RESUMO
We undertook an ultrastructural study of the spermiogenesis of the lanternfish Lampanyctus crocodilus (Myctophiformes, Myctophidae) with special emphasis on the condensation of chromatin and the biochemical characterisation of its sperm nuclear basic proteins (SNBPs). The round head of the early spermatid of L. crocodilus develops into a curved conical-shaped head in the spermatozoon. Two flagella, present even in the spermatid, are inserted laterally at the convex side of the sperm head. Both flagella possess an axoneme with a 9 + 0 instead of the typical 9 + 2 axonemal structure. Mitochondria undergo a characteristic redistribution during spermiogenesis. A reduced number of them are present lying away from the centrioles at both ends of the concave side of the sperm head. During the chromatin condensation stages in spermiogenesis, fibrogranular structures with granules of 25 ± 5 and 50 ± 5 nm can be observed in the early spermatid and develop into larger granules of about 150 ± 50 nm in the middle spermatid. The latter granules coalesce during the transition to the advanced spermatid and spermatozoon giving rise to highly condensed chromatin in the sperm cell. Protamines are the main SNBPs associated with this chromatin; however, they are unusually large and correspond to the largest protamines described in fish to date. Small stoichiometric amounts of histones and other basic proteins coexist with these protamines in the spermatozoon.
Assuntos
Proteínas de Peixes/ultraestrutura , Peixes/fisiologia , Proteínas Nucleares/ultraestrutura , Espermatogênese , Espermatozoides/ultraestrutura , Animais , Cromatina/metabolismo , Cromatina/ultraestrutura , Proteínas de Peixes/análise , Proteínas de Peixes/metabolismo , Masculino , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Espermátides/metabolismo , Espermátides/ultraestrutura , Espermatozoides/metabolismoRESUMO
During early-to-middle spermiogenesis in multicellular, internally fertilizing charalean green algae (Chara fibrosa, Chara vulgaris, Chara tomentosa, Nitella missouriensis), patterning of chromatin/nucleoplasm in developing spermatid nuclei changes from granules â fibers â contorted lamellae â condensed chromatin. Cytochemical, immunocytochemical, electrophoretic studies on C. vulgaris and C. tomentosa spermatids (Kwiatkowska, Poplonska) and amino acid analysis of protamines in Chara corallina sperm (Reynolds, Wolfe), indicate that more positively charged protamines replace histones directly during spermiogenesis, not indirectly through other intermediate transitional proteins as in internally fertilizing neogastropods and sharks with more ordered spermatid lamellae. We hypothesize that such lamellar-mediated patterning is due to liquid-liquid phase separation by spinodal decomposition. This is a spontaneous thermodynamic process that involves diffusive instability of a lamellar chromatin network, a dominant pattern repeat distance and bicontinuity of chromatin/nucleoplasm phases. C. vulgaris sperm show contorted lamellae in the posterior region, whereas C. corallina sperm display contorted peripheral lamellae and interior fibrils. Among internally fertilizing liverworts, which may have evolved from Zygnematales, mid-spermatid nuclei lack lamellae. Instead they display self-coiled chromatin rods in Blasia pusilla, contain short chromatin tubules in Haplomitrium hookeri resembling those in internally fertilizing mosses and a hornwort and indirectly replace histones with protamines in Marchantia polymorpha.
Assuntos
Histonas/metabolismo , Protaminas/metabolismo , Espermatogênese , Briófitas/crescimento & desenvolvimento , Briófitas/metabolismo , Núcleo Celular/metabolismo , Clorófitas/genética , Clorófitas/crescimento & desenvolvimento , Cromatina/genética , Histonas/genética , Protaminas/genética , TermodinâmicaRESUMO
An interesting characteristic of decapod crustacean sperm nuclei is that they do not contain highly packaged chromatin. In the present study we re-examine the presence of DNA-interacting proteins in sperm nuclei of the brachyuran Maja brachydactyla. Although previous reports have indicated that, unlike the majority of sperm cells, DNA of decapod sperm is not organized by basic proteins, in this work we show that: (1) histones are present in sperm of M. brachydactyla; (2) histones are associated with sperm DNA; (3) histone H3 appears in lower proportions than the other core histones, while histone H2B appears in higher proportions; and (4) histone H3 in sperm nuclei is acetylated. This work complements a previous study of sperm histones of Cancer pagurus and supports the suggestion that decapod crustacean sperm chromatin deserves further attention.
