Protection of expressed immunoglobulin genes against nuclease cleavage.

Fragmentation of the actively transcribed kappa immunoglobulin gene in mouse myeloma nuclei with micrococcal nuclease and the restriction nuclease Bsp RI reveals a chromatin structure without the regularity of repeating nucleosomes found in bulk chromatin. Such regularity is restored about 2.2 kb 3' of the coding region. An only moderately increased micrococcal nuclease sensitivity and a 65% average protection of the Bsp RI sites indicates a DNA-protein interaction in the transcribed region which is not very different from that of an inactive gene. As determined by indirect endlabeling the frequency of Bsp RI cleavage both, after very mild and exhaustive digestion, varied moderately from site to site along the gene. In addition, it was not in each case the same at analogous sites on both alleles which are both transcribed. Thus, the experiments demonstrate differences between the chromatin structures of the genes which may be related to regulatory phenomena and thereby corroborate earlier findings made with DNAase I.     

Footprint analysis of the bsp RI DNA methyltransferase-DNA interaction.

The interaction between the GGCC-specific Bsp RI DNA methyltransferase (M. Bsp RI) and substrate DNA was studied with footprinting techniques using a DNA fragment that was unmodified on both strands. Footprinting with DNase I revealed an approximately 14 bp protected region. Footprinting with dimethylsulfate detected major groove interactions with the guanine bases of the recognition sequence. Reaction with 1,10-phenanthroline-copper did not show protection, suggesting that minor groove interactions play little role in sequence-specific recognition by M. Bsp RI. Hydroxyl radical footprinting revealed a protected stretch of 6 nt. The hydroxyl radical footprint of M. Bsp RI differs markedly from the the footprint reported for the Hha I and Sss I methyltransferases. The pattern of protection from dimethylsulfate and hydroxyl radicals suggests that the interactions of M. Bsp RI with DNA are similar to those detected in the co-crystal structure of the Hae III methyltransferase.     

The role of H2B histones from the sea urchin sperm in the formation of supranucleosome structures

The structural role of histone H2B from sea urchin sperm (H2Bsp) has been examined in experiments on reconstitution of chromatin from DNA and core histones taken in three variants: (1) four core histones from sea urchin sperm; (2) four core histones from calf thymus; (3) (H3, H4, H2A) from calf thymus and H2Bsp. It is shown that H2Bsp when present in reconstituted chromatin induces its aggregation. Fidelity of the reconstitution of nucleosomes has been tested using DNase I probe, one- and two-dimensional electrophoresis and electron microscopy. The reconstitutes that contain H2Bsp appear under electron microscope mainly as regular closely spaced large granules, about 450 A in diameter, which are very similar to the granules found in "native" sea urchin sperm chromatin. The reconstitutes formed by four core histones from calf thymus appear as randomly arranged particles, about 100 A in diameter. We conclude that histone H2Bsp participates in interactions between nucleosomes and is involved in the formation of the condensed supranucleosomal structure in sea urchin sperm chromatin.     

Btc22 chaperone is required for secretion and stability of the type III secreted protein Bsp22 in Bordetella bronchiseptica

The type III secretion system (T3SS) is a sophisticated protein secretion machinery that delivers bacterial virulence proteins into host cells. A needle-tip protein, Bsp22 , is one of the secreted substrates of the T3SS and plays an essential role in the full function of the T3SS in Bordetella bronchiseptica. In this study, we found that BB1618 functions as a chaperone for Bsp22 . The deletion of BB1618 resulted in a dramatic impairment of Bsp22 secretion into the culture supernatants and Bsp22 stability in the bacterial cytosol. In contrast, the secretion of other type III secreted proteins was not affected by the BB1618 mutation. Furthermore, the BB1618 mutant strain could not induce cytotoxicity and displayed the same phenotypes as the Bsp22 mutant strain. An immunoprecipitation assay demonstrated that BB1618 interacts with Bsp22 , but not with BopB and BopD . Thus, we identified BB1618 as a specific type III chaperone for Bsp22 . Therefore, we propose that BB1618 be renamed Btc22 for the Bordetella type III chaperone for Bsp22 .     

