatin ultrastructure and aminoacid incorporation rate during spermiogenesis are reflected in basic nuclear protein changes.

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DNA Replication in Quiescent Cell Nuclei: Regulation by the Nuclear Envelope and Chromatin Structure

Molecular Biology of the Cell ◽

10.1091/mbc.10.12.4091 ◽

1999 ◽

Vol 10 (12) ◽

pp. 4091-4106 ◽

Cited By ~ 31

Author(s):

Zhi Hong Lu ◽

Hongzhi Xu ◽

Gregory H. Leno

Keyword(s):

Dna Replication ◽

Nuclear Envelope ◽

Chromatin Structure ◽

Cell Nuclei ◽

Free System ◽

Replication Complex ◽

Linker Histones ◽

Egg Extract ◽

Initiation Of Replication ◽

Stable Association

Quiescent nuclei from differentiated somatic cells can reacquire pluripotence, the capacity to replicate, and reinitiate a program of differentiation after transplantation into amphibian eggs. The replication of quiescent nuclei is recapitulated in extracts derived from activated Xenopus eggs; therefore, we have exploited this cell-free system to explore the mechanisms that regulate initiation of replication in nuclei from terminally differentiatedXenopus erythrocytes. We find that these nuclei lack many, if not all, pre-replication complex (pre-RC) proteins. Pre-RC proteins from the extract form a stable association with the chromatin of permeable nuclei, which replicate in this system, but not with the chromatin of intact nuclei, which do not replicate, even though these proteins cross an intact nuclear envelope. During extract incubation, the linker histones H1 and H10 are removed from erythrocyte chromatin by nucleoplasmin. We show that H1 removal facilitates the replication of permeable nuclei by increasing the frequency of initiation most likely by promoting the assembly of pre-RCs on chromatin. These data indicate that initiation in erythrocyte nuclei requires the acquisition of pre-RC proteins from egg extract and that pre-RC assembly requires the loss of nuclear envelope integrity and is facilitated by the removal of linker histone H1 from chromatin.

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The analysis of 40 kDa nuclear protein, p40, in interphase cells and mitotic cells

Journal of Cell Science ◽

10.1242/jcs.106.3.741 ◽

1993 ◽

Vol 106 (3) ◽

pp. 741-748 ◽

Cited By ~ 1

Author(s):

Y. Kaneda ◽

K. Kinoshita ◽

M. Sato ◽

K. Tanaka ◽

Y. Kaneda

Keyword(s):

Monoclonal Antibody ◽

Nuclear Envelope ◽

Nuclear Protein ◽

Dnase I ◽

High Salt ◽

The Other ◽

Interphase Nuclei ◽

Interphase Cells ◽

Detergent Treatment ◽

Sequential Treatments

We previously reported that the monoclonal antibody M108 recognized a 40 kDa protein both in the nucleus and the cytoplasm. This nuclear 40 kDa antigen was located in the nuclear envelope in interphase cells and in the perichromosomal region during mitosis. Now, we have analyzed this nuclear 40 kDa protein (p40) further, through morphological and biochemical approaches. At the beginning of mitosis, the perinuclear p40 detached from the nuclear envelope and moved to surround the condensing chromatin, while in the late stage of mitosis, the perichromosomal p40 moved back to the reassembled nuclear envelope. Most of the perichromosomal p40 on the metaphase chromosome was solubilized only by DNase I treatment, not by either high salt or detergent treatment. On the other hand, the perinuclear p40 was not solubilized by DNase1 alone, or high salt detergent alone. Sequential treatments with DNase I and high salt detergent were required to extract p40 in interphase nuclei. These results suggest that p40 was associated both with the nuclear envelope and chromatin DNA in interphase nuclei, while it bound only to chromatin DNA in mitosis.

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Coilin Shuttles between the Nucleus and Cytoplasm InXenopus Oocytes

Molecular Biology of the Cell ◽

10.1091/mbc.10.10.3425 ◽

1999 ◽

Vol 10 (10) ◽

pp. 3425-3434 ◽

Cited By ~ 14

Author(s):

Michel Bellini ◽

Joseph G. Gall

Keyword(s):

