Genetic polymorphism of erythrocyte histone H1 in Japanese quail

Histone H1 from erythrocytes of Japanese quail was resolved in a sodium dodecyl sulfate (SDS)-polyacrylamide gel into five fractions differing in apparent molecular weights. A polymorphism of histone H1.1, H1.2, and H1.3 bands was detected among quail individuals. While some birds possessed either a high (phenotype .3+) or a low (phenotype .3+/.3-) level of H1.3, at least half of the quail population lacked this H1 band (phenotype .3-). Appropriate genetic crosses demonstrated that H1.3 behaved as though it was coded by a gene with two codominant alleles at an autosomal locus. Using two-dimensional polyacrylamide gel electrophoresis (acid-urea followed by SDS gels), it was found that birds .3+ contained polypeptides H1.b1 and H1.b'1; birds .3-, polypeptides H1.b2 and H1.b'2 with lower apparent molecular weights; and birds .3+/.3-, both types of polypeptides in equal proportions. The H1.b2 + H1.b'2 complement was not discernible in SDS gels, for it migrated together with H1.c' within band H1.4. It was found that a small number of birds lacking the H1.2 band in SDS gels failed to express histone H1.a. Since birds with phenotype .2- with a defective allele of the gene H1.a were simultaneously lacking the H1.3 band, it seems that the imperfect allele of the H1.a gene might be closely linked to the alleles producing H1.b2 + H1.b'2.     

Chromatin-modifying complex component Nurf55/p55 associates with histones H3 and H4 and polycomb repressive complex 2 subunit Su(z)12 through partially overlapping binding sites

Drosophila Nurf55 is a component of different chromatin-modifying complexes, including the PRC2 (Polycomb repressive complex 2). Based on the 1.75-Å crystal structure of Nurf55 bound to histone H4 helix 1, we analyzed interactions of Nurf55 (Nurf55 or p55 in fly and RbAp48/46 in human) with the N-terminal tail of histone H3, the first helix of histone H4, and an N-terminal fragment of the PRC2 subunit Su(z)12 using isothermal calorimetry and pulldown experiments. Site-directed mutagenesis identified the binding site of histone H3 at the top of the Nurf55 WD40 propeller. Unmodified or K9me3- or K27me3-containing H3 peptides were bound with similar affinities, whereas the affinity for K4me3-containing H3 peptides was reduced. Helix 1 of histone H4 and Su(z)12 bound to the edge of the β-propeller using overlapping binding sites. Our results show similarities in the recognition of histone H4 and Su(z)12 and identify Nurf55 as a versatile interactor that simultaneously contacts multiple partners.     

A comparison of the histone H1 complements of avian erythrocytes.

1. Histone H1 from chicken, turkey, duck and goose erythrocytes was resolved into six bands and that from quail into seven bands in an acetic acid-urea polyacrylamide gel. 2. A fast migrating minor subtype H1.e was detected in avian erythrocytes using two-dimensional polyacrylamide gel electrophoresis. 3. Although histone subtype H1.z from quail, turkey and duck was well separated in acid-urea gel, a similar protein in goose was found only in two-dimensional gel. This spot was absent in chicken. 4. Histone H1 spots .c, .c' and .d migrate in two-dimensional gel in a relatively constant manner forming a triangle-shaped pattern that facilitates comparison of H1 subtypes among various avian species.     

In vivo - in vitro toxicogenomic comparison of TCDD-elicited gene expression in Hepa1c1c7 mouse hepatoma cells and C57BL/6 hepatic tissue

Background

By comparing the quail genome with that of chicken, chromosome rearrangements that have occurred in these two galliform species over 35 million years of evolution can be detected. From a more practical point of view, the definition of conserved syntenies helps to predict the position of genes in quail, based on information taken from the chicken sequence, thus enhancing the utility of this species in biological studies through a better knowledge of its genome structure. A microsatellite and an Amplified Fragment Length Polymorphism (AFLP) genetic map were previously published for quail, as well as comparative cytogenetic data with chicken for macrochromosomes. Quail genomics will benefit from the extension and the integration of these maps.

Results

The integrated linkage map presented here is based on segregation analysis of both anonymous markers and functional gene loci in 1,050 quail from three independent F2 populations. Ninety-two loci are resolved into 14 autosomal linkage groups and a Z chromosome-specific linkage group, aligned with the quail AFLP map. The size of linkage groups ranges from 7.8 cM to 274.8 cM. The total map distance covers 904.3 cM with an average spacing of 9.7 cM between loci. The coverage is not complete, as macrochromosome CJA08, the gonosome CJAW and 23 microchromosomes have no marker assigned yet. Significant sequence identities of quail markers with chicken enabled the alignment of the quail linkage groups on the chicken genome sequence assembly. This, together with interspecific Fluorescence In Situ Hybridization (FISH), revealed very high similarities in marker order between the two species for the eight macrochromosomes and the 14 microchromosomes studied.

