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Periprosthetic infections are one of the most serious complications in orthopedic surgeries, and those caused by Staphylococcus aureus ( S. aureus ) are particularly hard to treat due to their tendency to form biofilms on implants and their notorious ability to invade the surrounding bones. The existing prophylactic local antibiotic deliveries involve excessive drug loading doses that could risk the development of drug resistance strains. Utilizing an oligonucleotide linker sensitive to micrococcal nuclease (MN) cleavage, we previously developed an implant coating capable of releasing covalently tethered vancomycin, triggered by S. aureus -secreted MN, to prevent periprosthetic infections in the mouse intramedullary (IM) canal. To further engineer this exciting platform to meet broader clinical needs, here, we chemically modified the oligonucleotide linker by a combination of 2'-O-methylation and phosphorothioate modification to achieve additional modulation of its stability/sensitivity to MN and the kinetics of MN-triggered on-demand release. We found that when all phosphodiester bonds within the oligonucleotide linker 5'-carboxy-mCmGTTmCmG-3-acrydite, except for the one between TT, were replaced by phosphorothioate, the oligonucleotide (6PS) stability significantly increased and enabled the most sustained release of tethered vancomycin from the coating. By contrast, when only the peripheral phosphodiester bonds at the 5'- and 3'-ends were replaced by phosphorothioate, the resulting oligonucleotide (2PS) linker was cleaved by MN more rapidly than that without any PS modifications (0PS). Using a rat femoral canal periprosthetic infection model where 1000 CFU S. aureus was inoculated at the time of IM pin insertion, we showed that the prophylactic implant coating containing either 0PS- or 2PS-modified oligonucleotide linker effectively eradicated the bacteria by enabling the rapid on-demand release of vancomycin. No bacteria were detected from the explanted pins, and no signs of cortical bone changes were detected in these treatment groups throughout the 3 month follow-ups. With an antibiotic tethering dose significantly lower than conventional antibiotic-bearing bone cements, these coatings also exhibited excellent biocompatibility. These chemically modified oligonucleotides could help tailor prophylactic anti-infective coating strategies to meet a range of clinical challenges where the risks for S. aureus prosthetic infections range from transient to long-lasting.

Tembusu virus (TMUV), a member of the genus flavivirus, primarily causes egg-drop syndrome in ducks and is associated with low disease mortality but high morbidity. The commercially available live vaccines for treating TMUV currently include the main WF100, HB, and FX2010-180P strains, and efficient treatment and/or preventative measures are still urgently needed. Capsid-targeted viral inactivation (CTVI) is a conceptually powerful new antiviral strategy that is based on two proteins from the capsid protein of a virus and a crucial effector molecule. The effector molecule can destroy the viral DNA/RNA or interfere with the proper folding of key viral proteins, while the capsid protein mainly plays a role in viral integration and assembly; the fusion proteins are incorporated into virions during packaging. This study aimed to explore the potential use of this strategy in duck TMUV. Our results revealed that these fusion proteins can be expressed in susceptible BHK21 cells without cytotoxicity and possess excellent Ca 2+ -dependent nuclease activity, and their expression is also detectable in DF-1 cells. Compared to those in the negative controls (BHK21 and BHK21/pcDNA3.1(+) cells), the numbers of viral RNA copies in TMUV-infected BHK21/Cap-SNase and BHK21/Cap-Linker-SNase cells were reduced by 48 h, and the effect of Cap-Linker-SNase was superior to that of Cap-SNase. As anticipated, these results suggest that these fusion proteins contribute to viral resistance to treatment. Thus, CTVI might be applicable for TMUV inhibition as a novel antiviral therapeutic candidate during viral infection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aims: Recent studies have revealed that neutrophil extracellular traps (NETs) provide negative feedback in the progression to chronic inflammation and contribute to the pathogenesis of multiple autoimmune diseases including type 1 diabetes (T1D). In addition, accumulating evidences suggest that gut immunity play a key role in T1D pathogenesis. Our study aimed to evaluate whether staphylococcal nuclease (SNase) targeting intestinal NETs can ameliorate the intestinal inflammatory environment and protect against T1D development in non-obese diabetic(NOD) mice., Main Methods: Degradation of NETs with SNase in vitro was examined using SYTOX green assay. NOD/LtJ mice were oral administration of Lactococcus lactisl (L. lactis) pCYT: SNase and blood glucose levels were monitored weekly. Several biomarkers of NETs formation, gut leakage and inflammation were determined using a commercial ELISA kit. T Cell phenotypes in peripheral immune system were analyzed in flow cytometry and fecal samples were isolated to investigate intestinal microbiota., Key Findings: The oral delivery of SNase by L. lactis can decrease the NETs levels and ameliorate inflammation both in the intestine and pancreatic islets and finally effectively regulate the blood glucose levels of NOD mice. Meanwhile, zonulin and lipopolysaccharide levels also reduced in SNase-fed NOD mice, suggesting SNase could improve gut barrier function via intestinal NETs degradation. Furthermore, the abundances of the intestinal microbiota and butyrate-producing gut bacteria were also increased with SNase treatment., Significance: SNase shows potential for intestinal NETs to prevent T1D based on the gut-pancreas axis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Nucleosomes have structural and regulatory functions in all eukaryotic DNA-templated processes. The position of nucleosomes on DNA and the stability of the underlying histone-DNA interactions affect the access of regulatory proteins to DNA. Both stability and position are regulated through DNA sequence, histone post-translational modifications, histone variants, chromatin remodelers, and transcription factors. Here, we explored the functional implications of nucleosome properties on gene expression and development in Caenorhabditis elegans embryos. We performed a time-course of micrococcal nuclease (MNase) digestion and measured the relative sensitivity or resistance of nucleosomes throughout the genome. Fragile nucleosomes were defined by nucleosomal DNA fragments that were recovered preferentially in early MNase-digestion time points. Nucleosome fragility was strongly and positively correlated with the AT content of the underlying DNA sequence. There was no correlation between promoter nucleosome fragility and the levels of histone modifications or histone variants. Genes with fragile nucleosomes in their promoters tended to be lowly expressed and expressed in a context-specific way, operating in neuronal response, the immune system, and stress response. In addition to DNA-encoded nucleosome fragility, we also found fragile nucleosomes at locations where we expected to find destabilized nucleosomes, for example, at transcription factor binding sites where nucleosomes compete with DNA-binding factors. Our data suggest that in C. elegans promoters, nucleosome fragility is in large part DNA-encoded and that it poises genes for future context-specific activation in response to environmental stress and developmental cues., (© 2017 Jeffers and Lieb; Published by Cold Spring Harbor Laboratory Press.)