Assuntos
Braquiúros/genética , Núcleo Celular/química , Cromatina/ultraestrutura , Nucleossomos/ultraestrutura , Espermatozoides/química , Aminoácidos/análise , Animais , Cromatina/química , Histonas/análise , MasculinoRESUMO
During spermiogenesis of Sepia officinalis histones are directly substituted by a molecule of precursor protamine, which is later transformed into the protamine through a deletion of the amino terminal end. In the present work, it is shown that the pattern of spermiogenic chromatin condensation consists of a phase of "patterning" and a phase of "condensation." In the phase of patterning, three structural remodelings are produced in the chromatin structure: [somatic-like chromatin --> 18 nm granules --> 25 nm fibers --> 44 nm fibers]. The first remodeling of the chromatin into granules of 18 nm takes place without the entrance of specific proteins in the spermiogenic nuclei. The second remodeling [granules of 18 nm --> fibers of 25 nm] is due to the entrance of the precursor protamine and its interaction with the DNA-histone complex. The third remodeling [fibers of 25 nm --> fibers of 44 nm] occurs simultaneously with the disappearance of histones from the chromatin. In the phase of condensation, the fibers of 44 nm coalesce among themselves to form progressively larger aggregates of chromatin. In this phase there are no substantial variations in the nuclear proteins, so that the condensation of the chromatin must respond to posttranscriptional changes of the precursor protamine (dephosphorylation, deletion of the amino-terminal end).
Assuntos
Cromatina/química , Histonas/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Espermatogênese , Sequência de Aminoácidos , Animais , Cromatina/ultraestrutura , Histonas/química , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/genética , Protaminas/química , Protaminas/genética , Sepia , Análise de Sequência de Proteína , Espermatozoides/ultraestruturaRESUMO
The characterization of the involvement of different histone post-translational modifications (PTMs) and histone variants in chromatin structure has represented one of the most recurrent topics in molecular biology during the last decade (since 1996). The interest in this topic underscores the critical roles played by chromatin in such important processes as DNA packaging, DNA repair and recombination, and regulation of gene expression. The genomic information currently available has pushed the boundaries of this research a step further, from the study of local domains to the genome-wide characterization of the mechanisms governing chromatin dynamics. How the heterchromatin and euchromatin compartmentalization is established has been the subject of recent extensive research. Many PTMs, as well as histone variants have been identified to play a role, including the replacement of histone H2A by the histone variant H2A.Z. Several studies have provided support to a role for H2A.Z (known as Htz1 in yeast) in transcriptional regulation, chromosome structure, DNA repair and heterochromatin formation. Although the mechanisms by which H2A.Z defines different structural regions in the chromatin have long remained elusive, various reports published last year have shed new insight into this process. The present mini review focuses its attention on the genome-wide distribution of H2A.Z, with special attention to the mechanisms involved in its distribution and exchange as well as on the role of its N-terminal acetylation.
RESUMO
Syndromes of disordered 'chromatin remodeling' are unique in medicine because they arise from a general deregulation of DNA transcription caused by mutations in genes encoding enzymes which mediate changes in chromatin structure. Chromatin is the packaged form of DNA in the eukaryotic cell. It consists almost entirely of repeating units, called nucleosomes, in which short segments of DNA are wrapped tightly around a disk-like structure comprising two subunits of each of the histone proteins H2A, H2B, H3 and H4. Histone proteins are covalently modified by a number of different adducts (i.e. acetylation and phosphorylation) that regulate the tightness of the DNA-histone interactions. Mutations in genes encoding enzymes that mediate chromatin structure can result in a loss of proper regulation of chromatin structure, which in turn can result in deregulation of gene transcription and inappropriate protein expression. In this review we present examples of representative genetic diseases that arise as a consequence of disordered chromatin remodeling. These include: alpha-thalassemia/mental retardation syndrome, X-linked (ATR-X); Rett syndrome (RS); immunodeficiency-centromeric instability-facial anomalies syndrome (ICF); Rubinstein-Taybi syndrome (RSTS); and Coffin-Lowry syndrome (CLS).