Organization of Bsp-repeats in the fox genome

Bsp repeats comprise approximately 1% of silver for total DNA and are preferentially localized in centromeric regions. Two of Bsp fragments cloned earlier, such as non-homologous rsV1 and rsV3, make up together a Bsp unit (680 bp) and possess a set of sites known to have regulatory functions in eucaryotic genomes. In this work, tandem organization of Bsp repeats is demonstrated. A stretched Bsp sequence (approximately 1460 bp, dimeric form) flanked by BamHI sites was cloned and its restriction map was plotted. With use of rsV1 and rsV3 probes the new sequence exhibited linked structure: rsV1-rsV3-rsV1-rsV3-rsV1. Blot-hybridization with the restriction fragments of fox total DNA suggested hierarchical order of Bsp clustes in the genome. It is supposed that the processes of selective amplification of individual fragments had been of real importance during evolution of Bsp repeats.     

基于遗传算法酵母核小体定位性质预测

在DNA序列上,定位模糊的特殊核小体与定位良好的普通核小体同时存在于染色体区域内,但由于二者的化学性质差异不明显,区分较为困难。本文针对实验核小体在真核基因转录起始位点周围的分布规律和保守性建立了一个核小体分布模型,并在前人所做的预测核小体位置的工作基础上,利用遗传算法寻找模型上不同性质核小体的分布中心,构建核小体定位性质判别准则,最终确定了转录起始位点上、下游定位良好和模糊核小体的位置。     

Bordetella Bsp22 forms a filamentous type III secretion system tip complex and is immunoprotective in vitro and in vivo

Type III secretion system (T3SS) tip complexes serve as adaptors that bridge the T3SS needle and the pore-forming translocation apparatus. In this report we demonstrate that Bsp22, the most abundantly secreted substrate of the Bordetella T3SS, self-polymerizes to form the Bordetella bronchiseptica tip complex. Bsp22 is required for both T3SS-mediated cytotoxicity against eukaryotic cells and haemoglobin release from erythrocytes. Bacterial two-hybrid analysis and protein pull-down assays demonstrated the ability of Bsp22 to associate with itself and to bind BopD, a component of the Bordetella translocation pore. Immunoblot and cross-linking analysis of secreted proteins or purified Bsp22 showed extensive multimerization which was shown by transmission electron microscopy to lead to the formation of variable length flexible filaments. Immunoelectron microscopy revealed Bsp22 filaments on the surface of bacterial cells. Given its required role in secretion and cell-surface exposure, we tested the protective effects of antibodies against Bsp22 in vitro and in vivo . Polyclonal antisera against Bsp22 fully protected epithelial cells from T3SS-dependent killing and immunization with Bsp22 protected mice against Bordetella infection. Of the approximately 30 genes which encode the Bordetella T3SS apparatus, bsp22 is the only one without characterized orthologues in other well-characterized T3SS loci. A maximum likelihood phylogenetic analysis indicated that Bsp22 defines a new subfamily of T3SS tip complex proteins. Given its immunogenic and immunoprotective properties and high degree of conservation among Bordetella species, Bsp22 and its homologues may prove useful for diagnostics and next-generation subunit vaccines.     

Quantifying the role of steric constraints in nucleosome positioning

Statistical positioning, the localization of nucleosomes packed against a fixed barrier, is conjectured to explain the array of well-positioned nucleosomes at the 5′ end of genes, but the extent and precise implications of statistical positioning in vivo are unclear. We examine this hypothesis quantitatively and generalize the idea to include moving barriers as well as nucleosomes actively packed against a barrier. Early experiments noted a similarity between the nucleosome profile aligned and averaged across genes and that predicted by statistical positioning; however, we demonstrate that aligning random nucleosomes also generates the same profile, calling the previous interpretation into question. New rigorous results reformulate statistical positioning as predictions on the variance structure of nucleosome locations in individual genes. In particular, a quantity termed the variance gradient, describing the change in variance between adjacent nucleosomes, is tested against recent high-throughput nucleosome sequencing data. Constant variance gradients provide support for generalized statistical positioning in ∼50% of long genes. Genes that deviate from predictions have high nucleosome turnover and cell-to-cell gene expression variability. The observed variance gradient suggests an effective nucleosome size of 158 bp, instead of the commonly perceived 147 bp. Our analyses thus clarify the role of statistical positioning in vivo.     

