Microsatellite Organization in the Grasshopper Abracris flavolineata (Orthoptera: Acrididae) Revealed by FISH Mapping: Remarkable Spreading in the A and B Chromosomes

With the aim of acquiring deeper knowledge about repetitive DNAs chromosomal organization in grasshoppers, we used fluorescent in situ hybridization (FISH) to map the distribution of 16 microsatellite repeats, including mono-, di-, tri- and tetra-nucleotides, in the chromosomes of the species Abracris flavolineata (Acrididae), which harbors B chromosome. FISH revealed two main patterns: (i) exclusively scattered signals, and (ii) scattered and specific signals, forming evident blocks. The enrichment was observed in both euchromatic and heterochromatic areas and only the motif (C)₃₀ was absent in heterochromatin. The A and B chromosomes were enriched with all the elements that were mapped, being observed in the B chromosome more distinctive blocks for (GA)₁₅ and (GAG)₁₀. For A complement distinctive blocks were noticed for (A)₃₀, (CA)₁₅, (CG)₁₅, (GA)₁₅, (CAC)₁₀, (CAA)₁₀, (CGG)₁₀, (GAA)₁₀, (GAC)₁₀ and (GATA)₈. These results revealed an intense spreading of microsatellites in the A. flavolineata genome that was independent of the A+T or G+C enrichment in the repeats. The data indicate that the microsatellites compose the B chromosome and could be involved in the evolution of this element in this species, although no specific relationship with any A chromosome was observed to discuss about its origin. The systematic analysis presented here contributes to the knowledge of repetitive DNA chromosomal organization among grasshoppers including the B chromosomes.     

Contrasting the Chromosomal Organization of Repetitive DNAs in Two Gryllidae Crickets with Highly Divergent Karyotypes

A large percentage of eukaryotic genomes consist of repetitive DNA that plays an important role in the organization, size and evolution. In the case of crickets, chromosomal variability has been found using classical cytogenetics, but almost no information concerning the organization of their repetitive DNAs is available. To better understand the chromosomal organization and diversification of repetitive DNAs in crickets, we studied the chromosomes of two Gryllidae species with highly divergent karyotypes, i.e., 2n(♂) = 29,X0 (Gryllus assimilis) and 2n = 9, neo-X₁X₂Y (Eneoptera surinamensis). The analyses were performed using classical cytogenetic techniques, repetitive DNA mapping and genome-size estimation. Conserved characteristics were observed, such as the occurrence of a small number of clusters of rDNAs and U snDNAs, in contrast to the multiple clusters/dispersal of the H3 histone genes. The positions of U2 snDNA and 18S rDNA are also conserved, being intermingled within the largest autosome. The distribution and base-pair composition of the heterochromatin and repetitive DNA pools of these organisms differed, suggesting reorganization. Although the microsatellite arrays had a similar distribution pattern, being dispersed along entire chromosomes, as has been observed in some grasshopper species, a band-like pattern was also observed in the E. surinamensis chromosomes, putatively due to their amplification and clustering. In addition to these differences, the genome of E. surinamensis is approximately 2.5 times larger than that of G. assimilis, which we hypothesize is due to the amplification of repetitive DNAs. Finally, we discuss the possible involvement of repetitive DNAs in the differentiation of the neo-sex chromosomes of E. surinamensis, as has been reported in other eukaryotic groups. This study provided an opportunity to explore the evolutionary dynamics of repetitive DNAs in two non-model species and will contribute to the understanding of chromosomal evolution in a group about which little chromosomal and genomic information is known.     

Histone H3 Lysine 56 Acetylation: A New Twist in the Chromosome Cycle

Several recent reports have identified lysine 56 (K56) as a novel site of acetylation in yeast histone H3. K56 acetylation is predicted to disrupt some of the histone-DNA interactions at the entry and exit points of the nucleosome core particle. This modification occurs in virtually all the newly synthesised histones that are deposited into chromatin during S-phase. Cells with mutations that block K56 acetylation show increased genome instability and hypersensitivity to genotoxic agents that interfere with replication. Removal of K56 acetylation takes place in the G2/M phase of the cell cycle and is dependent upon Hst3 and Hst4, two proteins that are related to the NAD+-dependent histone deacetylase Sir2. In response to DNA damage checkpoint activation during S-phase, expression of Hst3/Hst4 is delayed to extend the window of opportunity in which K56 acetylation can act in the DNA damage response. The high abundance of histone H3 K56 acetylation, its regulation and strategic location in the nucleosome core particle raise a number of fascinating issues that we discuss here.     

