RESUMEN
After fertilization the sea urchin sperm nucleus transforms into the male pronucleus which later fuses with the female pronucleus re-establishing the diploid genome of the embryo. This process requires remodeling of the sperm chromatin structure including the replacement of the sperm histones by maternally derived cleavage stage histone variants. In recent years, a group of protein complexes that promote chromatin-remodeling in an ATP-dependent manner have been described. To gain understanding into the molecular mechanisms operating during sea urchin male pronuclei formation, we analyzed whether chromatin-remodeling activity was present in unfertilized eggs as well as during early embryogenesis. We report that in the sea urchin Tetrapygus niger, protein extracts from the cytoplasm but not from the nucleus, of unfertilized eggs exhibit ATP-dependent nucleosome remodeling activity. This cytosolic activity was not found at early stages of sea urchin embryogenesis. In addition, by using polyclonal antibodies in Western blot analyses, we found that an ISWI-related protein is primarily localized in the cytoplasm of the sea urchin eggs. Interestingly, SWI2/SNF2-related proteins were not detected neither in the nucleus nor in the cytoplasm of unfertilized eggs. During embryogenesis, as transcriptional activity is increased an ISWI-related protein is found principally in the nuclear fraction. Together, our results indicate that the cytoplasm in sea urchin eggs contains an ATP-dependent chromatin-remodeling activity, which may include ISWI as a catalytic subunit.
Asunto(s)
Citoplasma/metabolismo , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Óvulo/metabolismo , Erizos de Mar/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Dominio Catalítico , Cromatina/enzimología , Cromatina/metabolismo , Citoplasma/enzimología , Embrión no Mamífero/enzimología , Embrión no Mamífero/metabolismo , Femenino , Masculino , Nucleosomas/enzimología , Óvulo/enzimología , Erizos de Mar/enzimologíaRESUMEN
During sea urchin embryogenesis the early histone genes are temporally expressed to accommodate the high demand for histone proteins during DNA replication at early cleavage stages of development. The early histone genes are transcriptionally active from the 16-cell stage, reaching a peak in expression at the 128-cell stage that gradually decreases until expression is completely inhibited at the late blastula stage. We are studying the gene regulatory mechanisms that control early histone gene expression in sea urchins to understand the interrelationships between chromatin remodeling and transcriptional activation during development. Here, we have investigated chromatin organization and transcription factor interactions by analyzing nuclease hypersensitivity and protein binding in the promoter region of the early histone H3 gene from the sea urchin Tetrapygus niger. We have found a DNase I hypersensitive domain centered at -90 in the early histone H3 gene promoter which is only detected in embryos at the 128-cell stage expressing high levels of early histone H3 mRNA. This hypersensitive site (-110 to -70) encompasses two regulatory elements (TnH3NFH3.1 and TnH3CCAAT). The -94 to -77 region of the histone H3 promoter is recognized by a transcription factor complex in nuclear extracts from 128-cell embryos. Methylation interference analysis and competition studies demonstrated a specific interaction at the CCAAT sequence. Using specific antibodies we find that the homeodomain transcription factor CDP/cut is the DNA-binding component of the complex interacting with the early histone H3 gene promoter in T. niger. Our results provide further evidence for the functional role of CDP/cut in developmental regulation of histone gene expression in phylogenetically diverse eukaryotic species.