Assuntos
Montagem e Desmontagem da Cromatina/genética , Doenças Genéticas Inatas/complicações , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Mutação/genética , Montagem e Desmontagem da Cromatina/fisiologia , Síndrome de Coffin-Lowry/complicações , Síndrome de Coffin-Lowry/diagnóstico , Síndrome de Coffin-Lowry/genética , Humanos , Deficiência Intelectual Ligada ao Cromossomo X/complicações , Deficiência Intelectual Ligada ao Cromossomo X/diagnóstico , Deficiência Intelectual Ligada ao Cromossomo X/genética , Mutação/fisiologia , Síndrome de Rett/complicações , Síndrome de Rett/diagnóstico , Síndrome de Rett/genética , Síndrome de Rubinstein-Taybi/complicações , Síndrome de Rubinstein-Taybi/diagnóstico , Síndrome de Rubinstein-Taybi/genética , Talassemia alfa/complicações , Talassemia alfa/diagnóstico , Talassemia alfa/genéticaAssuntos
Cloroquina/análogos & derivados , Cromatina/química , Corantes/farmacologia , Substâncias Intercalantes/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Núcleo Celular/metabolismo , Cloroquina/farmacologia , Cromatina/metabolismo , Relação Dose-Resposta a Droga , Etídio/farmacologia , Células HeLa , Humanos , Células K562 , Nuclease do Micrococo/farmacologiaRESUMO
We have characterized for the first time the sperm nuclear basic proteins (SNBP) from two species of nemerteans: Cerebratulus californiensis and Cerebratulus lacteus. Gel electrophoretic and chromatographic (RP-HPLC) analysis of the nuclear sperm extracts indicate that histones are the major protein components which are present. The linker histones (histones of the H1 family) exhibit a rather unusual composition and some of them contain cysteine. Several histone H1 isoforms are present, one of which has a composition similar to that of other H1 histones found in the sperm chromatin of other groups of lower invertebrates.
Assuntos
Histonas/química , Invertebrados/fisiologia , Proteínas Nucleares/análise , Espermatozoides/química , Animais , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Ágar , Histonas/análise , MasculinoRESUMO
H2A.Z and H2A.1 nucleosome core particles and oligonucleosome arrays were obtained using recombinant versions of these histones and a native histone H2B/H3/H4 complement reconstituted onto appropriate DNA templates. Analysis of the reconstituted nucleosome core particles using native polyacrylamide gel electrophoresis and DNase I footprinting showed that H2A.Z nucleosome core particles were almost structurally indistinguishable from its H2A.1 or native chicken erythrocyte counterparts. While this result is in good agreement with the recently published crystallographic structure of the H2A.Z nucleosome core particle (Suto, R. K., Clarkson, M J., Tremethick, D. J., and Luger, K. (2000) Nat. Struct. Biol. 7, 1121-1124), the ionic strength dependence of the sedimentation coefficient of these particles exhibits a substantial destabilization, which is most likely the result of the histone H2A.Z-H2B dimer binding less tightly to the nucleosome. Analytical ultracentrifuge analysis of the H2A.Z 208-12, a DNA template consisting of 12 tandem repeats of a 208-base pair sequence derived from the sea urchin Lytechinus variegatus 5 S rRNA gene, reconstituted oligonucleosome complexes in the absence of histone H1 shows that their NaCl-dependent folding ability is significantly reduced. These results support the notion that the histone H2A.Z variant may play a chromatin-destabilizing role, which may be important for transcriptional activation.
