Chromatin structure of histone genes in sea urchin sperms and embryos.

The nucleosomal organization of active and repressed alpha subtype histone genes has been investigated by micrococcal nuclease digestion of P. lividus sperm, 32-64 cell embryo and mesenchyme blastula nuclei, followed by hybridization with 32P-labeled specific DNA probes. In sperms, fully repressed histone genes are regularly folded in nucleosomes, and exhibit a greater resistance to micrococcal nuclease cleavage than bulk chromatin. In contrast, both coding and spacer alpha subtype histone DNA sequences acquire an altered conformation in nuclei from early cleavage stage embryos, i.e., when these genes are maximally expressed. Switching off of the alpha subtype histone genes, in mesenchyme blastulae, restores the typical nucleosomal organization on this chromatin region. As probed by hybridization to D.melanogaster actin cDNA, actin genes retain a regular nucleosomal structure in all the investigated stages.     

Regulation of histone synthesis during early Urechis caupo (Echiura) development

The histones present in mature oocytes and embryos of Urechis caupo and their pattern of synthesis during early development have been characterized. Acid-soluble proteins extracted from mature oocyte germinal vesicles and from embryonic nuclei were analyzed by two-dimensional polyacrylamide gel electrophoresis. Histones are accumulated in the mature oocytes in amounts sufficient to provide for the assembly of chromatin through the 32- to 64-cell stage of embryogenesis. Two H1 histones, which appear to be variants, were found. Germinal vesicles and cleavage-stage nuclei are enriched in H1M (maternal). During late cleavage a faster-migrating H1, H1E (embryonic), appears among the nuclear histones and, as embryogenesis continues, replaces H1M as the predominant H1. No new core histone variants are detected during early development. Examination of [³H]lysine-labeled histones from germinal vesicles and embryonic nuclei reveals stage-specific patterns of histone synthesis. H1M is the major H1 species synthesized in mature oocytes. After fertilization, a switch to the predominant synthesis of H1E occurs. Comparison of the [³H]lysine incorporated into H1E and core histones indicates that H1E synthesis is disproportionately high from midcleavage through the midblastula stage. By the gastrula stage, a balanced synthesis of H1E and each core histone is established. The results indicate that there is noncoordinate regulation of H1 and core histone synthesis during Urechis development.     

Synchronization of cell division in eight-cell bovine embryos produced in vitro: Effects of nocodazole

Current methods of arresting and synchronizing cell division have not been very successful and have had few applications in embryo studies. Our objective was to determine the reliability of a metaphase arrest agent, nocodazole, for halting and synchronizing blastomere division in cleavage-stage bovine embryos, and to verify its reversibility and toxicity in vitro. Eight-cell-stage embryos obtained at 58 hr postinsemination were treated with varying concentrations of nocodazole for 12 hr. Treated embryos were assessed for cleavage arrest, chromatin morphology, DNA synthesis, and histone H1 and myelin basic protein (MBP) kinase activity, and were scored for blastocyst formation and hatching rate. They were subsequently fixed to count the number of nuclei. Complete arrest of cell division was observed at concentrations of 0.4 μg ml⁻¹ and above. Removal from nocodazole treatment led to immediate release from cleavage arrest, and was followed by synchronized mitosis, histone H1 kinase deactivation, and reentry into interphase within 3–5 hr. DNA synthesis was reinitiated at 6 hr after release. Although cell numbers and hatching rate decreased, the proportion of embryos reaching blastocyst stage was not significantly affected in nocodazole-treated embryos. It is concluded that nocodazole is a suitable choice for the cell-cycle synchronization of donor embryos for use in studies on the interactions between nucleus and cytoplasm during early embryogenesis. © 1996 Wiley-Liss, Inc.     

Site and stage specific action of endogenous nuclease and micrococcal nuclease on histone genes of sea urchin embryos

The early histone genes of sea urchin embryos are expressed exclusively during cleavage stages of embryogenesis. The chromatin containing these genes was examined by nuclease sensitivity. An endogenous nuclease active during cleavage, produces 1300-bp segments containing early histone genes. The cutting sites have been mapped; there are very sensitive sites close to the cap site for H1, H2A, H2B, and H4. Chromatin obtained from embryos of later stages, when the genes are not expressed, do not display this pattern of nuclease sensitivity. Micrococcal nuclease produces nucleosomes that contain histone genes when used with nuclei from later stages, but not with nuclei from cleavage stages.     