Asunto(s)
Cromatina/genética , Histonas/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Represoras/metabolismo , Erizos de Mar/genética , Animales , Secuencia de Bases , Cromatina/metabolismo , Clonación Molecular , ADN/química , ADN/genética , ADN/metabolismo , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Erizos de Mar/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The variability of sperm histones was compared in two species of sea urchin. Whole sperm specific histones (SpH), were isolated from Tetrapygus niger (Arbacoida) and Parechinus angulosus (Echinoida). Individual histones were purified by chromatography on BioGel P-60 followed by reverse high pressure liquid chromatography (HPLC). The heterogeneity of each major histone type from T. niger was established from their HPLC elution patterns and further confirmed by electrophoresis in polyacrylamide gels containing 6 mM Triton X-100 combined with a transverse urea gradient (0--8 M). In T. niger, as well as in P. angulosus, a single form of SpH1 and SpH2A were found. In contrast, SpH2B was found to be heterogeneous, but represented by one major form in both species. The relatedness between both sets of histones was determined by establishing their immunological cross-reactivity. In this context, polyclonal antibodies elicited against T. niger sperm histones were assayed against individual histones from P. angulosus. From the results obtained, it emerged that histone SpH2A was the more closely related protein between these two species, followed by histone SpH1. In contrast, histone SpH2B was found to be only moderately related. These results confirm that SpH2A did not co-evolve with SpH2B, as was predicted for most species.
Asunto(s)
Histonas/análisis , Erizos de Mar/química , Espermatozoides/química , Animales , Cromatografía Líquida de Alta Presión , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Inmunoglobulina G/inmunología , Masculino , ConejosRESUMEN
Chromatin remodeling at eukaryotic gene promoter sequences accompanies transcriptional activation. Both molecular events rely on specific protein-DNA interactions that occur within these promoter sequences. Binding of CBFalpha/AML/PEBP2alpha (core binding factor alpha/acute myelogenous leukemia/polyoma enhancer binding protein 2alpha) proteins is a key event in both tissue-specific and developmentally regulated osteocalcin (OC) promoter activity. To address linkage between chromatin organization and transcription factor binding, we reconstituted segments of the rat OC gene proximal promoter into mononucleosomes and studied binding of CBFalpha proteins. We analyzed binding of bacterially produced Cbfalpha2Alpha and Cbfalpha2B, two splice variants of the human CBFalpha2 gene, and determined the effect of heterodimerization with the Cbfbeta subunit on binding activity. Our results indicate that binding of the truncated Cbfalpha2A protein to naked DNA is independent of Cbfbeta whereas Cbfalpha2A binding to nucleosomal DNA was enhanced by Cbfbeta. In contrast, the Cbfalpha2B interaction with either naked or nucleosomal DNA was strongly dependent on heterodimerization with the Cbfbeta subunit. Additionally, our results demonstrate that both Cbfalpha2A alone and Cbfalpha2B complexed with Cbfbeta can interact with nucleosomal DNA only if there is a degree of flexibility in the positioning of the histone octamer on the DNA fragment and exposure of the CBFalpha site. This situation was achieved with a DNA segment of 182 bp from the rat OC promoter that preferentially positions mononucleosomes upstream of the CBFalpha binding site and leaves this element partially exposed. Taken together, these results suggest that nucleosomal translational positioning is a major determinant of the binding of CBFalpha factors to nucleosomal DNA.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Proteínas de Neoplasias , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Factores de Transcripción/metabolismo , Animales , Composición de Base , Sitios de Unión/genética , Pollos , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Subunidades alfa del Factor de Unión al Sitio Principal , Factores de Unión al Sitio Principal , ADN/metabolismo , Proteínas de Unión al ADN/genética , Histonas/genética , Nucleosomas/genética , Osteocalcina/genética , Osteocalcina/metabolismo , Ratas , Homología de Secuencia de Ácido Nucleico , Factor de Transcripción AP-2 , Factores de Transcripción/genéticaRESUMEN
Fertilization in sea urchins is followed by the replacement of sperm-specific histones by cleavage-stage histone variants recruited from maternal stores. Such remodelling of zygote chromatin involves a cysteine proteinase that degrades the sperm-specific histones in a selective manner, leaving the maternal cleavage-stage histone variants intact. The mechanism that determines the selectivity of the sperm-histone-selective proteinase (SpH-proteinase) was analysed by focusing on the post-translational modification status of both sets of histones. It has previously been reported that only native cleavage-stage histones are poly(ADP-ribosylated), whereas the sperm-specific histones are not modified. To determine whether the poly(ADP-ribose) moiety afforded protection from degradation, the ADP-ribose polymers were removed from the cleavage-stage histones in vitro; these proteins were then assayed as potential substrates of the SpH-proteinase. Strikingly, the cleavage-stage histone variants were extensively degraded after the enzymic removal of their ADP-ribose moieties. In addition, the SpH cysteine proteinase was not inhibited by isolated poly(ADP-ribose) polymers. Consequently, only poly(ADP-ribosylated) cleavage-stage histone variants are protected from proteolysis. These results demonstrate a novel role for this type of post-translational modification, namely the protection of nuclear proteins against nuclear proteinases after fertilization.