Japanese encephalitis virus (JEV) is a leading member of the mosquito-transmitted flavivirus family, and is mainly distributed in China, India and South East Asia, where it can cause the central nervous system disease with irreversible neurological damage in humans and animals. Few effective anti viral drugs are currently available against JEV infections. To explore the feasibility of using capsid-targeted viral inactivation (CTVI), as an anti viral strategy against JEV infection, a plasmid pcDNA-Cap-SNase was constructed for expressing a fusion protein of JEV capsid (Cap) and Staphylococcus aureus nuclease (SNase). Under G418 selection, a mammalian cell line BHK-21/Cap-SNase stably expressing Cap-SNase fusion proteins could be detected by rabbit antiserum against JEV and had good nuclease activity in degrading DNA or RNA. The viral titer from JEV-infected BHK-21/Cap-SNase cell line was reduced about 69.7% compared with that produced in control BHK-21 cells. It was clearly demonstrated that Cap-SNase fusion proteins could be use to efficiently inhibit JEV replication, resulting in a reduction of viral titer. Therefore, the CTVI approach might be applicable to JEV inhibition as a novel anti viral strategy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP-SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP-SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP-SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP-SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.

The packaging of DNA into nucleosomes imposes obstacles on gene transcription, and histone-modifying and nucleosome-remodeling complexes work in concert to alleviate these obstacles so as to facilitate transcription. Emerging evidence shows that chromatin-associated poly(ADP-ribose) polymerase 1 (PARP-1) and its enzymatic activity facilitate inflammatory gene transcription and modulate the inflammatory response in animal models. However, the molecular mechanisms by which PARP-1 enzymatic activity facilitates transcription are not well understood. Here we show that through an intracellular signaling pathway, lipopolysaccharide (LPS) stimulation induces PARP-1 enzymatic activity and the ADP-ribosylation of histones at transcriptionally active and accessible chromatin regions in macrophages. In vitro DNase I footprinting and restriction endonuclease accessibility assays reveal that histone ADP-ribosylation directly destabilizes histone-DNA interactions in the nucleosome and increases the site accessibility of the nucleosomal DNA to nucleases. Consistent with this, LPS stimulation-induced ADP-ribosylation at the nucleosome-occupied promoters of il-1β, mip-2, and csf2 facilitates NF-κB recruitment and the transcription of these genes in macrophages. Therefore, our data suggest that PARP-1 enzymatic activity facilitates gene transcription through increasing promoter accessibility by histone ADP-ribosylation.

Myostatin is a negative regulator of the growth and development of skeletal muscle mass. In fish, myostatin is expressed in several organs in addition to skeletal muscle. To understand the mechanisms regulating myostatin gene expression in the sea perch, Lateolabrax japonicus, we examined the methylation status of the myostatin gene promoter region in several tissues (liver, eye, kidney, brain, and heart) isolated from adult specimens. The frequency of methylated cytosines was very low in all tissues, regardless of the level of myostatin expression, suggesting that DNA methylation is not involved in the tissue-specific regulation of myostatin expression. Southern blot analysis of genomic DNA obtained from micrococcal nuclease-treated nuclei showed that chromatin digestion occurs in tissues where the myostatin gene is actively transcribed and that the myostatin gene is protected from micrococcal nuclease in tissues where myostatin is not expressed. The chromatin structure in the myostatin gene region appears to regulate its expression without DNA methylation.

The solvent accessible surface area (SASA) algorithm is conventionally used to characterize protein surfaces in electrostatic energy computations of proteins. Unfortunately, it often fails to find narrow cavities inside a protein. As a consequence pK(a) computations based on this algorithm perform badly. In this study a new cavity-algorithm is introduced, which solves this problem and provides improved pK(a) values. The procedure is applied to 20 pK(a) values of titratable groups introduced as point mutations in SNase variants, where crystal structures are available. The computations of these pK(a)s are particular challenging, since they are placed in a rather hydrophobic environment. For nine mutants, where the titratable residue is in contact with a large cavity, the RMSD(pKa) between computed and measured pK(a) values is 2.04, which is a considerable improvement as compared to the original results obtained with Karlsberg(+) (http://agknapp.chemie.fu-berlin.de/karlsberg/) that yielded an RMSD(pKa) of 8.8. However, for 11 titratable residues the agreement with experiments remains poor (RMSD(pKa) = 6.01). Considering 15 pK(a)s of SNase, which are in a more conventional less hydrophobic protein environment, the RMSD(pKa) is 2.1 using the SASA-algorithm and 1.7 using the new cavity-algorithm. The agreement is reasonable but less good than what one would expect from the general performance of Karlsberg(+) indicating that SNase belongs to the more difficult proteins with respect to pK(a) computations. We discuss the possible reasons for the remaining discrepancies between computed and measured pK(a)s., (Copyright © 2011 Wiley-Liss, Inc.)

Chromatin structure plays an important role in modulating the accessibility of genomic DNA to regulatory proteins in eukaryotic cells. We performed an integrative analysis on dozens of recent datasets generated by deep-sequencing and high-density tiling arrays, and we discovered an array of well-positioned nucleosomes flanking sites occupied by the insulator binding protein CTCF across the human genome. These nucleosomes are highly enriched for the histone variant H2A.Z and 11 histone modifications. The distances between the center positions of the neighboring nucleosomes are largely invariant, and we estimate them to be 185 bp on average. Surprisingly, subsets of nucleosomes that are enriched in different histone modifications vary greatly in the lengths of DNA protected from micrococcal nuclease cleavage (106-164 bp). The nucleosomes enriched in those histone modifications previously implicated to be correlated with active transcription tend to contain less protected DNA, indicating that these modifications are correlated with greater DNA accessibility. Another striking result obtained from our analysis is that nucleosomes flanking CTCF sites are much better positioned than those downstream of transcription start sites, the only genomic feature previously known to position nucleosomes genome-wide. This nucleosome-positioning phenomenon is not observed for other transcriptional factors for which we had genome-wide binding data. We suggest that binding of CTCF provides an anchor point for positioning nucleosomes, and chromatin remodeling is an important component of CTCF function., Competing Interests: The authors have declared that no competing interests exist.