Conclusion

Integrating the two microsatellite and the AFLP quail genetic maps greatly enhances the quality of the resulting information and will thus facilitate the identification of Quantitative Trait Loci (QTL). The alignment with the chicken chromosomes confirms the high conservation of gene order that was expected between the two species for macrochromosomes. By extending the comparative study to the microchromosomes, we suggest that a wealth of information can be mined in chicken, to be used for genome analyses in quail.     

Histones from Saccharomyces cerevisiae

Total histones and histone fractions isolated from Saccharomyces cerevisiae chromatin were analysed by polyacrylamide gel electrophoresis. The presence of the four histone fractions H2a, H2b, H3 and H4 was demonstrated. In addition, yeast chromatin contained a protein similar to histone H1 from mammals in molecular weight, charge and association properties with Triton X-100. However, it had a much lower lysine to arginine ratio, equal to about 3, as compared with H1 histones from higher eukaryotes. The order of electrophoretic mobilities of yeast histone fractions in acidic urea-polyacrylamide gels was similar to that observed for histones from plant sources, i.e. H4>H3>H2a>H2b>H1. Previously undetected protein (protein X) was extracted from yeast chromatin with 5 % HClO₄. The properties of this protein are under investigation.     

Distribution of Allelic Forms of Erythrocyte H1 Histones in Japanese Quail Populations Divergently Selected for Amount of Weight Loss After Transient Starvation

Three polymorphic subtypes of erythrocytehistone H1 (H1.a, H1.b, and H1.z) were analyzed using asodium dodecyl sulfate polyacrylamide gel in quailpopulations divergently selected for a high (line 1) or low (line 2) reduction in body massfollowing temporary food withdrawal. Both H1.b and H1.zhistone alleles were found to be differently distributedin these populations during the selection period. The frequency of b¹ in line 2 wasapproximately 1.9-2.8 times lower than in line 1 andapproached the values in line 1 when the selection wassuspended. Similarly, the frequency of allelez² at locus H1.z increased significantly (about 1.6-2.3 times)in line 2 during selection and returned to the initialvalues when selection was stopped. On the other hand,allele a⁰ at locus H1.a was kept atrelatively low levels (usually below 0.05) in both linesduring selection. At that time its level wasapproximately three to four times lower than in a randommating control population. When selection was suspended, the frequency of a⁰ in line 1increased significantly, approaching the values in thecontrol line, and remained essentially unchanged in line2. Thus, all three polymorphic histone H1 loci in quailresponded through changes in allele frequencies to thebreeding selection, which was directed at the amount ofbody weight loss upon transient starvation. It seemsthat either H1 histone locus could be linked to loci controlling the rate of body weightreduction following starvation or weight loss duringfasting might be influenced by a panel of H1 histonealleles that can contribute to functional differences in avian chromatin.     

AFLP linkage map of the Japanese quail Coturnix japonica

The quail is a valuable farm and laboratory animal. Yet molecular information about this species remains scarce. We present here the first genetic linkage map of the Japanese quail. This comprehensive map is based solely on amplified fragment length polymorphism (AFLP) markers. These markers were developed and genotyped in an F2 progeny from a cross between two lines of quail differing in stress reactivity. A total of 432 polymorphic AFLP markers were detected with 24 TaqI/EcoRI primer combinations. On average, 18 markers were produced per primer combination. Two hundred and fifty eight of the polymorphic markers were assigned to 39 autosomal linkage groups plus the ZW sex chromosome linkage groups. The linkage groups range from 2 to 28 markers and from 0.0 to 195.5 cM. The AFLP map covers a total length of 1516 cM, with an average genetic distance between two consecutive markers of 7.6 cM. This AFLP map can be enriched with other marker types, especially mapped chicken genes that will enable to link the maps of both species and make use of the powerful comparative mapping approach. This AFLP map of the Japanese quail already provides an efficient tool for quantitative trait loci (QTL) mapping.     