Asunto(s)
Fertilización , Histonas/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Animales , Biopolímeros , Femenino , Erizos de MarRESUMEN
At intermediate stages of male pronucleus formation, sperm-derived chromatin is composed of hybrid nucleoprotein particles formed by sperm H1 (SpH1), dimers of sperm H2A-H2B (SpH2A-SpH2B), and a subset of maternal cleavage stage (CS) histone variants. At this stage in vivo, the CS histone variants are poly(ADP-ribosylated), while SpH2B and SpH1 are phosphorylated. We have postulated previously that the final steps of sperm chromatin remodeling involve a cysteine-protease (SpH-protease) that degrades sperm histones in a specific manner, leaving the maternal CS histone variants unaffected. More recently we have reported that the protection of CS histones from degradation is determined by the poly(ADP-ribose) moiety of these proteins. Because of the selectivity displayed by the SpH-protease, the coexistence of a subset of SpH together with CS histone variants at intermediate stages of male pronucleus remodeling remains intriguing. Consequently, we have investigated the phosphorylation state of SpH1 and SpH2B in relation to the possible protection of these proteins from proteolytic degradation. Histones H1 and H2B were purified from sperm, phosphorylated in vitro using the recombinant alpha-subunit of casein kinase 2, and then used as substrates in the standard assay of the SpH-protease. The phosphorylated forms of SpH1 and SpH2B were found to remain unaltered, while the nonphosphorylated forms were degraded. On the basis of this result, we postulate a novel role for the phosphorylation of SpH1 and SpH2B that occurs in vivo after fertilization, namely to protect these histones against degradation at intermediate stages of male chromatin remodeling.
Asunto(s)
Fertilización/fisiología , Histonas/metabolismo , Espermatozoides/metabolismo , Secuencia de Aminoácidos , Animales , Cromatina/metabolismo , Endopeptidasas/metabolismo , Femenino , Histonas/genética , Histonas/aislamiento & purificación , Técnicas In Vitro , Masculino , Fosforilación , Erizos de MarRESUMEN
The role of proteolysis during fertilization has been investigated only to a very limited extent as compared with its role on the control of cell cycle progression. In this report, we discuss briefly the proteases involved in fertilization, their relevance in the egg-sperm interaction and in the chromatin remodeling that occurs before the reestablishment of the diploid condition of the zygote. We further emphasize how the post-translational modifications of target proteins modulate these proteolytic events. J. Cell. Biochem. Suppls. 32/33:149-157, 1999.