Chromosome segregation ensures that DNA is equally divided between daughter cells during each round of cell division. The centromere (CEN) is the specific locus on each chromosome that directs formation of the kinetochore, the multiprotein complex that interacts with the spindle microtubules to promote proper chromosomal alignment and segregation during mitosis. CENs are organized into a specialized chromatin structure due to the incorporation of an essential CEN-specific histone H3 variant (CenH3) in the centromeric nucleosomes of all eukaryotes. Consistent with its essential role at the CEN, the loss or up-regulation of CenH3 results in mitotic defects. Despite the requirement for CenH3 in CEN function, it is unclear how CenH3 nucleosomes structurally organize centromeric DNA to promote formation of the kinetochore. To address this issue, we developed a modified chromatin immunoprecipitation approach to analyze the number and position of CenH3 nucleosomes at the budding yeast CEN. Using this technique, we show that yeast CENs have a single CenH3 nucleosome positioned over the CEN-determining elements. Therefore, a single CenH3 nucleosome forms the minimal unit of centromeric chromatin necessary for kinetochore assembly and proper chromosome segregation.

Nucleosome positions within the chromatin landscape are known to serve as a major determinant of DNA accessibility to transcription factors and other interacting components. To delineate nucleosomal patterns in a model genetic organism, Caenorhabditis elegans, we have carried out a genome-wide analysis in which DNA fragments corresponding to nucleosome cores were liberated using an enzyme (micrococcal nuclease) with a strong preference for cleavage in non-nucleosomal regions. Sequence analysis of 284,091 putative nucleosome cores obtained in this manner from a mixed-stage population of C. elegans reveals a combined picture of flexibility and constraint in nucleosome positioning. As has previously been observed in studies of individual loci in diverse biological systems, we observe areas in the genome where nucleosomes can adopt a wide variety of positions in a given region, areas with little or no nucleosome coverage, and areas where nucleosomes reproducibly adopt a specific positional pattern. In addition to illuminating numerous aspects of chromatin structure for C. elegans, this analysis provides a reference from which to begin an investigation of relationships between the nucleosomal pattern, chromosomal architecture, and lineage-based gene activity on a genome-wide scale.

Epigenetic processes involve alterations in the covalent modification of histones, which are driven by sequence-specific transcription factors recruiting the enzymatic machinery performing these reactions. Such histone modifications alter the charge or biochemical surface of the chromatin fiber and also serve as "docking" sites for transcription factor and chromatin remodelling complexes. Examining histone modification patterns is vital if we want to understand how transcription complexes establish patterns of gene expression. Histone modification can be quite localized and sometimes only involves a few nucleosomes. We therefore established a chromatin immunoprecipitation procedure that uses crosslinked chromatin allowing the isolation of small chromatin fragments while simultaneously minimizing histone movement during chromatin preparation.

Dengue infection poses a serious public health problem in most tropical and subtropical areas. No effective antiviral drugs or vaccines are currently available against dengue infection. To explore the feasibility of using capsid-targeted viral inactivation (CTVI) as an antiviral strategy against dengue infection, we constructed a plasmid expressing a fusion protein consisting of staphylococcal nuclease (SN) fused to dengue 2 virus capsid protein (D2C), and investigated its effects on the production of infectious virions when introduced into BHK cells infected with dengue virus. The results indicated that D2C-SN can be expressed and tolerated in this mammalian cell culture. The enzymatically active SN moiety was incorporated into nascent virions during the process of viral assembly. By comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of wild-type SN, on the infectivity of progeny virions, we clearly demonstrated that nucleolytic activity was the major antiviral mechanism. Expression of D2C-SN fusion protein as a therapeutic agent resulted in a reduction in infectious titers of 12- to 60-fold. Therefore, dengue virus may be particularly vulnerable to a CTVI therapeutic approach.

The genotoxic potential of total particulate matter (TPM) from mainstream smoke (MS) and sidestream smoke (SS) of Indian smoking products, namely cigarettes and bidis, as well as a brand of US cigarettes, was studied by determining the levels of bulky aromatic DNA adducts in mouse tissues. TPM from MS or SS of various smoking products [equal weights (2.5 mg) or the amount derived from equal (0.25) cigarette/bidi] was applied topically to mouse skin once a day for four consecutive days and adduct levels were determined in DNA from skin and lung by (32)P-post-labelling analysis. Relatively higher levels of bulky aromatic DNA adducts were noted in mouse skin treated with MS from a single Indian non-filter (INF) cigarette when compared with MS of a single bidi (with about half the product weight and one-quarter the tobacco compared with a cigarette), while comparable adduct levels were noted with SS from these two products. Considering the differences in the yields of constituents of tobacco smoke from the different products analyzed, the genotoxic potential of INF, Indian filter king (IFK) and American filter (AF) cigarettes as well as bidis was determined by topically applying an equal amount of TPM (rather than equal product-derived TPM). SS-derived TPM from all the products showed relatively higher levels of total polycyclic aromatic hydrocarbons and induced relatively higher levels of bulky aromatic DNA adducts than those derived from MS. The data indicate that TPM (MS + SS) from cigarettes appears to be more genotoxic than that from bidis and the contribution of tendu leaf (a non-tobacco bidi wrapper) to the generation of bulky aromatic DNA adducts appears to be significant, particularly in SS of bidis. Topical pretreatment with curcumin decreased the levels of TPM-derived adducts while pretreatment with dietary turmeric failed to show such protection.

Characteristic steps during cellular apoptosis are the induction of chromatin condensation and subsequent DNA fragmentation, finally leading to the formation of oligomers of nucleosomes. We have examined the kinetics and local distribution of this nucleosomal fragmentation within different genomic regions. For the induction of apoptosis, HL60 cells were treated with the water-soluble camptothecin derivative topotecan (a topoisomerase I inhibitor). The genomic origin of the fragments was analysed by Southern blot hybridisation of the cleaved DNA. In these experiments we observed similar hybridisation patterns of the fragmented DNA, indicating a random and synchronous cleavage of the nuclear chromatin. However, hybridisation with a telomeric probe revealed that, in contrast to the other analysed genomic regions, the telomeric chromatin was not cleaved into nucleosomal fragments despite our observation that the telomeric DNA in HL60 cells is organised in nucleosomes. We determined just a minor shortening of the telomeric repeats early during apoptosis. These observations suggest that telomeric chromatin is excluded from internucleosomal cleavage during apoptosis.

DNA unwinding factor (DUF) was discovered as an essential DNA replication factor in Xenopus egg extracts. DUF consists of an HMG protein and a homolog of Cdc68p/Spt16p, and has the capability of unwinding dsDNA. Here we have examined the interaction of DUF with chromatin. DUF was incorporated into chromatin assembled from sperm heads and from plasmid DNA in egg extracts. It was revealed that the chromatin assembled in egg extracts immunodepleted of DUF is less sensitive to micrococcal nuclease (NNase) digestion than that assembled in control extracts, indicating that chromatin containing DUF has more decompact structure than that without DUF. Also we found that DUF has a high affinity for core histones in vitro. We suggest that the function of DUF may be to make the chromatin structure accessible to replication factors.