Maintenance of a "Parasitic" B Chromosome in the Grasshopper MELANOPLUS FEMUR-RUBRUM

About 10-15% of the males and females of the grasshopper Melanoplus femur-rubrum collected near Rochester, New York, possessed a supernumerary B chromosome. The frequency of the B chromosome remained fairly constant during the years 1971-1974. The B chromosome was shown previously to be transmitted at a rate of about 0.5 and 0.8 by 1B males and females, respectively. This study was designed to determine the forces preventing the B chromosome from increasing in frequency due to the high rate of transmission by the females. Eighty inseminated females collected in the wild were analyzed cytologically together with their embryos (10-20 per female). Ten of the 80 females had a B chromosome, and they transmitted it at a rate of about 0.75. Among the 983 embryos analyzed, 0.141 had one B, 0.007 had two, and the mean number of B chromosomes per embryo was 0.155. The frequency of the B chromosome in the sperm pool (0.061) was consistent with a 0.5 rate of transmission. Individuals with two B chromosomes apparently have low viability, because about six were expected, but none was found among 851 adult males and females examined. The data suggest that the viability of the 1B individuals was only about 0.86 that of the OB individuals. There was no evidence that the B chromosome increased the fecundity of either the 1B males or females. It was concluded, therefore, that the B chromosome reduced the fitness of all the individuals carrying it and was thus "parasitic," and that it was maintained in the population only because of its high transmission rate. The maintenance of other B chromosomes with high transmission rates is reviewed.     

Histone H2B Ubiquitylation Is Not Required for Histone H3 Methylation at Lysine 4 in Tetrahymena

Ubiquitylation of histone H2B and/or a component of the system that ubiquitylates H2B is required for methylation of histone H3 at lysine 4 (H3K4) in yeasts and probably in humans. In this study, the single ubiquitylation site was mapped to conserved lysine 115 of the C-terminal region of histone H2B in the single-cell model organism Tetrahymena thermophila. In strains lacking H2B ubiquitylation, H3K4 methylation was not detectably affected. As in other organisms, the E2 ubiquitin-conjugating enzyme Ubc2 and the E3 ubiquitin ligase Bre1 were required for H2B ubiquitylation. However, neither enzyme was required for H3K4 methylation. These studies argue that, in T. thermophila, the histone ubiquitylation mechanism is not required for H3K4 methylation, demonstrating that different organisms can speak different languages in the “cross-talk” among post-translational modifications on different histones.     

Genetic Mapping of a Possible New Alcohol Dehydrogenase Sequence to Mouse Chromosome 3 at the Adh-1/Adh-3 Complex

Southern blot analysis of mouse genomic DNA reveals two Eco RI fragments which faintly hybridize to mouse Adh-1 cDNA and are not part of the Adh-1 gene. These fragments were isolated from agarose gels, cloned, and characterized. Sequence analysis of the 2.1-kb Eco RI fragment suggests that it is likely a pseudogene since it does not contain a long open reading frame. However, the 2.0-kb Eco RI fragment contains a coding sequence with a long open reading frame which corresponds to exon 6 of the mouse Adh-1 gene. Comparison of the coding sequence with other known ADHs suggests that the sequence has diverged sufficiently from any currently known class of ADH to be a possible distinct class. Further confirmation awaits analysis of currently available genomic clones. Using these sequences as probe, restriction fragment length polymorphisms were identified for each sequence between C57Bl/6J and DBA/2J inbred mouse strains. The strain distribution pattern for these allelic differences was determined among the B × D recombinant inbred strains. This analysis revealed that the 2.1-kb Eco RI sequence is located on chromosome 3 but at a distance from the Adh-1/Adh-3 complex as previously reported. However, the new polymorphism identified in the 2.0-kb Eco RI fragment enabled this sequence to be mapped at the Adh-1/Adh-3 complex.     