纳米二氧化锆对人永生化角质形成细胞组蛋白H3修饰的影响

目的 阐明纳米二氧化锆暴露对人永生化角质形成细胞Ha Ca T组蛋白H3常见修饰位点的影响,探讨组蛋白H3修饰变化的潜在机制,为纳米材料的进一步安全应用提供理论基础。方法 在利用扫描电子显微镜、激光粒度仪、X射线衍射仪等技术对纳米二氧化锆进行详细表征的基础上,通过蛋白质免疫印迹及流式细胞术等方法评价纳米二氧化锆暴露对细胞生存率、细胞内蓄积量以及组蛋白H3修饰等的影响。结果 在分散介质中纳米二氧化锆明显团聚,比表面积减少,二次粒径增大,其短时间内（1 h）即诱导了组蛋白H3第10位丝氨酸的磷酸化、第9及14位赖氨酸的乙酰化、第4及27位赖氨酸的三甲基化修饰水平的升高。进一步分析发现,纳米二氧化锆的细胞内蓄积量及其引起的DNA损伤水平,与纳米二氧化锆诱导的组蛋白H3修饰水平均呈线性相关。结论 纳米二氧化锆暴露后诱导了Ha Ca T细胞组蛋白H3常见修饰位点的变化,其细胞内的蓄积是诱导组蛋白H3修饰变化的关键因素之一,且组蛋白H3修饰的调控机制可能涉及DNA损伤修复途径。     

Nucleosome compaction facilitates HP1γ binding to methylated H3K9

The α, β and γ isoforms of mammalian heterochromatin protein 1 (HP1) selectively bind to methylated lysine 9 of histone H3 via their chromodomains. Although the phenotypes of HP1-knockout mice are distinct for each isoform, the molecular mechanisms underlying HP1 isoform-specific function remain elusive. In the present study, we found that in contrast to HP1α, HP1γ could not bind tri-methylated H3 lysine 9 in a reconstituted tetra-nucleosomes when the nucleosomes were in an uncompacted state. The hinge region connecting HP1''s chromodomain and chromoshadow domain contributed to the distinct recognition of the nucleosomes by HP1α and HP1γ. HP1γ, but not HP1α, was strongly enhanced in selective binding to tri-methylated lysine 9 in histone H3 by the addition of Mg²⁺ or linker histone H1, which are known to induce compaction of nucleosomes. We propose that this novel property of HP1γ recognition of lysine 9 in the histone H3 tail in different nucleosome structures plays a role in reading the histone code.     

A first-generation microsatellite linkage map of the Japanese quail

A linkage map of the Japanese quail (Coturnix japonica) genome was constructed based upon segregation analysis of 72 microsatellite loci in 433 F(2) progeny of 10 half-sib families obtained from a cross between two quail lines of different genetic origins. One line was selected for long duration of tonic immobility, a behavioural trait related to fearfulness, while the other was selected based on early egg production. Fifty-eight of the markers were resolved into 12 autosomal linkage groups and a Z chromosome-specific linkage group, while the remaining 14 markers were unlinked. The linkage groups range from 8 cM (two markers) to 206 cM (16 markers) and cover a total map distance of 576 cM with an average spacing of 10 cM between loci. Through comparative mapping with chicken (Gallus gallus) using orthologous markers, we were able to assign linkage groups CJA01, CJA02, CJA05, CJA06, CJA14 and CJA27 to chromosomes. This map, which is the first in quail based solely on microsatellites, is a major step towards the development of a quality molecular genetic map for this valuable species. It will provide an important framework for further genetic mapping and the identification of quantitative trait loci controlling egg production and fear-related behavioural traits in quail.     

Heterochromatin Controls γH2A Localization in Neurospora crassa

In response to genotoxic stress, ATR and ATM kinases phosphorylate H2A in fungi and H2AX in animals on a C-terminal serine. The resulting modified histone, called γH2A, recruits chromatin-binding proteins that stabilize stalled replication forks or promote DNA double-strand-break repair. To identify genomic loci that might be prone to replication fork stalling or DNA breakage in Neurospora crassa, we performed chromatin immunoprecipitation (ChIP) of γH2A followed by next-generation sequencing (ChIP-seq). γH2A-containing nucleosomes are enriched in Neurospora heterochromatin domains. These domains are comprised of A·T-rich repetitive DNA sequences associated with histone H3 methylated at lysine-9 (H3K9me), the H3K9me-binding protein heterochromatin protein 1 (HP1), and DNA cytosine methylation. H3K9 methylation, catalyzed by DIM-5, is required for normal γH2A localization. In contrast, γH2A is not required for H3K9 methylation or DNA methylation. Normal γH2A localization also depends on HP1 and a histone deacetylase, HDA-1, but is independent of the DNA methyltransferase DIM-2. γH2A is globally induced in dim-5 mutants under normal growth conditions, suggesting that the DNA damage response is activated in these mutants in the absence of exogenous DNA damage. Together, these data suggest that heterochromatin formation is essential for normal DNA replication or repair.     