Changes of Chromosome Length and Constitutive Heterochromatin in Association with Cell Division during Early Development of Cynops pyrrhogaster embryo

The length of chromosomes in the presumptive ectoderms of Cynops embryos was measured at nine successive cell divisions from the 6th (cleavage stage) to the 14th (gastrula stage). Up to the 10th cell division (cleavage stage) the chromosome length remained constant. At the 11th cell division the chromosomes began to shorten and continued to shorten at every cell division up to the 14th cell division. The degree of shortening and the mode of variation in length corresponded to the respective developmental stages of cleavage, blastula and gastrula. During those periods when chromosomes became shortened, some fine C-bands of the paracentromeric region found in earlier stages fused together. The chromatin of interphase nuclei also showed considerable changes during chromosome shortening. Besides the size reduction of interphase nuclei, the number of C-band granules in an interphase nucleus decreased in parallel with chromosome shortening and fusion of C-bands in mitotic chromosome.     

Phosphorylation of nonhistone chromatin proteins during sea urchin development

The phosphorylation of nonhistone chromatin proteins during development was studied in the sea urchin, $\text{Strongylocentrotus purpuratus}$. The rate of phosphorylation was found to be maximal during gastrula, slightly lower during prism and almost 70% lower in pluteus stage embryos. Analysis of the phosphorylated nonhistone chromatin proteins by SDS-acrylamide gel electrophoresis showed significant variations in the labeling pattern during different stages of development. A specific protein which is actively phosphorylated during gastrula and prism stages is nearly absent from the pluteus stage.     

Electron microscopic analysis of chromatin replication in the cellular blastoderm drosophila melanogaster embryo

Transmission electron microscopic techniques were used to study the spatial distribution of replicons and the ultrastructure of chromatin in the S phase genome of cellular blastoderm Drosophila melanogaster embryos. We observed chromatin exhibiting distinct bifurcations along each fiber during the initial 20 min of the first cell cycle of blastulation. We interpreted the “bubble-like” configurations produced by adjacent bifurcations as intermediate structures in chromatin replication (that is, replicons). We observed homologous ribonucleoprotein (RNP) fiber arrays on both chromatid arms within some replicons, implying DNA sequence homology and reinforcing the identification of such arms as daughter chromatid fibers. We did not observe replicon configurations on chromatin obtained from embryos staged at more than 20 min into cellular blastulation. Daughter chromatid fibers, however, were identified by the presence of identical RNP fiber arrays on chromatid strands arranged in parallel on the electron microscope grid.We examined the distribution of replicon structures on the cellular blastoderm genome and compared it with electron microscopic data on DNA replication in cleavage embryos (Blumenthal, Kriegstein and Hogness, 1973). S phase is completed in slightly < 4 min during cleavage, or approximately one fifth the time required for DNA synthesis in cellular blastoderm embryos. The mean distance separating adjacent replication origins at cellularization was estimated to be 10.6 kilobases (kb), a value 35% greater than the 7.9 kb inter-origin average determined for cleavage embryos. In contrast to the near-simultaneous activation of replication origins during cleavage replication, we observed that replication origins are not activated synchronously at cellular blastulation. We concluded that the marked increase in the duration of S phase is effected by a reduction in the frequency of replication activation events which occur asynchronously during genome replication at cellularization.We found that the ultrastructure of newly replicated chromatin exhibited a morphology indistinguishable from nucleosomal chromatin. Unreplicated chromatin fibers separating adjacent replicons also exhibit spherical subunits. We inferred that the spherical structures on replicating chromatin are nucleosomes and concluded that histones are not disassociated from the DNA significantly prior to DNA replication, and that a very rapid reassociation of nucleosomes occurs on both daughter DNA molecules following replication.     

Cytological effects of the microinjection of antibody to ras p21 in early cleavage Xenopus embryos

