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

Embedding Phenomenological Quark-Lepton Mass Matrices into SU(5) Gauge Models

We construct phenomenological quark-lepton mass matrices based on S$_3$ permutation symmetry in a manner fully compatible with SU(5) grand unification. The Higgs particles we need are {\bf 5}, {\bf 45} and their conjugates. The model gives a charge $-$1/3 quark vs charged lepton mass relation, and also a good fit to mass-mixing relations for the quark sector, as well as an attractive mixing pattern for the lepton sector, explaining a large mixing angle between $\nu_\mu$ and $\nu_\tau$, and either large or small $\nu_e-\nu_\mu$ mixing angle, depending on the choice of couplings, consistent with the currently accepted solutions to the solar neutrino problem.Comment: 12 pages, LaTex file, no figure

Reactor Neutrino Experiments with a Large Liquid Scintillator Detector

We discuss several new ideas for reactor neutrino oscillation experiments with a Large Liquid Scintillator Detector. We consider two different scenarios for a measurement of the small mixing angle $\theta_{13}$ with a mobile $\bar{\nu}_e$ source: a nuclear-powered ship, such as a submarine or an icebreaker, and a land-based scenario with a mobile reactor. The former setup can achieve a sensitivity to $\sin^2 2\theta_{13} \lesssim 0.003$ at the 90% confidence level, while the latter performs only slightly better than Double Chooz. Furthermore, we study the precision that can be achieved for the solar parameters, $\sin^2 2\theta_{12}$ and $\Delta m_{21}^2$, with a mobile reactor and with a conventional power station. With the mobile reactor, a precision slightly better than from current global fit data is possible, while with a power reactor, the accuracy can be reduced to less than 1%. Such a precision is crucial for testing theoretical models, e.g. quark-lepton complementarity.Comment: 18 pages, 3 figures, 2 tables, revised version, to appear in JHEP, Fig. 1 extended, Formula added, minor changes, results unchange

The Isospin Makeup of the Giant Resonances from (p,n) Reaction Studies at Intermediate Energies

This work was supported by National Science Foundation Grant PHY 75-00289 and Indiana Universit

Universal behavior of localization of residue fluctuations in globular proteins

Localization properties of residue fluctuations in globular proteins are studied theoretically by using the Gaussian network model. Participation ratio for each residue fluctuation mode is calculated. It is found that the relationship between participation ratio and frequency is similar for all globular proteins, indicating a universal behavior in spite of their different size, shape, and architecture.Comment: 4 pages, 3 figures. To appear in Phys. Rev.

Strong Spin-Flip Transitions in (p,n) Reactions

This work was supported by National Science Foundation Grant PHY 76-84033 and Indiana Universit