Nuclear Envelope ◽

Pyruvate Kinase ◽

Western Blotting ◽

Nuclear Import ◽

Nuclear Protein ◽

Germinal Vesicle ◽

Marker Protein ◽

Coiled Bodies ◽

Antibody Complex ◽

Antigen Antibody

Coiled bodies are discrete nuclear organelles often identified by the marker protein p80-coilin. Because coilin is not detected in the cytoplasm by immunofluorescence and Western blotting, it has been considered an exclusively nuclear protein. In theXenopus germinal vesicle (GV), most coilin actually resides in the nucleoplasm, although it is highly concentrated in 50–100 coiled bodies. When affinity-purified anti-coilin antibodies were injected into the cytoplasm of oocytes, they could be detected in coiled bodies within 2–3 h. Coiled bodies were intensely labeled after 18 h, whereas other nuclear organelles remained negative. Because the nuclear envelope does not allow passive diffusion of immunoglobulins, this observation suggests that anti-coilin antibodies are imported into the nucleus as an antigen–antibody complex with coilin. Newly synthesized coilin is not required, because cycloheximide had no effect on nuclear import and subsequent targeting of the antibodies. Additional experiments with myc-tagged coilin and myc-tagged pyruvate kinase confirmed that coilin is a shuttling protein. The shuttling of Nopp140, NO38/B23, and nucleolin was easily demonstrated by the targeting of their respective antibodies to the nucleoli, whereas anti-SC35 did not enter the germinal vesicle. We suggest that coilin, perhaps in association with Nopp140, may function as part of a transport system between the cytoplasm and the coiled bodies.

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High-performance capillary electrophoresis of core histones and their acetylated modified derivatives

Biochemical Journal ◽

10.1042/bj2830467 ◽

1992 ◽

Vol 283 (2) ◽

pp. 467-471 ◽

Cited By ~ 58

Author(s):

H Lindner ◽

W Helliger ◽

A Dirschlmayer ◽

M Jaquemar ◽

B Puschendorf

Keyword(s):

Capillary Electrophoresis ◽

Capillary Zone Electrophoresis ◽

Rat Liver ◽

Phosphate Buffer ◽

High Performance ◽

Buffer System ◽

Zone Electrophoresis ◽

Core Histones ◽

Capillary Zone ◽

High Performance Capillary Electrophoresis

By using high-performance capillary electrophoresis, we have successfully separated rat liver core histones into several subfractions. Inconvenient interactions of the highly basic proteins with the capillary wall were eliminated by a phosphate buffer system containing 0.03% hydroxyprophylmethylcellulose. Sample amounts of a few nanolitres were analysed within about 20 min. Multiacetylated histones H4 and H3 from induced Friend erythroleukaemic cells prepurified by h.p.l.c. were clearly separated into their non-acetylated and distinct acetylated forms. Our results illustrate that the application of capillary zone electrophoresis on its own or in combination with h.p.l.c. to the analysis of histones provides an important new alternative to traditional gel electrophoreses.

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A compendium of the histone H1 family of somatic subtypes: An elusive cast of characters and their characteristics

Biochemistry and Cell Biology ◽

10.1139/o01-099 ◽

2001 ◽

Vol 79 (3) ◽

pp. 289-304 ◽

Cited By ~ 65

Author(s):

Missag H Parseghian ◽

Barbara A Hamkalo

Keyword(s):

Historical Context ◽

Histone H1 ◽

General Function ◽

Linker Histones ◽

Chromatin Compaction ◽

The Core ◽

The Past ◽

Current State ◽

Core Histones

The last 35 years has seen a substantial amount of information collected about the somatic H1 subtypes, yet much of this work has been overshadowed by research into highly divergent isoforms of H1, such as H5. Reports from several laboratories in the past few years have begun to call into question some of the traditional views regarding the general function of linker histones and their heterogeneity. Hence, the impression in some circles is that less is known about these ubiquitous nuclear proteins as compared with the core histones. The goal of the following review is to acquaint the reader with the ubiquitous somatic H1s by categorizing them and their characteristics into several classes. The reasons for our current state of misunderstanding is put into a historical context along with recent controversies centering on the role of H1 in the nucleus. Finally, we propose a model that may explain the functional role of H1 heterogeneity in chromatin compaction.Key words: histone H1, linker histones, chromatin organization, chromatin compaction, heat shock.

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Nuclear transport defects and nuclear envelope alterations are associated with mutation of the Saccharomyces cerevisiae NPL4 gene.

