Endogenous nuclease. Properties and effects on transcribed genes in chromatin

Nuclei from a variety of tissues displayed wide differences in the rate of cleavage of chromatin DNA by the endogenous nuclease(s). However, at the nucleosomal level of chromatin organization, both the linker DNA and the nucleosome core were cleaved during incubation of nuclei from all tissues examined, as well as in rat thymocytes following the injection of glucocorticoid. The transcribed ovalbumin gene in hen oviduct nuclei was cleaved selectively by the endogenous nuclease as compared to the bulk of the DNA and inactive globin genes as revealed by both solution and Southern blot hybridization analysis. Preferential digestion of the ovalbumin sequence was clearly apparent after 20 min of nuclear incubation. In addition, in nuclei from immature chick oviducts, the nuclease sensitivity of the ovalbumin gene paralleled the differential expression of this gene during primary estrogen stimulation, estrogen withdrawal, and secondary hormone stimulation, thus implying that transcribed oviduct chromatin is most accessible to this nuclease. Since the rapid autodigestion of DNA in isolated nuclei does not normally occur in intact cells, some type of nuclease activation process or change in the chromatin substrate must occur during nuclear preparation or incubation. The autodigestion process in nuclei was inhibited by spermine4+, spermidine3+,Ca2+, Mg2+, Na+, and K+ and the degree of this inhibition was proportional to the effectiveness of these cations in promoting the precipitation of nuclease-sheared chromatin. Incubation of nuclei in buffers containing cationic compositions that are thought to be similar to the in vivo state resulted in essentially complete inhibition of nuclease activity and maximal precipitation of chromatin. The rate of the selective fragmentation of the ovalbumin gene, like the bulk of the nuclear DNA, was reduced upon increasing the cationic concentration of the nuclear incubation medium. These results suggest that the activation of chromatin autodigestion during nuclear incubation results from a reduction in the cationic composition of nucleoplasm and, once activated, the endogenous nuclease selectively attacks transcribed genes in chromatin

Fractionation and characterization of chromosomal proteins by the hydroxyapatite dissociation method

A method was developed which enables the characterization and fractionation of chromosomal proteins according to their chromatin binding properties. The method is based on the ability of hydroxyapatite to bind native chromatin in solutions which do not dissociate chromosomal proteins from the DNA. The proteins are then selectively dissociated from the immobilized chromatin by treatment with NaCl, urea, or guanidine HCl. The hydroxyapatite dissociation method represents a rapid one-step fractionation procedure which results in the quantitative recovery of chromosomal proteins devoid of nucleic acids and is suitable for large preparations. The hydroxyapatite dissociation method provides a versatile procedure for the study and preparation of chromosomal proteins. The patterns of dissociation of both histones and nonhistone chromosomal proteins by NaCl and urea from chicken oviduct chromatin were characterized by this method. In addition, this technique enabled the purification of the major histone species in a single operation. Partial purification of specific nonhistone proteins, including the estrogen receptor, was also achieved. We suggest that this method will be a useful tool in elucidation of the chemical and biological properties of the proteins from chromatin

Conformation of ovalbumin and globin genes in chromatin during differential gene expression

Micrococcal nuclease and DNase I were used to study changes in the chromatin conformation of ovalbumin and globin genes during differential expression of these sequences. Oviduct nuclei, obtained from estrogen-treated chicks or chicks withdrawn from the hormone for 3, 4, or 6 days, were incubated with micrococcal nuclease until 1 to 3% of the DNA was rendered acid-soluble. The resulting DNA fragments then were separated into four size classes. In the stimulated oviducts, the concentration of the ovalbumin gene in mononucleosome-length DNA (165 to 200 base pairs) was 6-fold greater than in the fraction containing DNA fragments >1300 base pairs in length. This selective cleavage decreased progressively as a function of estrogen withdrawal time and correlated temporally with a decline in the concentration of oviduct nuclear estrogen receptors. The expressed globin genes in immature erythrocyte nuclei were also cleaved preferentially by micrococcal nuclease, whereas the transcriptionally silent globin sequences in mature erythrocyte nuclei were not. The globin genes in both immature and mature erythrocyte nuclei were destroyed by DNase I 3 times faster than the greater part of the nuclear DNA. To determine if the globin genes in the mature erythrocyte were cleaved preferentially by DNase I, nuclei were incubated with this enzyme until 1 to 3% of the DNA was rendered acid-soluble. The resulting DNA fragments then were separated into four size classes. The concentration of globin genes in the size class containing the smallest DNA fragments (less than 300 base pairs) from both immature and mature erythrocyte nuclei was about 6-fold greater than in undigested DNA, and about 15-fold greater than in the fraction containing DNA fragments >1200 base pairs in length. These results suggest that the micrococcal nuclease sensitive conformation of the ovalbumin and globin genes in chromatin is dynamically related to the expression of these sequences. The DNase I sensitive structure as determined by both nucleolytic destruction and cleavage, in contrast, remains associated with globin sequences following their inactivation