Asunto(s)
Endopeptidasas/metabolismo , Fertilización/fisiología , Procesamiento Proteico-Postraduccional , Animales , Cromatina/metabolismo , Cisteína Endopeptidasas/metabolismo , Femenino , Fertilización/genética , Histonas/metabolismo , Humanos , Masculino , Complejos Multienzimáticos/metabolismo , Óvulo/metabolismo , Complejo de la Endopetidasa Proteasomal , Espermatozoides/metabolismo , Ubiquitinas/metabolismoRESUMEN
We have identified a 60-kDa cysteine protease that is associated with chromatin in sea urchin zygotes. This enzyme was found to be present as a proenzyme in unfertilized eggs and was activated shortly after fertilization. At a pH of 7.8-8.0, found after fertilization, the enzyme degraded the five sperm-specific histones (SpH), while the native cleavage-stage (CS) histone variants remained unaffected. Based on its requirements for reducing agents, its inhibition by sulfhydryl blocking compounds and its sensitivity to the cysteine-type protease inhibitors (2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane-ethyl-es ter (E-64 d), cystatin and leupeptin, this protease can be defined as a cysteine protease. Consistently, this protease was not affected by serine-type protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and pepstatin. The substrate selectivity and pH modulation of the protease activity strongly suggest its role in the removal of sperm-specific histones, which determines sperm chromatin remodeling after fertilization. This suggestion was further substantiated by the inhibition of sperm histones degradation in vivo by E-64 d. Based on these three lines of evidence, we postulate that this cysteine protease is responsible for the degradation of sperm-specific histones which occurs during male pronucleus formation.
Asunto(s)
Núcleo Celular/metabolismo , Cisteína Endopeptidasas/metabolismo , Histonas/metabolismo , Espermatozoides/metabolismo , Animales , Núcleo Celular/enzimología , Cromatina/enzimología , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/aislamiento & purificación , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas del Huevo/química , Proteínas del Huevo/aislamiento & purificación , Proteínas del Huevo/metabolismo , Precursores Enzimáticos/química , Femenino , Concentración de Iones de Hidrógeno , Lisosomas/enzimología , Masculino , Peso Molecular , Sustancias Reductoras/farmacología , Erizos de Mar , Inhibidores de Serina Proteinasa/farmacología , Interacciones Espermatozoide-Óvulo , Especificidad por Sustrato , Reactivos de Sulfhidrilo/farmacología , Cigoto/enzimologíaRESUMEN
To determine the changes in chromatin organization during male pronucleus remodeling, we have compared the composition of nucleoprotein particles (NP-ps) resulting from digestion with endogenous nuclease (ENase) and with micrococcal nuclease (MNase). Whole nuclei were isolated from sea urchin gametes and zygotes containing partially decondensed (15 min postinsemination, p.i.) or a fully decondensed (40 min p.i.) male pronucleus and digested with nucleases. The NP-ps generated were analyzed in agarose gels, and their histone composition was determined. Sperm core histones (SpH) and cleavage stage (CS) variants were identified by Western immunoblots revealed with specific antibodies. A single NP-ps was generated after digestion of sperm nucleus with MNase, which migrated in agarose gels between DNA fragments of 1.78-1.26 Kb. Sperm chromatin remained undigested after incubation in ENases activating buffer, indicating that these nuclei do not contain ENases. One type of NP-ps was obtained by digestion of unfertilized egg nuclei, either with ENase or MNase; the NP-ps was located in the region of the agarose gel corresponding to DNA fragments of 3.4-1.95 Kb [Imschenetzky et al. (1989): Exp Cell Res 182:436-444]. When whole nuclei from zygotes containing the female pronucleus and a partially remodeled male pronucleus were digested with ENase, a single NP-ps was generated, which migrated between DNA fragments of 2.5-1.9 Kb. This particle contained only CS histone variants. Alternatively, when these nuclei were digested with MNase, two NP-ps were generated; the slower migrating NP-ps (s) was located in the same position of the agarose gel as those resulting from ENase digestion and the faster migrating NP-ps (f) migrated between DNA fragments of 1.95-1.26 Kb. It was found that NP-ps (s) contained only CS histone variants, whereas NP-ps (f) were formed by a subset of SpH and by CS histone variants. When nuclei from zygotes containing a fully decondensed male pronucleus were digested either with ENase or MNase, a single type of NP-ps was observed, which migrated in the same position as NP-ps (s) in agarose gels. This particle contained only CS histone variants. On the basis of the histone compositions and on electrophoretic similarities, it was concluded that NP-ps (s) originated from the female pronucleus and that NP-ps (f) were generated from the partially remodeled male pronucleus. Consequently, our results indicate that at an intermediate stage of male pronucleus remodeling the chromatin is formed by NP-ps containing a subset of both SpH and of CS histone variants, whereas at final stages of male pronucleus decondensation chromatin organization is similar to that of the female pronucleus.