Assuntos
Cromatina/química , Histonas/química , Nucleossomos/química , Dobramento de Proteína , Ativação TranscricionalRESUMO
Proteins can exist in a trinity of structures: the ordered state, the molten globule, and the random coil. The five following examples suggest that native protein structure can correspond to any of the three states (not just the ordered state) and that protein function can arise from any of the three states and their transitions. (1) In a process that likely mimics infection, fd phage converts from the ordered into the disordered molten globular state. (2) Nucleosome hyperacetylation is crucial to DNA replication and transcription; this chemical modification greatly increases the net negative charge of the nucleosome core particle. We propose that the increased charge imbalance promotes its conversion to a much less rigid form. (3) Clusterin contains an ordered domain and also a native molten globular region. The molten globular domain likely functions as a proteinaceous detergent for cell remodeling and removal of apoptotic debris. (4) In a critical signaling event, a helix in calcineurin becomes bound and surrounded by calmodulin, thereby turning on calcineurin's serine/threonine phosphatase activity. Locating the calcineurin helix within a region of disorder is essential for enabling calmodulin to surround its target upon binding. (5) Calsequestrin regulates calcium levels in the sarcoplasmic reticulum by binding approximately 50 ions/molecule. Disordered polyanion tails at the carboxy terminus bind many of these calcium ions, perhaps without adopting a unique structure. In addition to these examples, we will discuss 16 more proteins with native disorder. These disordered regions include molecular recognition domains, protein folding inhibitors, flexible linkers, entropic springs, entropic clocks, and entropic bristles. Motivated by such examples of intrinsic disorder, we are studying the relationships between amino acid sequence and order/disorder, and from this information we are predicting intrinsic order/disorder from amino acid sequence. The sequence-structure relationships indicate that disorder is an encoded property, and the predictions strongly suggest that proteins in nature are much richer in intrinsic disorder than are those in the Protein Data Bank. Recent predictions on 29 genomes indicate that proteins from eucaryotes apparently have more intrinsic disorder than those from either bacteria or archaea, with typically > 30% of eucaryotic proteins having disordered regions of length > or = 50 consecutive residues.
Assuntos
Conformação Proteica , Proteínas/química , Proteínas/fisiologia , Modelos Moleculares , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas/genética , Relação Estrutura-AtividadeRESUMO
The MgCl2-induced folding of defined 12-mer nucleosomal arrays, in which ubiquitinated histone H2A (uH2A) replaced H2A, was analyzed by quantitative agarose gel electrophoresis and analytical centrifugation. Both types of analysis showed that uH2A arrays attained a degree of compaction similar to that of control arrays in 2 mM MgCl2. These results indicate that attachment of ubiquitin to H2A has little effect on the ability of nucleosomal arrays to form higher order folded structures in the ionic conditions tested. In contrast, uH2A arrays were found to oligomerize at lower MgCl2 concentrations than control nucleosomal arrays, suggesting that histone ubiquitination may play a role in nucleosomal fiber association.
Assuntos
Histonas/metabolismo , Cloreto de Magnésio/metabolismo , Nucleossomos/metabolismo , Ubiquitinas/metabolismo , Animais , Bovinos , Centrifugação , Galinhas , Eletroforese em Gel de ÁgarRESUMO
The folding ability of chromatin fractions containing approximately identical nucleosome numbers and the same linker histone composition, but with different extents of core histone acetylation, were analyzed by analytical ultracentrifugation. It was found that the acetylated fractions consistently exhibited a relatively small but significantly lower extent of compaction than that of their native nonacetylated counterparts. This was regardless of the extent of the size distribution heterogeneity of the fractions analyzed. Furthermore the acetylated chromatin fibers exhibited an enhanced solubility in both NaCl and MgCl(2), which is neither the result of a differential binding affinity of the linker histones to chromatin nor of an alteration in the relative amounts of the histone H1 variants.