质谱法分析大鼠精子发生过程中组蛋白H1变异体的多样性

目的为探究连接组蛋白H1在精子发生过程染色体重构中的功能,了解一共有多少种连接组蛋白H1参与各期生精细胞的染色体的构建。方法分离高纯度的SD大鼠的各期生精细胞,提取组蛋白,应用SDS-PAGE分离组蛋白的各组分,组蛋白（H1）经过蛋白酶（Glu-c和Arg-c）酶切,应用质谱进行检测。结果鉴定了组蛋白H1的体细胞亚型（H1.1-H1.5）和睾丸特异的连接组蛋白亚型（H1t）。组蛋白H1t分别表达在精原细胞,精母细胞和圆形精子细胞中。结论大鼠精子发生过程中,其主要连接组蛋白H1的种类是：H1.1-H1.5和H1t。     

High-Resolution Mapping of H1 Linker Histone Variants in Embryonic Stem Cells

H1 linker histones facilitate higher-order chromatin folding and are essential for mammalian development. To achieve high-resolution mapping of H1 variants H1d and H1c in embryonic stem cells (ESCs), we have established a knock-in system and shown that the N-terminally tagged H1 proteins are functionally interchangeable to their endogenous counterparts in vivo. H1d and H1c are depleted from GC- and gene-rich regions and active promoters, inversely correlated with H3K4me3, but positively correlated with H3K9me3 and associated with characteristic sequence features. Surprisingly, both H1d and H1c are significantly enriched at major satellites, which display increased nucleosome spacing compared with bulk chromatin. While also depleted at active promoters and enriched at major satellites, overexpressed H1⁰ displays differential binding patterns in specific repetitive sequences compared with H1d and H1c. Depletion of H1c, H1d, and H1e causes pericentric chromocenter clustering and de-repression of major satellites. These results integrate the localization of an understudied type of chromatin proteins, namely the H1 variants, into the epigenome map of mouse ESCs, and we identify significant changes at pericentric heterochromatin upon depletion of this epigenetic mark.     

Independent Mechanisms Target SMCHD1 to Trimethylated Histone H3 Lysine 9-Modified Chromatin and the Inactive X Chromosome

The chromosomal protein SMCHD1 plays an important role in epigenetic silencing at diverse loci, including the inactive X chromosome, imprinted genes, and the facioscapulohumeral muscular dystrophy locus. Although homology with canonical SMC family proteins suggests a role in chromosome organization, the mechanisms underlying SMCHD1 function and target site selection remain poorly understood. Here we show that SMCHD1 forms an active GHKL-ATPase homodimer, contrasting with canonical SMC complexes, which exist as tripartite ring structures. Electron microscopy analysis demonstrates that SMCHD1 homodimers structurally resemble prokaryotic condensins. We further show that the principal mechanism for chromatin loading of SMCHD1 involves an LRIF1-mediated interaction with HP1γ at trimethylated histone H3 lysine 9 (H3K9me3)-modified chromatin sites on the chromosome arms. A parallel pathway accounts for chromatin loading at a minority of sites, notably the inactive X chromosome. Together, our results provide key insights into SMCHD1 function and target site selection.     

Distinct Features of the Histone Core Structure in Nucleosomes Containing the Histone H2A.B Variant

Nucleosomes containing a human histone variant, H2A.B, in an aqueous solution were analyzed by small-angle neutron scattering utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome are detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails are compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Therefore, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.     

Distinct Features of the Histone Core Structure in Nucleosomes Containing the Histone H2A.B Variant

Nucleosomes containing a human histone variant, H2A.B, in an aqueous solution were analyzed by small-angle neutron scattering utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome are detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails are compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Therefore, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.     

Evolution of Histone H4 and H3 Genes in Different Ciliate Lineages

The histones H4 are known as highly conserved proteins. However, in ciliates a high degree of variation was found compared both to other eukaryotes and between the ciliate species. To date, only H4 histones of species belonging to two distantly related classes have been investigated. In order to obtain more detailed information on histone H4 variation in ciliates we undertook a comprehensive sequence analysis of PCR-amplified internal H4 fragments from 12 species belonging to seven out of the nine currently recognized ciliate classes. In addition, we used PCR primers to amplify longer fragments of H3 and H4 genes including the intergenic region. The encoded amino acid sequences reveal a high number of differences when compared with those of other eukaryotes and the ciliate species investigated. Furthermore, in some species H4 gene variants were detected, which result in amino acid differences. The greatest number of substitutions and insertions found was in the amino terminal region of the H4 histones. However, all sequences possess a conserved region corresponding to those of all other eukaryotic H4 histones. The histone gene variations were used to reconstruct phylogenetic relationships. The tree from our data matches perfectly with the ribosomal RNA data: The heterotrichs, which were considered as a late branching lineage, diverge at the base of the ciliate tree and groups formerly thought to represent ancestral lineages now appear as highly derived ciliates. Received: 4 April 1997 / Accepted: 1 August 1997     