Asunto(s)
Núcleo Celular/química , Histonas/análisis , Nucleosomas/química , Espermatozoides/química , Cigoto/química , Animales , Western Blotting , Cromatina/metabolismo , ADN/aislamiento & purificación , Electroforesis en Gel de Agar , Endonucleasas/farmacología , Femenino , Variación Genética , Histonas/genética , Histonas/aislamiento & purificación , Masculino , Nucleasa Microcócica/farmacología , Nucleosomas/metabolismo , Oocitos/química , Erizos de MarRESUMEN
Sea urchin CS histone variants are electrophoretically heterogeneous when analyzed in two dimensional polyacrylamide gels (2D-PAGE). Previous results suggested that this heterogeneity is due to the poly (ADP-ribosylation) of these proteins. Consequently, native CS histone variants were subjected to different treatments to remove the ADP-ribose moiety. The incubation in 1 M hydroxylamine was not effective in eliminating the polymers of ADP-ribose from CS variants, and the treatment with sodium hydroxide was deleterious to the proteins. In contrast, the ADP-ribose moiety was successfully removed from the CS variants by incubation with phosphodiesterase (PDE). To eliminate contamination of CS histone variants with PDE extract, the enzyme was covalently bound to Sepharose 4B prior to its utilization. Treatment of native CS histone variants with this immobilized phosphodiesterase removed around 85% of the total ADP-ribose moiety from these proteins. After S-PDE treatment the complex electrophoretic pattern of CS histone variants in 2-D PAGE decreases to five major fractions. From these results we conclude that the electrophoretic heterogeneity of native CS histone variants is mainly due to the extent to which five main CS histone variants are poly(ADP)-ribosylated).
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Histonas/metabolismo , Animales , Western Blotting , Fase de Segmentación del Huevo , Compuestos de Dansilo/farmacología , Electroforesis en Gel de Poliacrilamida , Femenino , Colorantes Fluorescentes , Variación Genética , Glicosilación/efectos de los fármacos , Hidrazinas/farmacología , Hidrólisis , Hidroxilamina , Hidroxilaminas/farmacología , Masculino , Ácido Peryódico/farmacología , Fosfodiesterasa I , Hidrolasas Diéster Fosfóricas/farmacología , Poli Adenosina Difosfato Ribosa/análisis , Erizos de Mar , Hidróxido de Sodio/farmacologíaRESUMEN
To investigate changes in chromatin organization associated with DNA replication during the first stages of development of the sea urchin Tetrapygus niger, we compared micrococcal nuclease (MNase) digestion patterns of chromatin from zygotes harvested during the first S phase and from unfertilized eggs. We observed that the majority of DNA fragments derived from MNase digested zygote nuclei were similar to or smaller than a mononucleosome, while those derived from unfertilized egg nuclei were larger (1,500 to 410 bp). This result indicates that in zygotes, where active DNA replication is occurring, the major chromatin fraction is represented as unfolded nucleosomes. In contrast, in unfertilized eggs chromatin appears to be organized into polynucleosomes. To determine if the unfolded structure of nucleosomes observed during S phase is related to the level of poly (ADP-ribosylation) of cleavage stage (CS) histone variants, zygotes were treated with 20 mM 3-Amino Benzamide (3 ABA) during the interval between 3 and 30 min post-insemination (p.i.). This treatment with 3 ABA decreases the poly (ADP-ribosylation) of CS histone variants and inhibits the first S phase in zygotes [Imschenetzky et al. (1991): J Cell Biochem 46:234-241; Imschenetzky et al. (1993): J Cell Biochem 51:198-205]. When the MNase digested patterns of chromatin from these 3 ABA treated and control zygotes were compared, we found that the unfolded structure of the nucleosomes remains unaltered by the inhibition of the poly(ADP-ribose) synthetase with 3 ABA. This result indicates that the unfolded nucleosomal structure, particular to the chromatin of S phase zygotes, is not contemporaneous to DNA replication and is independent of the normal level of poly(ADP-ribosylation) of CS histone variants.