Levels of specifically bound [3H]ketanserin compared with levels of serotonin (5HT) in the brain regions of juvenile and sexually recrudescing female rainbow trout, Oncorhynchus mykiss

This study compared the distribution of specifically bound [3H]ketanserin (Bsp) with serotonin (5HT) in brain regions of juvenile and sexually recrudescing female trout. Amounts of Bsp varied widely among brain regions and consistently differed between juvenile and sexually recrudescing females. Levels of Bsp were significantly greater in the hypothalamus than the olfactory lobe, which were at least threefold greater than in all other tissues examined (Kruskal-Wallis test, p < 0.05). Bsp densities in the hypothalamus, preoptic area, and optic lobe were significantly greater in juveniles compared with corresponding tissues from sexually recrudescing females (Mann-Whitney U test, p < 0.05); in contrast, Bsp in olfactory lobe and spinal cord did not differ significantly between the two classes of fish. 5HT concentration was determined by high performance liquid chromatography - electrochemical detection (HPLC-EC) analysis. Biogenic amine standards eluted in a stereotypic pattern, with peaks consistently separable in time. 5HT concentration was significantly greater in hypothalamus than in olfactory lobe and undetectable in the pituitary (Kruskal-Wallis test, p < 0.05). Trends in distribution of Bsp and 5HT were comparable in the hypothalamus and preoptic area in juvenile and sexually recrudescing females. In general, density of specific [3H]ketanserin binding sites was directly related to 5HT content of brain regions in juvenile and sexually recrudescing females. 5HT concentrations (pmol/g tissue) were approximately 900-fold greater than Bsp (fmol/g tissue) in all brain regions, and approximately 300-fold greater than Bsp in the olfactory lobe. These results suggest important regulatory role(s) for 5HT in the trout preoptic-hypothalamo-hypophysial axis, which may differ from 5HT role(s) in trout olfactory lobe.     

The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly

Two very similar H3 histones-differing at only four amino acid positions-are produced in Drosophila cells. Here we describe a mechanism of chromatin regulation whereby the variant H3.3 is deposited at particular loci, including active rDNA arrays. While the major H3 is incorporated strictly during DNA replication, amino acid changes toward H3.3 allow replication-independent (RI) deposition. In contrast to replication-coupled (RC) deposition, RI deposition does not require the N-terminal tail. H3.3 is the exclusive substrate for RI deposition, and its counterpart is the only substrate retained in yeast. RI substitution of H3.3 provides a mechanism for the immediate activation of genes that are silenced by histone modification. Inheritance of newly deposited nucleosomes may then mark sites as active loci.     

Unfolding of nucleosome cores induced by chemical acetylation of histones

Relative accessibility of nucleosomal histones to acetic anhydride during acetylation has been studied as a function of concentration, pH and ionic strength of the solution using high-resolution gel-electrophoresis. It was shown that about 80% of lysine residues in nucleosomal histones and 100% of the same residues in histone complexes without DNA in 2 M NaCl are accessible to the modification, which is proved by the localization of the majority of lysine residues in nucleosomes near the surface of the histone octamer, by their participation in ionic interactions with DNA and, probably, in histone-histone contacts. Gel-electrophoretic experiments with nucleosomes and studies of the histone resistance to mild trypsinolysis indicated that neither nucleosomes themselves nor histone octamers are affected even though 50% of lysine residues in histones have been acetylated. The process of acetylation is accompanied by the growing tendency of histones to participate in mild trypsinolysis and by a gradual decline in electrophoretic mobility and in the value of the sedimentation constant. The circular dichroism spectra and the microscopic appearance of nucleosomes are also markedly changed. These results suggest that a gradual unfolding of nucleosomes occurs when 5 or more lysine residues in the nucleosomal histones have been acetylated.     

Inference of relationships in the 'twilight zone' of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII

Thus far, identification of functionally important residues in Type II restriction endonucleases (REases) has been difficult using conventional methods. Even though known REase structures share a fold and marginally recognizable active site, the overall sequence similarities are statistically insignificant, unless compared among proteins that recognize identical or very similar sequences. Bsp6I is a Type II REase, which recognizes the palindromic DNA sequence 5′GCNGC and cleaves between the cytosine and the unspecified nucleotide in both strands, generating a double-strand break with 5′-protruding single nucleotides. There are no solved structures of REases that recognize similar DNA targets or generate cleavage products with similar characteristics. In straightforward comparisons, the Bsp6I sequence shows no significant similarity to REases with known structures. However, using a fold-recognition approach, we have identified a remote relationship between Bsp6I and the structure of PvuII. Starting from the sequence–structure alignment between Bsp6I and PvuII, we constructed a homology model of Bsp6I and used it to predict functionally significant regions in Bsp6I. The homology model was supported by site-directed mutagenesis of residues predicted to be important for dimerization, DNA binding and catalysis. Completing the picture of sequence–structure–function relationships in protein superfamilies becomes an essential task in the age of structural genomics and our study may serve as a paradigm for future analyses of superfamilies comprising strongly diverged members with little or no sequence similarity.     