Assuntos
Cromatina/fisiologia , Cromatina/ultraestrutura , Histonas/metabolismo , Acetilação , Fracionamento Celular/métodos , Cromatografia Líquida de Alta Pressão , Células HeLa , Humanos , Cinética , Cloreto de Magnésio , Nucleossomos/fisiologia , Nucleossomos/ultraestrutura , Cloreto de Sódio , SolubilidadeRESUMO
In which taxa did H1 linker histones appear in the course of evolution? Detailed comparative analysis of the histone H1 and histone H1-related sequences available to date suggests that the origin of histone H1 can be traced to bacteria. The data also reveal that the sequence corresponding to the 'winged helix' motif of the globular structural domain, a domain characteristic of all metazoan histone H1 molecules, is evolutionarily conserved and appears separately in several divergent lines of protists. Some protists, however, appear to have only a lysine-rich basic protein, which has compositional similarity to some of the histone H1-like proteins from eubacteria and to the carboxy-terminal domain of the H1 linker histones from animals and plants. No lysine-rich basic proteins have been described in archaebacteria. The data presented in this review provide the surprising conclusion that whereas DNA-condensing H1-related histones may have arisen early in evolution in eubacteria, the appearance of the sequence motif corresponding to the globular domain of metazoan H1s occurred much later in the protists, after and independently of the appearance of the chromosomal core histones in archaebacteria.
Assuntos
Evolução Molecular , Histonas/química , Histonas/metabolismo , Filogenia , Sequência de Aminoácidos , Animais , Bactérias/química , Cromatina/química , Cromatina/metabolismo , Eucariotos/química , Fungos/química , Sequências Hélice-Alça-Hélice , Lisina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
In this review, we briefly analyze the current state of knowledge on histone variants and their posttranslational modifications. We place special emphasis on the description of the structural component(s) defining and determining their functional role. The information available indicates that this histone "variability" may operate at different levels: short-range "local" or long-range "global", with different functional implications. Recent work on this topic emphasizes an earlier notion that suggests that, in many instances, the functional response to histone variability is possibly the result of a synergistic structural effect.
Assuntos
Autoantígenos , Histonas/química , Processamento de Proteína Pós-Traducional/fisiologia , Acetilação , Sequência de Aminoácidos , Proteína Centromérica A , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Variação Genética , Histonas/metabolismo , Dados de Sequência Molecular , Fosforilação , Poli Adenosina Difosfato Ribose/metabolismo , Conformação Proteica , Proteínas/metabolismo , Ubiquitinas/metabolismoRESUMO
Proaerolysin, the proform of the channel-forming protein aerolysin, is secreted as a dimer by Aeromonas sp. The protein also exists as a dimer in the crystal, as well as in solution, at least at concentrations in the region of 500 microg/ml. Recently it has been argued that proaerolysin becomes monomeric at concentrations below 100 microg/ml and that only the monomeric form of the protoxin can bind to cell surface receptors (Fivaz, M., Velluz, M.-C., and van der Goot, F. G. (1999) J. Biol. Chem. 274, 37705-37708). Here we show, using non-denaturing polyacrylamide electrophoresis, chemical cross-linking, and analytical ultracentrifugation, that proaerolysin remains dimeric at the lowest concentrations of the protein that we measured (less than 5 microg/ml) and that the dimeric protoxin is quite capable of receptor binding.
Assuntos
Aeromonas/química , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Canais Iônicos/química , Toxinas Bacterianas/genética , Membrana Celular/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Dimerização , Eletroforese em Gel de Poliacrilamida , Eritrócitos/citologia , Eritrócitos/metabolismo , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Peso Molecular , Mutação , Proteínas Citotóxicas Formadoras de Poros , Ligação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Succinimidas/metabolismo , UltracentrifugaçãoRESUMO
This mini review consists of two parts. The first part will provide a brief overview of the theoretical aspects involved in the two kinds of experiments that can be conducted with the analytical ultracentrifuge (sedimentation velocity and sedimentation equilibrium) as they pertain to the study of chromatin. In the following sections, I describe the analytical ultracentrifuge experiments which, in my opinion, have contributed the most to our understanding of chromatin. Few other biophysical techniques, with the exception of X-ray scattering and diffraction, have contributed as extensively as the analytical ultracentrifuge to the characterization of so many different aspects of chromatin structure. In the course of his scientific career, Professor Henryk Eisenberg has made many important contributions to the theoretical aspects underlying ultracentrifuge analysis, especially in the analysis of solutions of polyelectrolytes and biological macromolecules [H. Eisenberg, Biological macromolecules and polyelectrolytes in solution, Clarendon Press, Oxford, 1976]. As an example he has devoted some of his research effort to the characterization of chromatin in solution. This review includes these important contributions.