We review micromechanical experiments studying mechanoelastic properties of mitotic chromosomes. We discuss the history of this field, starting from the classic in vivo experiments of Nicklas (1983). We then focus on experiments where chromosomes were extracted from prometaphase cells and then studied by micromanipulation and microfluidic biochemical techniques. These experiments reveal that chromosomes have a well-behaved elastic response over a fivefold range of stretching, with an elastic modulus similar to that of a loosely tethered polymer network. Perturbation by microfluidic "spraying" of various ions reveals that the mitotic chromosome can be rapidly and reversibly decondensed or overcondensed, i.e., that the native state is not maximally compacted. We compare our results for chromosomes from cells to results of experiments by Houchmandzadeh and Dimitrov (1999) on chromatids reconstituted using Xenopus egg extracts. Remarkably, while the stretching elastic response of reconstituted chromosomes is similar to that observed for chromosomes from cells, reconstituted chromosomes are far more easily bent. This result suggests that reconstituted chromatids have a large-scale structure that is quite different from chromosomes in somatic cells. Finally, we discuss microspraying experiments of DNA-cutting enzymes, which reveal that the element that gives mitotic chromosomes their mechanical integrity is DNA itself. These experiments indicate that chromatin-condensing proteins are not organized into a mechanically contiguous "scaffold," but instead that the mitotic chromosome is best thought of as a cross-linked network of chromatin. Preliminary results from restriction enzyme digestion experiments indicate a spacing between chromatin "cross-links" of roughly 15 kb, a size similar to that inferred from classical chromatin loop isolation studies. These results suggest a general strategy for the use of micromanipulation methods for the study of chromosome structure.

Isolated newt (Notophthalmus viridescens) chromosomes were studied by using micromechanical force measurement during nuclease digestion. Micrococcal nuclease and short-recognition-sequence blunt-cutting restriction enzymes first remove the native elastic response of, and then to go on to completely disintegrate, single metaphase newt chromosomes. These experiments rule out the possibility that the mitotic chromosome is based on a mechanically contiguous internal non-DNA (e.g., protein) "scaffold"; instead, the mechanical integrity of the metaphase chromosome is due to chromatin itself. Blunt-cutting restriction enzymes with longer recognition sequences only partially disassemble mitotic chromosomes and indicate that chromatin in metaphase chromosomes is constrained by isolated chromatin-crosslinking elements spaced by approximately 15 kb.

Poly(ADP-ribose) polymerase (PARP) is a major NAD-dependent modifying enzyme that mediates important steps in DNA repair, transcription, and apoptosis, but its role during development is poorly understood. We found that a single Drosophila Parp gene spans more than 150 kb of transposon-rich centromeric heterochromatin and produces several differentially spliced transcripts, including a novel isoform, PARP-e, predicted to encode a protein lacking enzymatic activity. An insertion mutation near the upstream promoter for Parp-e disrupts all Parp expression. Heterochromatic but not euchromatic sequences become hypersensitive to micrococcal nuclease, nucleoli fail to form, and transcript levels of the copia retrotransposon are elevated more than 50-fold; the variegated expression of certain transgenes is dominantly enhanced. Larval lethality can be rescued and PARP activity restored by expressing a cDNA encoding PARP-e. We propose that PARP-e autoregulates Parp transcription by influencing the chromatin structure of its heterochromatic environment. Our results indicate that Parp plays a fundamental role organizing the structure of Drosophila chromatin.

In meiosis I sister centromeres are unified in their polarity on the spindle, and this unique behavior is known to require the function of meiosis-specific factors that set some intrinsic property of the centromeres. The fission yeast, Schizosaccharomyces pombe, possesses complex centromeres consisting of repetitive DNA elements, making it an excellent model in which to study the behavior of complex centromeres. In mitosis, during which sister centromeres mediate chromosome segregation by establishing bipolar chromosome attachments to the spindle, the central core of the S. pombe centromere chromatin has a unique irregular nucleosome pattern. Deletion of repeats flanking this core structure have no effect on mitotic chromosome segregation, but have profound effects during meiosis. While this demonstrates that the outer repeats are critical for normal meiotic sister centromere behavior, exactly how they function and how monopolarity is established remains unclear. In this study we provide the first analysis of the chromatin structure of a complex centromere during meiosis. We show that the nature and extent of the unique central core chromatin structure is maintained with no measurable expansion. This demonstrates that monopolarity of sister centromeres, and subsequent reversion to bipolarity, does not involve a global change to the centromeric chromatin structure.

We review micromechanical experiments on mitotic chromosomes. We focus on work where chromosomes were extracted from prometaphase amphibian cells, and then studied by micromanipulation and microfluidic biochemical techniques. These experiments reveal that chromosomes have well-behaved elastic response over a fivefold range of stretching, with an elastic modulus similar to that of a loosely tethered polymer network. Perturbation by microfluidic 'spraying' of various ions reveals that the mitotic chromosome can be rapidly and reversibly decondensed or overcondensed, i.e. that the native state is not maximally compacted. Finally, we discuss microspraying experiments of DNA-cutting enzymes which reveal that the element which gives mitotic chromosomes their mechanical integrity is DNA itself. These experiments indicate that chromatin-condensing proteins are not organized into a mechanically contiguous 'scaffold', but instead that the mitotic chromosome is best thought of as a cross-linked network of chromatin. Preliminary results from restriction-enzyme digestion experiments indicate a spacing between chromatin 'cross-links' of roughly 15 kb, a size similar to that inferred from classical chromatin-loop-isolation studies. We compare our results to similar experiments done by Houchmandzadeh and Dimitrov (J Cell Biol 145: 215-213 (1999)) on chromatids reconstituted using Xenopus egg extracts. Remarkably, while the stretching elastic response of the reconstituted chromosomes is similar to that observed for chromosomes from cells, the reconstituted chromosomes are far more easily bent. This result suggests that reconstituted chromatids have a large-scale structure which is quite different from chromosomes in somatic cells. More generally our results suggest a strategy for the use of micromanipulation methods for the study of chromosome structure.