Crystal structure and substrate-binding mode of a novel pectate lyase from alkaliphilic Bacillus sp. N16-5

The pectate lyase (Bsp165PelA) from Bacillus sp. N16-5 has great potential in industrial applications because it shows high specific activity under extremely alkaline conditions. Besides, activity measurement of Bsp165PelA does not require addition of calcium, in a way different from the other pectate lyases. Here we report crystal structures of Bsp165PelA in apo-form and in complex with trigalacturonate. The parallel β-helix, active site residues and substrate binding cleft are similar to those in the other pectate lyases from Polysaccharide Lyase family 1. However, some of the highly conserved Ca(2+) binding residues and secondary structures are altered in Bsp165PelA, making it difficult to coordinate with Ca(2+) as in the other pectate lyases. We found Bsp165PelA forms some direct enzyme-substrate interactions instead of using Ca(2+) ions bridging in the extremely alkaline environment.     

Theoretical analysis of epigenetic cell memory by nucleosome modification

Chromosomal regions can adopt stable and heritable alternative states resulting in bistable gene expression without changes to the DNA sequence. Such epigenetic control is often associated with alternative covalent modifications of histones. The stability and heritability of the states are thought to involve positive feedback where modified nucleosomes recruit enzymes that similarly modify nearby nucleosomes. We developed a simplified stochastic model for dynamic nucleosome modification based on the silent mating-type region of the yeast Schizosaccharomyces pombe. We show that the mechanism can give strong bistability that is resistant both to high noise due to random gain or loss of nucleosome modifications and to random partitioning upon DNA replication. However, robust bistability required: (1) cooperativity, the activity of more than one modified nucleosome, in the modification reactions and (2) that nucleosomes occasionally stimulate modification beyond their neighbor nucleosomes, arguing against a simple continuous spreading of nucleosome modification.     

Characterization of plant satellite DNA using restriction nucleases

The structural organization of satellite DNAs of mustard Brassica nigra and lemon Citrus limon has been studied by digestion with restriction nucleases. Analysis of DNA products produced by EcoRI and Bam I shows that two satellite DNAs contain long range periodicities belonging to several repeated sequences. The periodicities in two satellite DNAs differ characteristically, however, they have been found to contain common homologous sequences. Using the restriction nuclease Bsp I, a highly periodical fractions has been found in Citrus satellite DNA, composed of Bsp I fragments ranging from 80 to 1240 basepain. The major repeat units comprise five Bsp I fragments ranging from 80 to 200 bp. These fractions characterized by a high content of 5-methyl-cytosine.     

Repair of UV lesions in nucleosomes--intrinsic properties and remodeling

Nucleotide excision repair and reversal of pyrimidine dimers by photolyase (photoreactivation) are two major pathways to remove UV-lesions from DNA. Here, it is discussed how lesions are recognized and removed when the DNA is condensed into nucleosomes. During the recent years it was shown that nucleosomes inhibit photolyase and excision repair in vitro and slow down repair in vivo. The correlation of DNA-repair rates with nucleosome positions in yeast suggests that intrinsic properties of nucleosomes such as mobility and transient unwrapping of nucleosomal DNA facilitate damage recognition. Moreover, it was shown that nucleosome remodeling activities can act on UV-damaged DNA in vitro and facilitate repair suggesting that random remodeling of chromatin might contribute to damage recognition in vivo. Recent work on nucleosome structure and mobility is included to evaluate how nucleosomes accommodate DNA lesions and how nucleosome mobility and remodeling can take place on damaged DNA.     

Relation of nucleosomes to nucleotide sequences in the rat.

The relation of nucleosomes to nucleotide sequences is random for most single copy sequences in rat liver. This could be due to variation in the DNA content of nucleosomes, and a procedure for detecting such variation is described.