Assuntos
Cromatina/química , Ultracentrifugação/métodos , Animais , Cromatina/genética , DNA/química , DNA/metabolismo , Histonas/química , Histonas/metabolismo , Modelos Teóricos , Conformação Molecular , Nucleossomos/química , Nucleossomos/genética , Ultracentrifugação/instrumentaçãoRESUMO
We have characterized the sperm nuclear basic proteins (SNBPs) of the sticklebacks in the suborder Gasterosteoidei. The complete amino acid sequence of the protamines from Aulorhynchus flavidus, Pungitius pungitius, Gasterosteus aculeatus, (anadromous) and G. wheatlandi, as well as the sequences of the protamines of several species pairs of freshwater G. aculeatus, have been determined. Analysis of the primary structure of these proteins has shown that: a) despite the relatively low amino acid complexity and small molecular mass of these basic proteins, they are very good molecular markers at the generic level. The bootstrap parsimony analysis using their sequences provides a phylogenetic relationship for the old anadromous species of Gasterosteoidei which is identical to that obtained from morphological and behavioral analysis; b) the comparison of the sequences also suggests that protamines from the suborder Gasterosteoidei have most likely evolved from a common gene in the early Acanthopterygii by an extension of the carboxy terminal portion of the molecule; c) protamines are not good markers for recent postglacial freshwater isolates of G. aculeatus. However, in the unique case of Enos Lake (British Columbia), we have been able to detect an additional minor protamine component in the benthic forms of G. aculeatus that is not present in the limnetic forms. Thus, this new protamine must have appeared during the past 12,000 years concomitantly with the speciation of benthics and limnetics in this lake.
Assuntos
Evolução Molecular , Peixes/genética , Proteínas Nucleares/genética , Protaminas/genética , Espermatozoides/química , Sequência de Aminoácidos , Animais , Peixes/metabolismo , Marcadores Genéticos , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/química , Fosforilação , Protaminas/química , Homologia de Sequência de AminoácidosRESUMO
In the multicelled filamentous ascomycete Ascolobus immersus, the single copy gene for histone H1 can be silenced by methylation in the process known as methylation-induced premeiotically (MIP). The results of a recent paper using this unique system(1) have shown that histone H1 silencing results in an enhanced DNA accessibility to nucleases and an increase in the overall extent of DNA methylation. Interestingly, while none of these effects appear to decrease the immediate viability of this fungus, silencing of histone H1 results in a significant decrease in its overall life span. These results suggest that while linker histones may be dispensable for the relatively short life span of an individual cell, they are most likely indispensable for survival of higher eukaryote organisms.
Assuntos
Ascomicetos/fisiologia , Metilação de DNA , Inativação Gênica , Histonas/genética , Animais , Ascomicetos/genética , Cromatina , Genes Fúngicos , Fatores de TempoRESUMO
The nature of the structural changes induced by histone acetylation at the different levels of chromatin organization has been very elusive. At the histone level, it has been proposed on several occasions that acetylation may induce an alpha-helical conformation of their acetylated N-terminal domains (tails). In an attempt to provide experimental support for this hypothesis, we have purified and characterized the tail of histone H4 in its native and mono-, di-, tri-, and tetra- acetylated form. The circular dichroism analysis of these peptides shows conclusively that acetylation does increase their alpha-helical content. Furthermore, the same spectroscopic analysis shows that this is also true for both the acetylated nucleosome core particle and the whole histone octamer in solution. In contrast to the native tails in which the alpha-helical organization appears to be dependent upon interaction of these histone regions with DNA, the acetylated tails show an increase in alpha-helical content that does not depend on such an interaction.