Hormonal regulation of the conformation of the ovalbumin gene in chick oviduct chromatin

We have examined the effects of steroid hormones on the chromatin sensitivity of the ovalbumin gene to micrococcal nuclease and have attempted to define the importance of the nucleosome core, higher order chromatin folding, and transcription in the maintenance of the nuclease-sensitive conformation of the ovalbumin chromatin. Solution hybridization studies demonstrated that the sensitivity of the ovalbumin gene in oviduct nuclei to micrococcal nuclease paralleled the hormone-dependent transcription of the ovalbumin gene in the immature chick. Blot hybridization analysis also revealed a hormone-dependent change in this chromatin region since ovalbumin DNA fragments from nuclease-treated hen and estrogen-stimulated chick oviduct nuclei exhibited nucleosomal repeat patterns that were less discrete than those observed for the ovalbumin specific fragments from liver and hormone-withdrawn oviducts. This transcription-related conformation was not the result of the enhanced sensitivity of the ovalbumin-containing nucleosomal cores since the bulk of the nucleosomes associated with the ovalbumin chromatin were not preferentially cleaved internally by micrococcal nuclease. Rather, an analysis of the fragmentation of the ovalbumin chromatin as a function of digestion extent suggested a mechanism in which the heightened sensitivity resulted from the collective expansion of the nuclease cutting sites in the linker regions of the ovalbumin chromatin because the gene was in an unfolded conformation. The transcription-specific conformation was not merely a consequence of RNA synthesis per se since the selective sensitivity of the gene was unaffected by treatment of oviduct nuclei with a-amanitin, actinomycin D, or RNase. In addition, the presence of the transcriptional complex on the ovalbumin chromatin was presumably not required for selective nuclease recognition since preferential cleavage was observed under conditions expected to deplete oviduct nuclei of template-bound RNA polymerase and nascent RNA chains. These results are consistent with a model in which the expressed ovalbumin gene is in an unfolded polynucleosomal structure whose formation is related to transcriptional activity but not dependent on the transcriptional process

A Full PFPF Shell Model Study of a~=~48 Nuclei

Exact diagonalizations with a minimally modified realistic force lead to detailed agreement with measured level schemes and electromagnetic transitions in $^{48}$Ca, $^{48}$Sc, $^{48}$Ti, $^{48}$V, $^{48}$Cr and $^{48}$Mn. Gamow-Teller strength functions are systematically calculated and reproduce the data to within the standard quenching factor. Their fine structure indicates that fragmentation makes much strength unobservable. As a by-product, the calculations suggest a microscopic description of the onset of rotational motion. The spectroscopic quality of the results provides strong arguments in favour of the general validity of monopole corrected realistic forces, which is discussed.Comment: 30 pages, LaTeX with epsf.sty, 14 Postscript figures included and compressed using uufiles. Completely new version of previous preprint nucl-th/9307001. FTUAM-93/01, CRN/PT 93-3

Gamma-Ray Bursts: The Underlying Model

A pedagogical derivation is presented of the ``fireball'' model of gamma-ray bursts, according to which the observable effects are due to the dissipation of the kinetic energy of a relativistically expanding wind, a ``fireball.'' The main open questions are emphasized, and key afterglow observations, that provide support for this model, are briefly discussed. The relativistic outflow is, most likely, driven by the accretion of a fraction of a solar mass onto a newly born (few) solar mass black hole. The observed radiation is produced once the plasma has expanded to a scale much larger than that of the underlying ``engine,'' and is therefore largely independent of the details of the progenitor, whose gravitational collapse leads to fireball formation. Several progenitor scenarios, and the prospects for discrimination among them using future observations, are discussed. The production in gamma- ray burst fireballs of high energy protons and neutrinos, and the implications of burst neutrino detection by kilometer-scale telescopes under construction, are briefly discussed.Comment: In "Supernovae and Gamma Ray Bursters", ed. K. W. Weiler, Lecture Notes in Physics, Springer-Verlag (in press); 26 pages, 2 figure