Histone H3 variants in male gametic cells of lily and H3 methylation in mature pollen

Histones are vital structural proteins of chromatin that influence its dynamics and function. The tissue-specific expression of histone variants has been shown to regulate the expression of specific genes and genomic stability in animal systems. Here we report on the characterization of five histone H3 variants expressed in Lilium generative cell. The gcH3 and leH3 variants show unique sequence diversity by lacking a conserved lysine residue at position 9 (H3K9). The gH3 shares conserved structural features with centromeric H3 of Arabidopsis. The gH3 variant gene is strongly expressed in generative cells and gH3 histone is incorporated in to generative cell chromatin. The lysine residue of H3 at position 4 (H3K4) is highly methylated in the nuclei of generative cells of mature pollen, while methylation of H3K4 is low in vegetative cell nuclei. Taken together, these results suggest that male gametic cells of Lilium have unique chromatin state and histone H3 variants and their methylation might be involved in gene regulation of male gametic cells.Accession numbers for the sequence data The sequences reported in this paper have been deposited in the DDBJ database gcH3 GC1174 (accession no. AB195644), gH3 GC1008 (accession no. AB195646), leH3 GC1126 (accession no. AB195648), soH3-1 GC0075 (accession no. AB195650), soH3-2 GC1661 (accession no. AB195652), genomic sequence of gcH3 (accession no. AB195645), genomic sequence of gH3 (accession no. AB195647), genomic sequence of leH3 (accession no. AB195649), genomic sequence of soH3-2 (accession no. AB195651), genomic sequence of soH3-2 (accession no. AB195653).     

Histone Variant H2A.Z Regulates Centromere Silencing and Chromosome Segregation in Fission Yeast

The incorporation of histone variant H2A.Z into nucleosomes plays essential roles in regulating chromatin structure and gene expression. A multisubunit complex containing chromatin remodeling protein Swr1 is responsible for the deposition of H2A.Z in budding yeast and mammals. Here, we show that the JmjC domain protein Msc1 is a novel component of the fission yeast Swr1 complex and is required for Swr1-mediated incorporation of H2A.Z into nucleosomes at gene promoters. Loss of Msc1, Swr1, or H2A.Z results in loss of silencing at centromeres and defective chromosome segregation, although centromeric levels of CENP-A, a centromere-specific histone H3 variant that is required for setting up the chromatin structure at centromeres, remain unchanged. Intriguingly, H2A.Z is required for the expression of another centromere protein, CENP-C, and overexpression of CENP-C rescues centromere silencing defects associated with H2A.Z loss. These results demonstrate the importance of H2A.Z and CENP-C in maintaining a silenced chromatin state at centromeres.     

Histone H3 localizes to the centromeric DNA in budding yeast

During cell division, segregation of sister chromatids to daughter cells is achieved by the poleward pulling force of microtubules, which attach to the chromatids by means of a multiprotein complex, the kinetochore. Kinetochores assemble at the centromeric DNA organized by specialized centromeric nucleosomes. In contrast to other eukaryotes, which typically have large repetitive centromeric regions, budding yeast CEN DNA is defined by a 125 bp sequence and assembles a single centromeric nucleosome. In budding yeast, as well as in other eukaryotes, the Cse4 histone variant (known in vertebrates as CENP-A) is believed to substitute for histone H3 at the centromeric nucleosome. However, the exact composition of the CEN nucleosome remains a subject of debate. We report the use of a novel ChIP approach to reveal the composition of the centromeric nucleosome and its localization on CEN DNA in budding yeast. Surprisingly, we observed a strong interaction of H3, as well as Cse4, H4, H2A, and H2B, but not histone chaperone Scm3 (HJURP in human) with the centromeric DNA. H3 localizes to centromeric DNA at all stages of the cell cycle. Using a sequential ChIP approach, we could demonstrate the co-occupancy of H3 and Cse4 at the CEN DNA. Our results favor a H3-Cse4 heterotypic octamer at the budding yeast centromere. Whether or not our model is correct, any future model will have to account for the stable association of histone H3 with the centromeric DNA.