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleus and imported from the cytosol. Incubation of two in vitro-transcribed tRNAs, tRNA(Ile)(UAU) and tRNA(Gln)(CUG), with isolated mitochondria from Leishmania tarentolae, in the absence of any added cytosolic fraction, resulted in a protease-sensitive, ATP-dependent importation, as measured by nuclease protection. Evidence that nuclease protection represents importation was obtained by the finding that Bacillus subtilis pre-tRNA(Asp) was protected from nuclease digestion and was also cleaved by an intramitochondrial RNase P-like activity to produce the mature tRNA. The presence of a membrane potential is not required for in vitro importation. A variety of small synthetic RNAs were also found to be efficiently imported in vitro. The data suggest that there is a structural requirement for importation of RNAs greater than approximately 17 nt, and that smaller RNAs are apparently nonspecifically imported. The signals for importation of folded RNAs have not been determined, but the specificity of the process was illustrated by the higher saturation level of importation of the mainly mitochondria-localized tRNA(Ile) as compared to the level of importation of the mainly cytosol-localized tRNA(Gln). Furthermore, exchanging the D-arm between the tRNA(Ile) and the tRNA(Gln) resulted in a reversal of the in vitro importation behavior and this could also be interpreted in terms of tertiary structure specificity.

Various complexes that contain the core subunits of RNA polymerase II associated with different transcription factors have been isolated from eukaryotes; their precise molecular constitution depends on the purification procedure. We estimated the numbers of various components of such complexes in an HeLa cell by quantitative immunoblotting. The cells were lysed with saponin in a physiological buffer; approximately 140,000 unengaged polymerases (mainly of form IIA) were released. Only approximately 4,000 of these soluble molecules sedimented in glycerol gradients as holoenzyme-sized complexes. About 180,000 molecules of polymerases (approximately 110,000 molecules of form IIO) and 10,000 to 30,000 molecules of each of TFIIB, TFIIEalpha, TFIIEbeta, TFIIF-RAP74, TFIIF-RAP30, and TFIIH-MAT1 remained tightly associated with the nuclear substructure. Most proteins and run-on activity were retained when approximately 50% of the chromatin was detached with a nuclease, but approximately 45,000 molecules of bound TATA binding protein (TBP) were detached. Similar results were obtained after cross-linking living cells with formaldehyde. The results provide little support for the existence of a large pool of soluble holoenzyme; they are consistent with TBP-promoter complexes in nuclease-sensitive chromatin being assembled into preinitiation complexes attached to the underlying structure.

Ribosomes prepared from somatic tissue of Xenopus laevis inhibit transcription by RNA polymerase III. This observation parallels an earlier report that a high speed fraction from activated egg extract, which is enrichedin ribosomes, inhibits RNA polymerase III activityand destabilizes putative transcription complexes assembled on oocyte 5S rRNA genes. Transcription of somatic- and oocyte-type 5S rRNA genes and a tRNA gene are all repressed in the present experiments. We find that 5S rRNA genes incubated in S150 extract prepared from immature oocytes exhibit an extensive DNase I protection pattern that is nearly identical to that of the ternary complex of TFIIIA and TFIIIC bound to a somatic 5S rRNA gene. The complexes formed in this extract are stable at concentrations of ribosomes that completely repress transcription, indicating that formation of the TFIII(A+C) complex is not the target of inhibition. Ribosomes taken through a high salt treatment no longer repress transcription of class III genes, establishing that the inhibition is due to an associated factor and not the particle itself. The inhibitory activity released from ribosomes is inactivated by treatment with proteinase K, but not micrococcal nuclease. Preincubation of ribosomes with a general protein kinase inhibitor, 6-dimethylaminopurine, eliminates repression of transcription. Western blot analysis demonstrates that p34(cdc2), which is known to mediate repression of transcription by RNA polymerase III, is present in these preparations of ribosomes and can be released from the particles upon extraction with high salt. These results establish that a kinase activity, possibly p34(cdc2), is the actual agent responsible for the observed inhibition of transcription by ribosomes.

The hypermodified DNA base beta-D-glucosylhydroxymethyluracil, also called J, is a naturally occurring DNA modification. J was initially detected by 32P-postlabeling in Trypanosoma brucei and was recently also found in several other eukaryotic parasites. To use 32P-postlabeling as a method to quantitate the absolute levels of J in DNA we have tested the postlabeling efficiency of J using various synthesized standard oligonucleotides containing J. It is known that modified nucleotides, especially bulky ones, are often partially recovered by postlabeling and they are poor substrates for some of the enzymes used. We found that on average only 50% of J is recovered, which shows that the amount of J in T. brucei DNA has been twofold underestimated. Experiments with a short oligomer and defined pyrimidine tracts showed that the incomplete recovery of J is caused at least in part by resistance of J-containing DNA to degradation by micrococcal nuclease.

The fungal toxin gliotoxin induces apoptotic cell death in a variety of cells. Apoptosis induced in thymocytes by gliotoxin is rapid, and DNA fragmentation is observable within 4 h treatment. Apoptosis induced by gliotoxin is calcium-independent and unaffected by protein synthesis inhibitors. We have previously shown that gliotoxin results in phosphorylation of a 16.3-kDa protein within 10 min treatment of thymocytes. Here we show that this protein is histone H3 and phosphorylation occurs on Ser-10. Cyclic AMP levels and activity of protein kinase A (PKA) are raised in cells treated with gliotoxin. Apoptosis is inhibited by genistein which also inhibits PKA and histone H3 phosphorylation. Apoptosis is also inhibited by a number of specific inhibitors of PKA suggesting apoptosis induced by gliotoxin is modulated by this kinase. The agents forskolin and cholera toxin do not induce rapid phosphorylation of H3 although some increase in phosphorylation of H3 does occur after 8 h with these agents. Forskolin and cholera toxin also induce apoptosis but over a longer time course than gliotoxin. In all cases levels of apoptosis correlate with degree of H3 phosphorylation. Cells treated with gliotoxin show an early sensitivity to micrococcal nuclease and DNase I digestion indicating a functional relationship between DNA fragmentation and H3 phosphorylation.

Nuclease susceptibility of the satellite DNA-containing chromatin of the liver of young (18 +/- 2 weeks) and old (100 +/- 5 weeks) rats was analysed using nick-translated rat 185 bp satellite I DNA fragment cloned in pBR322. With increasing concentration of DNaseI and micrococcal nuclease (MNase), multimeric forms of the satellite ladder gradually disappear in both the ages. The rate of disappearance is faster in young rats as compared to old ones. Such age-dependent decrease in the susceptibility of satellite DNA-containing chromatin reflects its condensation towards heterochromatization in old age.