Relativistic Hydrodynamic Evolutions with Black Hole Excision

We present a numerical code designed to study astrophysical phenomena involving dynamical spacetimes containing black holes in the presence of relativistic hydrodynamic matter. We present evolutions of the collapse of a fluid star from the onset of collapse to the settling of the resulting black hole to a final stationary state. In order to evolve stably after the black hole forms, we excise a region inside the hole before a singularity is encountered. This excision region is introduced after the appearance of an apparent horizon, but while a significant amount of matter remains outside the hole. We test our code by evolving accurately a vacuum Schwarzschild black hole, a relativistic Bondi accretion flow onto a black hole, Oppenheimer-Snyder dust collapse, and the collapse of nonrotating and rotating stars. These systems are tracked reliably for hundreds of M following excision, where M is the mass of the black hole. We perform these tests both in axisymmetry and in full 3+1 dimensions. We then apply our code to study the effect of the stellar spin parameter J/M^2 on the final outcome of gravitational collapse of rapidly rotating n = 1 polytropes. We find that a black hole forms only if J/M^2<1, in agreement with previous simulations. When J/M^2>1, the collapsing star forms a torus which fragments into nonaxisymmetric clumps, capable of generating appreciable ``splash'' gravitational radiation.Comment: 17 pages, 14 figures, submitted to PR

The Theory/Applications Balance in Management Pedagogy: Where Do We Stand?

Business schools are expected to be "professional" in the sense that their mission is primarily to prepare people to practice their skills in the business world. Various critics, however, claim that management professors overemphasize theory and research and neglect the practice and applications students need to transfer classroom theory to the world of practice. This study compared an earlier sample with a more recent sample of Academy of Management members concerning the relative emphasis they believed should be placed on theory and applications in management pedagogy and the techniques they used to bring applications into the classroom. Current respondents believed that more emphasis should be placed on applications than the earlier respondents. An unexpected finding, however, was that the more recent respondents reported a lower mean usage of pedagogical techniques that are appropriate for developing students' ability to apply course concepts than the previous group. Possible reasons for these incongruent findings are discussed as well as the implications for management pedagogy.Yeshttps://us.sagepub.com/en-us/nam/manuscript-submission-guideline

Search for the standard model Higgs boson decaying into two photons in pp collisions at sqrt(s)=7 TeV

A search for a Higgs boson decaying into two photons is described. The analysis is performed using a dataset recorded by the CMS experiment at the LHC from pp collisions at a centre-of-mass energy of 7 TeV, which corresponds to an integrated luminosity of 4.8 inverse femtobarns. Limits are set on the cross section of the standard model Higgs boson decaying to two photons. The expected exclusion limit at 95% confidence level is between 1.4 and 2.4 times the standard model cross section in the mass range between 110 and 150 GeV. The analysis of the data excludes, at 95% confidence level, the standard model Higgs boson decaying into two photons in the mass range 128 to 132 GeV. The largest excess of events above the expected standard model background is observed for a Higgs boson mass hypothesis of 124 GeV with a local significance of 3.1 sigma. The global significance of observing an excess with a local significance greater than 3.1 sigma anywhere in the search range 110-150 GeV is estimated to be 1.8 sigma. More data are required to ascertain the origin of this excess.Comment: Submitted to Physics Letters

Search for a W' boson decaying to a bottom quark and a top quark in pp collisions at sqrt(s) = 7 TeV

Results are presented from a search for a W' boson using a dataset corresponding to 5.0 inverse femtobarns of integrated luminosity collected during 2011 by the CMS experiment at the LHC in pp collisions at sqrt(s)=7 TeV. The W' boson is modeled as a heavy W boson, but different scenarios for the couplings to fermions are considered, involving both left-handed and right-handed chiral projections of the fermions, as well as an arbitrary mixture of the two. The search is performed in the decay channel W' to t b, leading to a final state signature with a single lepton (e, mu), missing transverse energy, and jets, at least one of which is tagged as a b-jet. A W' boson that couples to fermions with the same coupling constant as the W, but to the right-handed rather than left-handed chiral projections, is excluded for masses below 1.85 TeV at the 95% confidence level. For the first time using LHC data, constraints on the W' gauge coupling for a set of left- and right-handed coupling combinations have been placed. These results represent a significant improvement over previously published limits.Comment: Submitted to Physics Letters B. Replaced with version publishe