The presence of two ras-related proteins (22 and 23 kDa) was demonstrated in Xenopus embryonic extracts by selective immunoprecipitation using anti-ras monoclonal antibodies 142-24E05 and Y13-259. We further describe the cytological effects of the microinjection of anti-ras monoclonal antibody Y13-259 into early cleavage blastomeres of Xenopus embryos. Injection of the antibody into a blastomere at the two-, four-, or eight-cell stage caused cleavage arrest in the descendants of the injected blastomere. Light microscopy (LM) of cleavage-arrested cells revealed extensive deformation of the cells as well as heterogeneity of distribution of yolk platelets and pigment granules. LM analysis of serial sections of cleavage-arrested cells revealed the presence of multiple nuclei. Although the nuclei expressed similar morphological properties, indicating that they were probably in the same stage of the nuclear cycle, they revealed highly variable chromatin densities. Electron microscope (EM) analysis of the cytoplasm of cleavage-arrested cells revealed the accumulation of vesicles and large membranous elements coincident with cleavage arrest. Furthermore, endoplasmic reticulum (ER) existed in two forms, as closed, circular profiles and as long, linear arrays. Mitochondria were characteristically aligned in single file on both sides of the two types of ER cisternae. EM analysis of nuclei confirmed variations in chromatin organization and suggested the occurrence of unique nuclear envelope fusion among micronuclei in cleavage-arrested cells. Cleavage arrest and changes in cytological features were not observed in the cytoplasm of cells microinjected with normal rat IgG. Thus the immunochemical data and microinjection experiments suggest that ras-like or ras antigenicity exists within rapidly replicating Xenopus blastomeres and may be involved in the organization of a number of its cytoplasmic elements.     

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) activates the maternal program of apoptosis shortly after MBT in Xenopus embryos

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) mRNA in 1- and 2-cell stage Xenopus embryos induces cell autonomous dissociation at the late blastula stage and developmental arrest at the early gastrula stage. The induction of cell dissociation took place "punctually" at the late blastula stage in the SAMDC-overexpressing cells, irrespective of the stage of the microinjection of SAMDC mRNA. When we examined the cells undergoing the dissociation, we found that they were TUNEL-positive and contained fragmented nuclei with condensed chromatin and fragmented DNA. Furthermore, by injecting Xenopus Bcl-2 mRNA together with SAMDC mRNA, we showed that SAMDC-overexpressing embryos are rescued completely by Bcl-2 and becometadpoles. These results indicatethat cell dissociation induced by SAMDC overexpression is due to apoptotic cell death. Since the level of S-adenosylmethionine (SAM) is greatly reduced in SAMDC-overexpressing embryos and this induces inhibition of protein synthesis accompanied by the inhibition of DNA and RNA syntheses, we conclude that deficiency in SAM induced by SAMDC overexpression activates the maternal program of apoptosis in Xenopus embryos at the late blastula stage, but not before. We propose that this mechanism serves as a surveillance mechanism to check and eliminate cells physiologically damaged during the cleavage stage.     

Histone acetylation and hSWI/SNF remodeling act in concert to stimulate V(D)J cleavage of nucleosomal DNA

The ordered assembly of immunoglobulin and TCR genes by V(D)J recombination depends on the regulated accessibility of individual loci. We show here that the histone tails and intrinsic nucleosome structure pose significant impediments to V(D)J cleavage. However, alterations to nucleosome structure via histone acetylation or by stable hSWI/SNF-dependent remodeling greatly increase the accessibility of nucleosomal DNA to V(D)J cleavage. Moreover, acetylation and hSWI/SNF remodeling can act in concert on an individual nucleosome to achieve levels of V(D)J cleavage approaching those observed on naked DNA. These results are consistent with a model in which regulated recruitment of chromatin modifying activities is involved in mediating the lineage and stage-specific control of V(D)J recombination.     

Topoisomerase II cleavage in chromatin

We have examined the effect of the anti-tumor drug VM-26 on purified Drosophila topoisomerase II, and used this drug to map (putative) topoisomerase II cleavage sites in chromatin. These studies indicate that VM-26 interferes with the strand breakage-rejoining catalytic cycle. VM-26 appears to stabilize the topoisomerase-II-cleavable complex and markedly enhances the formation of double-strand breaks in naked DNA. VM-26 also stimulates the formation of double-strand breaks in isolated Drosophila nuclei. Analysis of the parameters of the VM-26-stimulated cleavage reaction in nuclei strongly suggests that the double-strand scissions are generated by endogenous topoisomerase II. Finally, we have examined the distribution of (putative) cleavage sites for endogenous topoisomerase II in the chromatin of the 87A7 heat shock locus and the histone repeat unit. We have found that there are prominent VM-26-induced cleavage products from the 5' ends of the 87A7, the two heat shock protein 70 genes, and in the intergenic spacer separating these genes. Moreover, the pattern of VM-26-induced cleavage products is altered in nuclei prepared from heat-shocked cells. In the case of the histone repeat unit, only minor VM-26-induced cleavage products are observed in nuclei (in spite of the fact that experiments on naked DNA indicate that the histone repeat contains many major cleavage sites for purified topoisomerase II). These findings suggest that the nucleoprotein organization of different DNA segments may be important in determining whether specific sites are accessible to endogenous topoisomerase II in nuclei.