The G -->T transversion mutation, ade6-M26, creates the heptanucleotide sequence ATGACTG, which lies close to the 5' end of the open reading frame of the ade6 gene in Schizosaccharomyces pombe. The mutation generates a meiosis-specific recombination hot spot and a binding site for the Mts1/Mts2 protein. We examined the chromatin structure at the ade6 locus in the M26 strain and compared it to that of the wild-type and hot spot-negative control M375. Micrococcal nuclease (MNase) digestion and indirect end-labeling methods were applied. In the M26 strain, we detected a new MNase-hypersensitive site at the position of the M26 mutation and no longer observed the phasing of nucleosomes seen in the wild-type and the M375 strains. Quantitative comparison of MNase sensitivity of the chromatin in premeiotic and meiotic cultures revealed a small meiotic induction of MNase hypersensitivity in the ade6 promoter region of the wild-type and M375 strains. The meiotic induction of MNase hypersensitivity was enhanced significantly in the ade6 promoter region of the M26 strain and also occurred at the M26 mutation site. The formation of the MNase-sensitive region around the heptamer sequence was abolished by the introduction of single-nucleotide substitutions in the heptamer sequence, which also abolish hot spot activity and binding of Mts1/Mts2. These data suggest that Mts1/Mts2 binding to the heptamer sequence results in a chromatin structure suitable for the recruitment of a meiosis-specific recombination function or functions.

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.

We have previously reported repeat-induced gene silencing (RIGS) in Arabidopsis, in which transgene expression may be silenced epigenetically when repeated sequences are present. Among an allelic series of lines comprising a primary transformant and various recombinant progeny carrying different numbers of drug resistance gene copies at the same locus, silencing was found to depend strictly on repeated sequences and to correlate with an absence of steady-state mRNA. We now report characterization, in nuclei isolated from the same transgenic lines, of gene expression by nuclear run-on assay and of chromatin structure by nuclease protection assay. We find that silencing is correlated with absence of run-on transcripts, indicating that expression is silenced at the level of transcription. We find further that silencing is also correlated with increased resistance to both DNase I and micrococcal nuclease, indicating that the silenced state reflects a change in chromatin configuration. We propose that silencing results when a locally paired region of homologous repeated nucleotide sequences is flanked by unpaired heterologous DNA, which leads chromatin to adopt a local configuration that is difficult to transcribe, and possibly akin to heterochromatin.

The relationship between chromatin structure and endonuclease sensitivity was investigated. The cells used in this study were a) human myelogenous leukemic cell lines (HL-60, ML-I, U-937, THP-I) (Group I), which produced internucleosomal DNA cleavage, and b) human T-cell leukemia (MOLT-4), erythroleukemia (K562), glioblastoma (T98G, U87MG) and glioma (KG-1-C) cell lines (Group II), which produced no internucleosomal DNA cleavage, upon treatment with various apoptosis-inducing agents. When the nuclei, isolated from these cells were digested with micrococcal nuclease, chromatin DNA was cleaved into oligonucleosomal units. Although sensitivity to micrococcal nuclease considerably differed from cell to cell, Group I cells were generally more sensitive to micrococcal nuclease digestion than Group II cells. Similar sensitivity to DNase I was observed in both groups of cells. Acid-urea polyacrylamide gel electrophoresis of histone fractions from control and apoptosing HL-60 cells (induced either by hydrogen peroxide or UV irradiation) revealed no significant change in the relative composition of five major histones, indicating the absence of selective degradation of histone HI, but rather the nonspecific degradation of many nuclear proteins. These data suggest a difference in a chromatin structure between Group I and II cells, which might result in the selective production of internucleosomal DNA cleavage only in Group I cells.

Protein disulfide isomerase (PDI) alkylated at thiols of the thioredoxin-like -CHC- active sites is devoid of isomerase activity, but its chaperone-like activity to increase the reactivation yield and prevent the aggregation of guanidine hydrochloride-denatured D-glyceraldehyde-3-phosphate dehydrogenase upon dilution is unimpaired. A peptide of 28 amino acids markedly inhibits both the enzyme and the chaperone activities of PDI. The above results indicate that the -CGHC- active site is necessary for the isomerase activity but not required for the chaperone activity of PDI, whereas the peptide binding site is essential for both activities.

The chromatin structure of the dihydrofolate reductase (DHFR) gene was examined by DNA/protein cross-linking, chemical DNA methylation, and micrococcal nuclease digestion. The 5' promoter region of the gene displays two nucleosome-free zones (-550 to -300 and -150 to +100 bp relative to the ATG codon), each of which contains a number of micrococcal nuclease-hypersensitive sites. Regions upstream from the distal hypersensitive zone (-900 to -550 bp), downstream from the proximal hypersensitive zone (+100 to +400 bp), and between these two zones (-300 to -150 bp) appear either to be more than 80% histone-free or to contain histones whose globular domains have lost most of their contacts with DNA. Overall, a broad zone extending from -4300 to +4700 bp is altered relative to bulk chromatin, and within this region there are positioned nucleosomes and/or nucleosome-free zones in which the DNA appears to interact with a number of different non-histone proteins. By comparison, the chromatin in the 3' end of the gene (including the right end of the 5th intron and the 6th exon) contains randomly positioned nucleosomes, and its structure is intermediate between that of the 5' end of the gene and a downstream matrix attachment region that contains regularly organized chromatin. A 2.3 kb zone in the central part of the 5th intron reveals some features similar to the 5' end of the gene, suggesting a hitherto unrecognized functional role.

Capsid-targeted viral inactivation is an antiviral strategy in which toxic fusion proteins are targeted to virions, where they inhibit viral multiplication by destroying viral components. These fusion proteins consist of a virion structural protein moiety and an enzymatic moiety such as a nuclease. Such fusion proteins can severely inhibit transposition of yeast retrotransposon Ty1, an element whose transposition mechanistically resembles retroviral multiplication. We demonstrate that expression of a murine retrovirus capsid-staphylococcal nuclease fusion protein inhibits multiplication of the corresponding murine leukemia virus by 30- to 100-fold. Staphylococcal nuclease is apparently inactive intracellularly and hence nontoxic to the host cell, but it is active extracellularly because of its requirement for high concentrations of Ca2+ ions. Virions assembled in and shed from cells expressing the fusion protein contain very small amounts of intact viral RNA, as would be predicted for nuclease-mediated inhibition of viral multiplication.