Asunto(s)
Cromatina/química , Nucleosomas/química , Fase S , Cigoto/ultraestructura , Animales , Núcleo Celular/metabolismo , Replicación del ADN , Nucleasa Microcócica , Erizos de MarRESUMEN
Tyrosine protein kinase (TPK) activity is associated to malignant cellular transformation. This work compares TPK activity in 27 surgical biopsy samples of mammary carcinoma, 10 samples of fibroadenomas, 13 samples of fibrocystic breast disease and 27 samples of normal mammary tissue. TPK activity was determined in tissue homogenates using (Val5) angiotensin II as exogenous substrate. In samples of mammary carcinoma, TPK activity was 33.86 +/- 31.98 pmol P32/mg protein/30 min. This value was significantly higher that those observed in fibrocystic disease (3.92 +/- 2.35), fibroadenomas (13.86 +/- 10.9) and normal tissue (3.56 +/- 3.02).
Asunto(s)
Neoplasias de la Mama/enzimología , Mama/enzimología , Fibroadenoma/enzimología , Enfermedad Fibroquística de la Mama/enzimología , Proteínas Tirosina Quinasas/metabolismo , Adulto , Anciano , Análisis de Varianza , Neoplasias de la Mama/patología , Transformación Celular Neoplásica , Femenino , Fibroadenoma/patología , Enfermedad Fibroquística de la Mama/patología , Humanos , Persona de Mediana Edad , Análisis de RegresiónRESUMEN
To analyze the temporal relationship of poly(adenosine diphosphate [ADP]-ribosylation) signal with DNA replication and cell divisions, the effect of 3 aminobenzamide (3ABA), an inhibitor of the poly(ADP-ribose)synthetase, was determined in vivo during the first cleavage division of sea urchins. The incorporation of 3H-thymidine into DNA was monitored and cleavage division was examined by light microscopy. The poly(ADP-ribose) neosynthesized on CS histone variants was measured by labeling with 3H-adenosine during the two initial embryonic cell cycles and the inhibitory effect of 3ABA on this poly(ADP-ribosylation) was determined. The results obtained indicate that the CS histone variants are poly(ADP-ribosylated) de novo during the initial cell cycles of embryonic development. The synthesis of poly(ADP-ribose) is decreased but not abolished by 20 mM of 3ABA. The incubation of zygotes in 3ABA at the entrance into S1 phase decreased 3H-thymidine incorporation into DNA in phase S2, while S1 was unaltered. Alternatively, when the same treatment was applied to zygotes at the exit of S1 phase, a block of the first cleavage division and a retardation of S2 phase were observed. The inhibitory effect of 3ABA on both DNA replication and cell division was totally reversible by a release of the zygotes from this inhibition. Taking together these observations it may be concluded that the poly(ADP-ribosylation) signals related to embryonic DNA replication are not contemporaneous with S phase progression but are a requirement before its initiation.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
División Celular/fisiología , Replicación del ADN/fisiología , Poli(ADP-Ribosa) Polimerasas/fisiología , Transducción de Señal/fisiología , Animales , Benzamidas/farmacología , División Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Histonas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/biosíntesis , Fase S/efectos de los fármacos , Fase S/fisiología , Erizos de Mar/citología , Erizos de Mar/embriología , Factores de TiempoRESUMEN
To obtain information on the remodeling of sperm chromatin during male pronuclei formation, we have followed the sperm specific histones (SpH) that form the nucleosomal core by Western immunoblot analysis with polyclonal antibodies directed against the core SpH. The results obtained indicate that the complete set of SpH is absent from zygote chromatin at the beginning of the first S phase. The disappearance of SpH is not coincidental for the five histone classes: SpH4 and SpH3 are lost 5-15 min post insemination (p.i.), SpH2B and SpH2A disappear 20-40 min p.i., and SpH1 is progressively diminished up to 30 min p.i. This order of sperm chromatin remodeling is not affected by the inhibition of protein synthesis by emetine, indicating that the factor(s) responsible for SpH disappearance are present in unfertilized eggs. The lost SpH's are not replaced by newly synthesized CS variants, since the basic proteins synthesized de novo during male pronuclei formation are not incorporated into chromatin remaining in the cytoplasm. These newly synthesized proteins are different from the CS variants as judged by their electrophoretic migration.