Molecular Biology of the Cell ◽

10.1091/mbc.7.11.1835 ◽

1996 ◽

Vol 7 (11) ◽

pp. 1835-1855 ◽

Cited By ~ 46

Author(s):

C DeHoratius ◽

P A Silver

Keyword(s):

Nuclear Envelope ◽

Nuclear Pore Complex ◽

Protein Import ◽

Nuclear Protein ◽

Nuclear Transport ◽

Nuclear Pore ◽

Temperature Sensitive ◽

Nonpermissive Temperature ◽

Temperature Sensitive Mutants ◽

Nuclear Protein Import

To identify components involved in nuclear protein import, we used a genetic selection to isolate mutants that mislocalized a nuclear-targeted protein. We identified temperature-sensitive mutants that accumulated several different nuclear proteins in the cytoplasm when shifted to the semipermissive temperature of 30 degrees C; these were termed npl (nuclear protein localization) mutants. We now present the properties of yeast strains bearing mutations in the NPL4 gene and report the cloning of the NPL4 gene and the characterization of the Np14 protein. The npl4-1 mutant was isolated by the previously described selection scheme. The second allele, npl4-2, was identified from an independently derived collection of temperature-sensitive mutants. The npl4-1 and npl4-2 strains accumulate nuclear-targeted proteins in the cytoplasm at the nonpermissive temperature consistent with a defect in nuclear protein import. Using an in vitro nuclear import assay, we show that nuclei prepared from temperature-shifted npl4 mutant cells are unable to import nuclear-targeted proteins, even in the presence of cytosol prepared from wild-type cells. In addition, npl4-2 cells accumulate poly(A)+ RNA in the nucleus at the nonpermissive temperature, consistent with a failure to export mRNA from the nucleus. The npl4-1 and npl4-2 cells also exhibit distinct, temperature-sensitive structural defects: npl4-1 cells project extra nuclear envelope into the cytoplasm, whereas npl4-2 cells from nuclear envelope herniations that appear to be filled with poly(A)+ RNA. The NPL4 gene encodes an essential M(r) 64,000 protein that is located at the nuclear periphery and localizes in a pattern similar to nuclear pore complex proteins. Taken together, these results indicate that this gene encodes a novel nuclear pore complex or nuclear pore complex-associated component required for nuclear membrane integrity and nuclear transport.

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Interaction of Maize Chromatin-Associated HMG Proteins with Mononucleosomes: Role of Core and Linker Histones

Biological Chemistry ◽

10.1515/bc.2003.114 ◽

2003 ◽

Vol 384 (7) ◽

pp. 1019-1027 ◽

Cited By ~ 5

Author(s):

J. Lichota ◽

K. D. Grasser

Keyword(s):

Dna Binding ◽

High Mobility ◽

Micrococcal Nuclease ◽

Hmg Proteins ◽

Binding Motifs ◽

Linker Histones ◽

Dna Binding Motifs ◽

Core Histones ◽

Hmgb Proteins ◽

Nucleosome Binding

AbstractTwo groups of plant chromatin-associated high mobility group (HMG) proteins, namely the HMGA family, typically containing four A/T-hook DNA-binding motifs, and the HMGB family, containing a single HMG-box DNA-binding domain, have been identified. We have examined the interaction of recombinant maize HMGA and five different HMGB proteins with mononucleosomes (containing approx. 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin. The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA in the presence of H1 differed from that observed in the absence of H1. HMGA and the HMGB proteins bound H1-containing nucleosome particles with similar affinity. The plant HMG proteins could also bind nucleosomes that were briefly treated with trypsin (removing the N-terminal domains of the core histones), suggesting that the histone N-termini are dispensable for HMG protein binding. In the presence of untreated nucleosomes and trypsinised nucleosomes, HMGB1 could be chemically crosslinked with a core histone, which indicates that the trypsin-resistant part of the histones within the nucleosome is the main interaction partner of HMGB1 rather than the histone N-termini. In conclusion, these results indicate that specific nucleosome binding of the plant HMGB proteins requires simultaneous DNA and histone contacts.

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Association of cytoplasmic dynein with manchette microtubules and spermatid nuclear envelope during spermiogenesis in rats

Journal of Cell Science ◽

10.1242/jcs.107.3.625 ◽

1994 ◽

Vol 107 (3) ◽

pp. 625-633 ◽

Cited By ~ 5

Author(s):

T. Yoshida ◽

S.O. Ioshii ◽

K. Imanaka-Yoshida ◽

K. Izutsu

Keyword(s):