The involvement of ribosomal RNA in the binding of eukaryotic elongation factor eEF-2 to the ribosome was investigated. eEF-2 was complexed to empty reassociated 80S ribosomes in the presence of the nonhydrolyzable GTP analogue GuoPP[CH2]P. The formed complex was treated with dimethyl sulfate, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate, and micrococcus nuclease to allow specific modification at single-stranded regions of the rRNAs. The sites of modification were localized by primer extension using complementary deoxynucleotide primers and reverse transcriptase. The modification pattern was compared to that obtained from 80S ribosomes lacking bound eEF-2. Binding of the factor to the ribosome resulted in the protection of specific sites in both 18S and 28S rRNA, while the reactivity of 5.8S rRNA was unchanged. In 18S rRNA, the affected nucleotides were localized to the 5'- and 3'-domains, and in 28S rRNA the protected nucleotides were seen in domains II, IV, and V. The alpha-sarcin/ricin loop in domain VI of 28S rRNA was inaccessible for chemical modification even in the absence of bound eEF-2. However, the bound factor protected A4256, located in the alpha-sarcin/ricin loop, from ricin-induced depurination.

PCR amplification of the sec+ gene for staphylococcal enterotoxin C1 (SEC1) can be achieved from as little as 10 fg total genomic DNA (equivalent to less than 10 cells) using two nested primer pairs. The presence of bacterial cells, particularly thermonuclease-producing staphylococci, and the thermonuclease enzyme (TNase) itself, were found to be factors which individually and together reduced the sensitivity of PCR amplification.

To determine the replicative mechanism for human cytomegalovirus (HCMV) DNA, field inversion gel electrophoresis was used to separate HCMV replicative DNAs during lytic infection. Unit-length circular HCMV genomes lacking terminal restriction fragments were detected starting 4 h after infection even when cells were treated with aphidicolin, phosphonoacetic acid, or cycloheximide. Viral DNA synthesis began 24 h after infection and produced large amounts of high-molecular-weight replicative DNA that was a precursor of progeny genomes. Replicative DNA contained rare terminal restriction fragments, and long-arm termini were much less frequent than short-arm termini. Replicative DNA was not composed of unit-length circles because low-dose gamma irradiation of replicative DNA generated numerous random high-molecular-weight fragments rather than unit-length molecules. PacI digestion of replicative DNA from a recombinant HCMV with two closely spaced PacI sites revealed that replicative DNA is concatemeric and genome segment inversion occurs after concatemer synthesis. These results show that after circularization of the parental genome, DNA synthesis produces concatemers and genomic inversion occurs within concatemeric DNA. The results further suggest that concatemers acquire genomic termini during the cleavage/packaging process which preferentially inserts short-arm termini into empty capsids, causing a predominance of short-arm termini on the concatemer.

Chromatin organization of eukaryotic promoters is increasingly recognized as an important factor in the regulation of transcription in vivo. To determine the role of chromatin in HIV-1 expression, we have examined the nucleosome organization of the promoter of HIV-1 under low and high transcription rates. Independently of the cell line examined, nucleosomes are precisely positioned in the viral 5' long terminal repeat (5' LTR) and define two large nucleosome-free regions encompassing nt 200-450 and 610-720. A nucleosome positioned between these two regions, immediately after the transcription initiation site (nuc-1), is disrupted following TPA or TNF-alpha treatment. The disruption of nuc-1 from DNA is independent of DNA replication since it is completed in 20 min and independent of transcription as it is alpha-amanitin insensitive. A model is proposed in which nuc-1 plays an organizing role in the HIV-1 promoter to bring in close proximity factors bound to DNA in the two nucleosome-free regions, upstream and downstream of the site of transcription initiation. These results define chromatin as an integral component of the HIV-1 transcriptional regulatory machinery and identify a chromatin transition associated with activation of viral gene expression.

We show that the mechanism by which chromatin displaying higher-order structure is usually isolated from nuclei involves a transition to an extended nucleosomal arrangement. After being released from nuclei, chromatin must refold in order to produce the typical chromatin fibers observed in solution. For starfish sperm chromatin with a long nucleosome repeat (222 bp), isolated fibers are significantly wider than those in the nucleus, indicating that the refolding process does not regenerate the native higher-order structure. We also propose that for typical eukaryotic nuclei, the concept that the native state of the (inactive) bulk of the genome is a chromatin fiber with defined architecture be reconsidered.

In mouse hepatoma cells, both the regulatory and the transcribed regions of the cyp1a1 gene assume a nucleosomal configuration when the gene is silent; two nucleosomes occupy specific sites at the transcriptional promoter. Activation of transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin is accompanied by changes in chromatin structure, which depend upon a functional aromatic hydrocarbon (Ah) receptor. In the transcribed region of the gene, nucleosome disruption occurs as a consequence of RNA elongation. In contrast, at the promoter, loss of positioned nucleosome sis independent of transcription and represents an event in the mechanism by which the liganded Ah receptor enhances transcriptional initiation.

An oligonucleosome 12-mer was reconstituted in the absence of linker histones, onto a DNA template consisting of 12 tandemly arranged 208-base pair fragments of the 5 S rRNA gene from the sea urchin Ly-techinus variegatus (Simpson, R. T., Thoma, F. S., and Burbaker, J. M. (1985) Cell 42, 799-808). The ionic strength-dependent folding of this nucleohistone complex was compared with that of a native oligonucleosome fraction obtained from chicken erythrocyte chromatin, which had been carefully stripped of linker histones and fractionated in sucrose gradients. The DNA of this native fraction exhibited a narrow size distribution centered around the length of the 208-12 DNA template used in the reconstituted complex. These two complexes displayed very similar hydrodynamic behavior as judged by sedimentation velocity analysis. By combining these data with electron microscopy analysis, it was shown that the salt-dependent folding of oligonucleosomes in the absence of linker histones involves the bending of the linker DNA region connecting adjacent nucleosomes. It was also found that selective removal by trypsin of the N-terminal regions ("tails") of the core histones prevents the oligonucleosome chains from folding. Thus, in the absence of these histone domains, the bending of the linker DNA necessary to bring the nucleosomes in contact is completely abolished. In addition to the complete lack of folding, removal of the histone tails results in an unwinding at low salt of a 20-base pair region at each flanking side of the nucleosome core particle. The possible functional relevance of these results is discussed.