Asunto(s)
Núcleo Celular/metabolismo , Histonas/metabolismo , Espermatozoides/metabolismo , Cigoto/metabolismo , Animales , Western Blotting , Electroforesis , Electroforesis en Gel Bidimensional , Emetina/farmacología , Femenino , Masculino , Biosíntesis de Proteínas , Erizos de Mar , Interacciones Espermatozoide-Óvulo , Espermatozoides/ultraestructuraRESUMEN
The patterns of poly(ADP-ribosylation) in vivo of CS (cleavage stage) histone variants were compared in sea urchin zygotes at the entrance and the exit of S1 and S2 in the initial developmental cell cycles. This post-translational modification was detected by Western immunoblots with rabbit sera anti-poly(ADP-ribose) that was principally reactive against ADP-ribose polymers and slightly against ADP-ribose oligomers. The effect of 3 aminobenzamide (3-ABA), an inhibitor of the poly(ADP-ribose) synthetase, on S phase progression was determined in vivo by measuring the incorporation of 3H thymidine into DNA. The results obtained indicate that the CS histone variants are poly(ADP-ribosylated) in a cell cycle dependent manner. A significantly positive reaction of several CS variants with sera anti-poly(ADP-ribose) was found at the entrance into S phase, which decreases after its completion. The incubation of zygotes in 3-ABA inhibited the poly(ADP-ribosylation) of CS variants and prevented both the progression of the first S phase and the first cleavage division. These observations suggest that the poly(ADP-ribosylation) of atypical CS histone variants is relevant for initiation of sea urchin development and is required for embryonic DNA replication.
Asunto(s)
Histonas/química , Poli Adenosina Difosfato Ribosa/metabolismo , Cigoto/metabolismo , Animales , Benzamidas/farmacología , ADN/biosíntesis , Replicación del ADN/fisiología , Histonas/metabolismo , Biosíntesis de Proteínas , Fase S , Erizos de Mar , Timidina/metabolismo , Cigoto/citologíaRESUMEN
Two discrete supranucleosomal particles that differ in their electrophoretic migration on 1% agarose gels were isolated from unfertilized sea urchin eggs. Both particles contain the same complement of cleavage stage (CS) chromosomal proteins, which is identical to the complete set of basic proteins isolated directly from chromatin by extraction with 0.25 N HCl. DNA fragments between 210 and 1500 bp were found in both particles, and the basic unit of DNA repeat length determined by micrococcal nuclease digestion was 126 +/- 3 bp. The isolated nucleoparticles are electrophoretically stable over a wide range of DNA sizes (126-1500 bp) indicating that their structure is maintained by internal interactions among the CS chromosomal proteins, previously designated CS A through CS G. Based on these results we conclude that the CS chromosomal proteins are functionally equivalent to classical histones in their ability to direct higher ordered structures of chromatin.