Nuclear Envelope ◽

Mature Spermatozoon ◽

Cytoplasmic Dynein ◽

Bovine Brain ◽

Nuclear Surface ◽

Double Immunofluorescence ◽

Nuclear Shaping ◽

Spermatid Nucleus ◽

Microtubular Network ◽

Inner Region

During spermiogenesis, the shape of the spermatid nucleus, which is spherical, changes and it becomes the sperm head. A microtubular structure called a manchette is thought to be involved in this morphogenetic process. In this report, we demonstrate the localization of cytoplasmic dynein and manchette development by a double immunofluorescence technique using anti-bovine brain MAP 1C and anti-tubulin. Before step 6 of the Leblond and Clermont staging, the microtubules showed a fine reticular network, and the dynein staining was homogeneous. In step 6, the microtubular network was concentrated around the nucleus. The manchette developed in step 7 spermatids, and was fully formed, with a skirt-like appearance, covering the nuclear surface in step 8. Dynein fluorescence was associated with the microtubular manchette in steps 7–10. During these steps, the nucleus was protruded from the cytoplasm. In steps 11–13, the most active stages in nuclear shaping, the dynein was densely localized at the nuclear surface covered by the manchette. As the nucleus acquired a shape similar to the mature spermatozoon at step 14, the dynein fluorescence was localized only at the concave side of the nuclear caudal edge. The manchette became narrower and elongated. In step 15, the manchette extended into the elongated cytoplasm, diminishing during steps 16–18. The localization of the dynein was limited to the ventral aspect of the caudal head in these steps. There was little dynein fluorescence in mature spermatozoa. Immunoelectron microscopy showed positive reactions in the nuclear envelope and the inner region of the microtubular manchette. These observations suggest that cytoplasmic dynein, possibly bound to the nuclear envelope, and manchette microtubules are involved in the protrusion of the spermatid nucleus from the cytoplasm.

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Primate-specific histone variants

Genome ◽

10.1139/gen-2020-0094 ◽

2020 ◽

pp. 1-10

Author(s):

Dongbo Ding ◽

Thi Thuy Nguyen ◽

Matthew Y.H. Pang ◽

Toyotaka Ishibashi

Keyword(s):

Dna Repair ◽

Genomic Dna ◽

Expression Profiles ◽

Histone Variants ◽

Amino Acid Sequences ◽

Specific Expression ◽

Linker Histones ◽

Cellular Processes ◽

Core Histones ◽

And Function

Canonical histones (H2A, H2B, H3, and H4) are present in all eukaryotes where they package genomic DNA and participate in numerous cellular processes, such as transcription regulation and DNA repair. In addition to the canonical histones, there are many histone variants, which have different amino acid sequences, possess tissue-specific expression profiles, and function distinctly from the canonical counterparts. A number of histone variants, including both core histones (H2A/H2B/H3/H4) and linker histones (H1/H5), have been identified to date. Htz1 (H2A.Z) and CENP-A (CenH3) are present from yeasts to mammals, and H3.3 is present from Tetrahymena to humans. In addition to the prevalent variants, others like H3.4 (H3t), H2A.Bbd, and TH2B, as well as several H1 variants, are found to be specific to mammals. Among them, H2BFWT, H3.5, H3.X, H3.Y, and H4G are unique to primates (or Hominidae). In this review, we focus on localization and function of primate- or hominidae-specific histone variants.

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Unintegrated HIV-1 DNAs are loaded with core and linker histones and transcriptionally silenced

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1912638116 ◽

2019 ◽

Vol 116 (47) ◽

pp. 23735-23742 ◽

Cited By ~ 11

Author(s):

Franziska K. Geis ◽

Stephen P. Goff

Keyword(s):

Time Course ◽

Leukemia Virus ◽

Moloney Murine Leukemia Virus ◽

Linker Histones ◽

Immunodeficiency Virus ◽

Core Histones ◽

Posttranslational Histone Modifications ◽

Inactive Genes ◽

H3 Acetylation ◽

Hiv 1

Upon delivery into the nucleus of the host cell, linear double-stranded retroviral DNAs are either integrated into the host genome to form the provirus or act as a target of the DNA damage response and become circularized. Little is known about the chromatinization status of the unintegrated retroviral DNAs of the human immunodeficiency virus type 1 (HIV-1). In this study, we used chromatin immunoprecipitation to investigate the nature of unintegrated HIV-1 DNAs and discovered that core histones, the histone variant H3.3, and H1 linker histones are all deposited onto extrachromosomal HIV-1 DNA. We performed a time-course analysis and determined that the loading of core and linker histones occurred early after virus application. H3.3 and H1 linker histones were also found to be loaded onto unintegrated DNAs of the Moloney murine leukemia virus. The unintegrated retroviral DNAs are potently silenced, and we provide evidence that the suppression of extrachromosomal HIV-1 DNA is histone-related. Unintegrated DNAs were marked by posttranslational histone modifications characteristic of transcriptionally inactive genes: high levels of H3K9 trimethylation and low levels of H3 acetylation. These findings reveal insights into the nature of unintegrated retroviral DNAs.

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