Single-stranded DNA binding proteins (SSBs) have been isolated from many organisms, including Escherichia coli, Saccharomyces cerevisiae and humans. Characterization of these proteins suggests they are required for DNA replication and are active in homologous recombination. As an initial step towards understanding the role of the eukaryotic SSBs in DNA replication and recombination, we examined the DNA binding and strand exchange stimulation properties of the S. cerevisiae single-strand binding protein y-RPA (yeast replication protein A). y-RPA was found to bind to single-stranded DNA (ssDNA) as a 115,000 M(r) heterotrimer containing 70,000, 36,000 and 14,000 M(r) subunits. It saturated ssDNA at a stoichiometry of one heterotrimer per 90 to 100 nucleotides and binding occurred with high affinity (K omega greater than 10(9) M-1) and co-operativity (omega = 10,000 to 100,000). Electron microscopic analysis revealed that y-RPA binding was highly co-operative and that the ssDNA present in y-RPA-ssDNA complexes was compacted fourfold, arranged into nucleosome-like structures, and was free of secondary structure. y-RPA was also tested for its ability to stimulate the yeast Sepl and E. coli RecA strand-exchange proteins. In an assay that measures the pairing of circular ssDNA with homologous linear duplex DNA, y-RPA stimulated the strand-exchange activity of Sepl approximately threefold and the activity of RecA protein to the same extent as did E. coli SSB. Maximal stimulation of Sepl occurred at a stoichiometry of one y-RPA heterotrimer per 95 nucleotides of ssDNA. y-RPA stimulated RecA and Sepl mediated strand exchange reactions in a manner similar to that observed for the stimulation of RecA by E. coli SSB; in both of these reactions, y-RPA inhibited the aggregation of ssDNA and promoted the co-aggregation of single-stranded and double-stranded linear DNA. These results demonstrate that the E. coli and yeast SSBs display similar DNA-binding properties and support a model in which y-RPA functions as an E. coli SSB-like protein in yeast.

We have used new methods for chromatin isolation, together with conventional methods for measuring the nucleosome repeat length, to determine the repeat length of Schizosaccharomyces pombe chromatin. We obtain a result of 156(+/- 2) bp. Equivalent results are obtained using a psoralen crosslinking method for measuring the repeat length in viable spheroplasts. That result, together with other control experiments, rules out many possible artifacts. The measured value of 156(+/- 2) bp is smaller than the length of DNA found in the chromatosome. Thus, the chromatosome cannot be the fundamental unit of chromatin structure in all eukaryotes. The crossed linker model of chromatin higher order structure is incompatible with a nucleosome repeat length of 156 bp, and thus cannot apply to all eukaryotes. The solenoid model of higher order structure is compatible with this repeat length only if the solenoid is right-handed. We note two other properties of this chromatin. (1) Early in digestion, the DNA length of mononucleosomes from S. pombe and Aspergillus nidulans exceeds the nucleosome repeat length. (2) Many methods for isolating chromatin from S. pombe yield an apparent nucleosome repeat length of less than or equal to 140 bp; this result is found to be an artifactual consequence of nucleosome sliding.

An intermediate form of Abutilon mosaic geminivirus, a complex of viral double-stranded DNA (dsDNA) and protein, was characterized by two different techniques. Cesium sulphate gradient centrifugation was used to show that the majority of this form comigrates with host chromatin. Micrococcus nuclease digestion experiments with isolated nuclei demonstrated that the viral dsDNA is organized in a manner comparable to that of host nucleosomes. Results from a previous electron microscopical work (Abouzid, A. M., Frischmuth, T., and H. Jeske, 1988, Mol. Gen. Genet. 212, 252-258) suggested to us that a part of the viral dsDNA must be free of nucleosomes. Whether this nucleosome-free space corresponds to the intergenic region which contains most of the promotor sequences and the putative origin of replication is discussed.

Nucleosome formation on inverted repeats or on some alternations of purines and pyrimidines can be inhibited in vitro by DNA supercoiling through their supercoiling-induced structural transitions to cruciforms or Z-form DNA, respectively. We report here, as a result of study of single nucleosome reconstitutions on a DNA minicircle, that a physiological level of DNA supercoiling can also enhance nucleosome sequence preference. The 357 base-pair minicircle was composed of a promoter of phage SP6 RNA polymerase joined to a 256 base-pair fragment containing a sea urchin 5 S RNA gene. Nucleosome formation on the promoter was found to be enhanced on a topoisomer with in vivo superhelix density when compared to topoisomers of lower or higher superhelical densities, to the nicked circle, or to the linear DNA. In contrast, nucleosomes at other positions appeared to be insensitive to supercoiling. This observation relied on a novel procedure for the investigation of nucleosome positioning. The reconstituted circular chromatin was first linearized using a restriction endonuclease, and the linear chromatin so obtained was electrophoresed as nucleoprotein in a polyacrylamide gel. The gel showed well-fractionated bands whose mobilities were a V-like function of nucleosome positions, with the nucleosome near the middle migrating less. This behavior is similar to that previously observed for complexes of sequence-specific DNA-bending proteins with circularly permuted DNA fragments, and presumably reflects the change in the direction of the DNA axis between the entrance and the exit of the particle. Possible mechanisms for such supercoiling-induced modulation of nucleosome formation are discussed in the light of the supercoiling-dependent susceptibility to cleavage of the naked minicircle with S1 and Bal31 nucleases; and a comparison between DNase I cleavage patterns of the modulated nucleosome and of another, non-modulated, overlapping nucleosome.

To determine whether diabetes alters chromatin structure in vivo, micrococcal nuclease digestion kinetics were analyzed in cerebral cortical and hepatic nuclei of streptozotocin-induced diabetic rats. Cerebral nuclei of diabetic rats maintained for 6 weeks were less susceptible to micrococcal nuclease digestion compared with control rats. Insulin treatment reversed diabetes-related changes in nuclease digestion kinetics. There were no changes in the kinetics of digestion in hepatic nuclei. The reduced digestibility of cerebral DNA in diabetes could not be attributed to altered DNA fluorescence spectra, or altered distribution of most abundant chromatin proteins that were either solubilized or that remained insoluble immediately following nuclease digestion. It is concluded that chronic, uncontrolled hyperglycemia can alter chromatin structure of some tissues in vivo, and this change is probably related to subtle alterations in DNA-protein interactions.

Simian virus 40 chromosomes can be replicated in vitro with the same set of purified proteins required for the replication of naked DNA containing the viral origin. With these reconstituted systems, the fate of parental histones during replication was examined in vitro. The assembly of nucleosomes on replicating chromosomes was hardly affected by the presence of simultaneously replicating naked DNA competitor, suggesting that replication forks can traverse nucleosomes without the displacement of histones. Moreover, we demonstrate that the nascent nucleosomes were distributed almost equally between the leading and lagging strands. This distributive mode of nucleosome segregation favors the propagation of parental chromatin structures to both daughter cells, which can maintain cellular functions dictated by these structures during cell proliferation.