Complete genetic linkage maps from an interspecific cross between Fusarium circinatum and Fusarium subglutinans

The Gibberella fujikuroi complex includes many plant pathogens of agricultural crops and trees, all of which have anamorphs assigned to the genus Fusarium. In this study, an interspecific hybrid cross between Gibberella circinata and Gibberella subglutinans was used to compile a genetic linkage map. A framework map was constructed using a total of 578 AFLP markers together with the mating type (MAT-1 and MAT-2) genes and the histone (H3) gene. Twelve major linkage groups were identified (n=12). Fifty percent of the markers showed significant deviation from the expected 1:1 transmission ratio in a haploid F(1) cross (P <0.05). The transmission of the markers on the linkage map was biased towards alleles of the G. subglutinans parent, with an estimated 60% of the genome of F(1) individuals contributed by this parent. This map will serve as a powerful tool to study the genetic architecture of interspecific differentiation and pathogenicity in the two parental genomes.     

Divergent Residues Within Histone H3 Dictate a Unique Chromatin Structure in Saccharomyces cerevisiae

Histones are among the most conserved proteins known, but organismal differences do exist. In this study, we examined the contribution that divergent amino acids within histone H3 make to cell growth and chromatin structure in Saccharomyces cerevisiae. We show that, while amino acids that define histone H3.3 are dispensable for yeast growth, substitution of residues within the histone H3 α3 helix with human counterparts results in a severe growth defect. Mutations within this domain also result in altered nucleosome positioning, both in vivo and in vitro, which is accompanied by increased preference for nucleosome-favoring sequences. These results suggest that divergent amino acids within the histone H3 α3 helix play organismal roles in defining chromatin structure.     

DNA replication and H5 histone exchange during reactivation of chick erythrocyte nuclei in heterokaryons

Fusion of terminally differentiated chick erythrocytes (CE) with replicating quail myoblasts or established L6J1 rat myoblasts results in reactivation of DNA synthesis in the dormant CE nuclei and in suppression of DNA synthesis in the myoblast nuclei. The nuclei of primary quail myoblasts are more effectively inhibited than the nuclei of established rat myoblasts. Inhibition of DNA replication occurs not only by preventing G1 nuclei from entering S-phase but also by blocking nuclei in S-phase and by delaying nuclei in G2 from undergoing mitosis and starting a new DNA replication cycle. No inhibition of DNA synthesis could be observed when mouse erythrocytes, i.e., erythrocytes lacking nuclei, were fused with rat myoblasts to generate mouse-globin-containing L6J1 cybrids. — Reactivation of CE nuclei is associated with a loss of the tissuespecific H5 histone variant. Complete elimination of H5 histone, however, does not seem to be a necessary prerequisite for the initiation or completion of DNA replication in CE nuclei since H5 antigens are found on reactivated G1, S, and G2 nuclei.     

Properties of the complex between histone H1 and poly(ADP-ribose synthesised in HeLa cell nuclei

Preparations of H1 histone from HeLa cell nuclei incubated with [3H]NAD to permit poly(ADP-ribose) synthesis were electrophoresed on polyacrylamide gels. The incorporated radioactivity migrated as a sharply defined peak in association with a protein band which moved more slowly than H1, the major protein component. The following observations indicate that this complex is composed of two molecules of H1 and a single chain of poly(ADP-ribose) with one detectable covalent linkage of polymer to protein. 1. The [14C]arginine/[3H]lysine ratio is identical in H1 histone and in the protein moiety of the complex. 2. Protein is displaced from H1 histone to the complex during poly(ADP-ribose) synthesis. At least 90% of the protein in the complex (stainable protein and labelled protein) is derived from H1. 3. Sedimentation rate studies indicate a molecular weight of the complex about twice that of H1 histone. 4. The average chain length of the polymer is 15 ADP-ribose units and there are 7--8 ADP-ribose units for each molecule of H1 histone in the 'complex'. 5. Poly(ADP-ribose) glycohydrolase, which hydrolyses the polymer exoglycosidically from the AMP terminus, degrades the complex producing ADP-ribose and mono-ADP-ribosylated H1 histone which co-electrophoreses with unmodified H1. Although only one covalent linkage between protein and polymer has been detected, the 'complex' does not dissociate when electrophoresed on dodecylsulfate gels. Nor can the noncovalently linked H1 histone of the complex readily exchange with free H1. Complex formation does not occur when purified poly(ADP-ribose) and H1 are mixed.