Asunto(s)
Núcleo Celular/ultraestructura , ADN/aislamiento & purificación , Proteínas Nucleares/aislamiento & purificación , Nucleosomas/ultraestructura , Óvulo/ultraestructura , Animales , Núcleo Celular/análisis , Electroforesis en Gel de Agar , Electroforesis en Gel Bidimensional , Femenino , Peso Molecular , Nucleosomas/análisis , Óvulo/análisis , Erizos de MarRESUMEN
After fertilization the cone shaped sperm nucleus is transformed into a spheroidal male pronucleus which fuses with the female pronucleus reestablishing the diploid genome. These morphological changes are correlated with biochemical transitions of sperm-specific chromosomal proteins. To obtain information on the rearrangements of chromosomal proteins after fertilization, we have followed sperm-specific nonhistones (Sp NHCP) and the sperm-specific histones (SpH) in zygotes harvested at different times post-insemination (p.i.). The acquisition of proteins synthesized de novo was determined during the time of male pronucleus formation, estimated to occur until 30 min p.i. The results obtained may be summarized as follows: 1. The fate of Sp NHCP, followed by Western blot analysis with polyclonal antibodies obtained in rabbits against the whole complement of Sp NHCP, indicates that the majority of Sp NHCP are lost shortly p.i., except two proteins of 58 Kd and 61 Kd that were not distinguishable from eggs NHCP because a crossreaction was obtained. Acidic proteins synthesized de novo were bound to chromatin between 3 and 30 min p.i., and the majority of these proteins comigrated with egg NHCP in SDS/PAGE. 2. The histones from sperm differ from their counterparts found in unfertilized eggs, both in their amino acid composition and their microheterogeneity in bidimensional gels. Sperm contain the five major SpH whereas eggs have seven major histone variants, distinct from each other, with an electrophoretic behaviour consistent with histones but a different amino acid composition. The total complement of histone variants found in zygotes collected at amphimixis is identical with that found in unfertilized eggs, suggesting that SpH should be lost before that time.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Cromatina/metabolismo , Erizos de Mar/fisiología , Espermatozoides/fisiología , Cigoto/fisiología , Aminoácidos/análisis , Animales , Cromatina/análisis , Proteínas Cromosómicas no Histona/metabolismo , Femenino , Histonas/análisis , Masculino , Espermatozoides/análisis , Cigoto/análisisRESUMEN
To determine the transitions of sea urchin sperm specific non-histone chromosomal proteins (Sp NHCP) following fertilization, their presence in zygotes during and after male pronuclear formation was investigated by comparing the electrophoretic patterns of NHCP obtained from gametes with those isolated from zygotes. Polyclonal antibodies directed against whole Sp NHCP were used to detect, by Western immunoblots, the Sp NHCP among the NHCP present in zygotes at different times post-insemination. The results obtained from immunological and electrophoretic studies indicate that shortly after fertilization the majority of Sp NHCP are lost. The electrophoretic patterns of NHCP obtained from unfertilized eggs were almost identical with those observed for NHCP isolated from zygotes. This similarity strongly suggests that the NHCP of unfertilized eggs are conserved after fertilization.
Asunto(s)
Proteínas Cromosómicas no Histona/análisis , Fertilización , Erizos de Mar/fisiología , Espermatozoides/ultraestructura , Animales , Western Blotting , MasculinoRESUMEN
The five main histones were isolated from mature sperm of the sea urchin Tetrapygus niger. The electrophoretic mobility and amino acid composition of these proteins were compared with data from the literature on histones from sperms of other species of sea urchins. Electrophoretic analysis revealed that H1, H2A, H2B and H3 migrate as a single protein; but different from other species, H4 was resolved into two variants. The amino acid composition of the nucleosomal core histones differed markedly from the analogous fractions of other species; the most important of these differences are a lower content in lysine and arginine and a higher amount of glutamic acid and serine residues. In contrast with the nucleosomal core histones, sperm H1 histone is highly conserved among the different species of sea urchins, suggesting that this histone might be involved in the maintenance of the higher order structure of sperm chromatin.