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Nuclear envelope-limited chromatin sheets (ELCS) form during excessive interphase nuclear envelope growth in a variety of cells. ELCS appear as extended sheets within the cytoplasm connecting distant nuclear lobes. Cross-section stained images of ELCS, viewed by transmission electron microscopy, resemble a sandwich of apposed nuclear envelopes separated by ∼30 nm, containing a layer of parallel chromatin fibers. In this study, the ultrastructure of ELCS was compared by three different methods: (1) aldehyde fixation/dehydration/plastic embedding/sectioning and staining, (2) high-pressure freezing/freeze substitution into plastic/sectioning and staining, and (3) high-pressure freezing/cryo-sectioning/cryo-electron microscopy. ELCS could be clearly visualized by all three methods and, consequently, must exist in vivo and are not fixation artifacts. The ∼30-nm chromatin fibers could only be observed following aldehyde fixation; none were seen in cryo-sections. Electron microscopic tomography tangential views of aldehyde-fixed ELCS suggested an ordering of the separate chromatin fibers adjacent to the nuclear envelope. Possible mechanisms of this chromatin ordering are discussed.

The depressor activity of a novel nonpeptidic angiotensin II (AII) receptor antagonist, SC-51316 (2,5-dibutyl-2,4-dihydro-4-[[2-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4 '- yl]-methyl]-3H-1,2,4-triazol-3-one), is described. In anesthetized, ganglion-blocked rats, intravenous administration of SC-51316 inhibited the pressor response to an infusion of AII. To determine antihypertensive efficacy, conscious, spontaneously hypertensive rats were administered SC-51316 (30 mg/kg intragastrically) daily for 5 days. Blood pressure was reduced in a similar manner to that observed with the angiotensin converting enzyme inhibitor enalapril (10 mg/kg intragastrically). SC-51316 had no effect on heart rate. In conscious, sodium-deficient dogs, administration of SC-51316 (30 mg/kg orally) or enalapril (10 mg/kg orally) lowered blood pressure similarly over a 24 h observation period. Thus, SC-51316 antagonizes the activity of AII in vivo and is an orally active, antihypertensive agent.

We examined the interaction of a non-guanylate cyclase-linked atriopeptin (AP) binding site ligand, SC-46542 (des[Phe106,Gly107,Ala115,Gln116]AP-(103-126], and an endopeptidase 24.11 inhibitor, thiorphan, on mean arterial pressure, urinary sodium excretion, urinary cyclic guanosine monophosphate (cGMP) excretion, plasma cGMP concentration, and plasma AP immunoreactivity (ir) in conscious spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Compared to vehicle control rats, coadministration of SC-46542 and thiorphan increased urinary sodium excretion in SHR from 2.1 +/- 0.3 to 11.6 +/- 0.7 microEq/min/100 g body weight and in WKY from 1.6 +/- 0.4 to 4.4 +/- 0.4 microEq/min/100 g body weight, and increased urinary cGMP excretion in SHR from 2.7 +/- 0.5 to 79.0 +/- 17.5 pmol/min/100 g body weight and in WKY from 7.0 +/- 3.0 to 72.4 +/- 10.6 pmol/min/100 g body weight. The change in urinary sodium excretion was greater in SHR than WKY. The coadministration of SC-46542 and thiorphan had greater effects on urinary sodium excretion and urinary cGMP excretion than administration of either compound alone. Coadministration of thiorphan and SC-46542 had no effect on glomerular filtration rate or plasma cGMP concentration, suggesting that the urinary cGMP excretion response was nephrogenous. Compared to vehicle control rats, plasma APir was increased during coadministration of SC-46542 and thiorphan in both SHR (998 +/- 76 v 5.10 +/- 116 pg/mL) and WKY (775 +/- 36 v 414 +/- 36 pg/mL).(ABSTRACT TRUNCATED AT 250 WORDS)

Euplotes eurystomus is a hypotrichous ciliate containing a transcriptionally active macronucleus (MAC) and a transcriptionally inactive micronucleus. Soluble MAC chromatin contains a normal complement of inner histones, an H1-like protein which is very sensitive to proteolysis, and a considerable proportion of non-histone proteins. A combination of N-Tosyl lysine chloromethyl ketone (TLCK) and PMSF was found to be most effective in preventing proteolysis. Microccocal nuclease digestion yielded an average nucleosome repeat length of 187 +/- 25 bp for soluble chromatin; and 190 +/- 9 bp for isolated macronuclei. Thermal denaturation profiles of MAC chromatin in 0.25 mM EDTA display two main transitions at about 76 and 83 degrees C, resembling the melting of soluble chicken erythrocyte chromatin. Circular dichroic spectra of MAC chromatin were compared to soluble chicken erythrocyte chromatin under the same ionic strength conditions and were found to be very similar. 2D chromatin/DNA agarose gel electrophoresis resulted in a diagonal line of DNA staining, which establishes a strict correlation between DNA and chromatin electrophoretic mobility.

Euplotes eurystomus is a hypotrichous ciliated protozoan possessing within one cytoplasm a transcriptionally-inactive micronucleus with chromosomal-size DNA and a transcriptionally active macronucleus with "gene-size" DNA fragments. The chromatin in the macronucleus can be isolated in a soluble form without prior treatment by nucleases. In this study, macronuclear, soluble chromatin was subfractionated using isokinetic sucrose density gradient ultracentrifugation in a buffer consisting of 50 mM NaCl, 1 mM Na2 EDTA, 1 mM TEA HCl, pH 7.0, 0.1 mM TLCK and 0.1 mM PMSF. Fractions were collected and analyzed by DNA and protein gel electrophoresis, dot blot hybridization with specific gene probes, and modified Miller chromatin spreads. Analysis of the chromatin spreads showed that the sizes of the chromatin fragments in the various fractions correlate with the DNA size of the fragments. When dot blots of the fractions were hybridized with 5S rRNA, tubulin and rRNA gene probes we obtained about a 6 to 14-fold enrichment of hybridizable sequences in individual fractions. There appear to be differences in the non-histones present on each fraction as well as some overall similarities in histone and non-histone proteins.

Chicken-erythrocyte inner histone tetramer has been complexed with several natural and synthetic DNA duplexes by salt-gradient dialysis at various protein/DNA ratios. The resulting complexes, in low-ionic-strength buffer, have been examined by electron microscopy, circular dichroism, and thermal denaturation. Electron microscopy reveals nucleosomes (nu bodies) randomly arranged along DNA fibers, including poly(dA-dT)-poly(dA-dT), poly(dI-dC)-poly(dI-dC), but not poly(dA)-poly(dT). Circular dichroism studies showed prominent histone alpha-helix and "suppression" of nucleic acid ellipticity (lambda less than 240 nm). Thermal denaturation experiments revealed Tm behavior comparable to that of H1- (or H5-) depleted chromatin. Tm III and Tm IV increased linearly with G + C%(natural DNAs), but were virtually independent of the histone/DNA ratio; therefore, the melting of nucleosomes along a DNA chain is insensitive to adjacent "spacer" DNA lengths. This suggests that Tm III and Tm IV arise from the melting of different domains of DNA associated with the core nu body.

Hydrodynamic, spectroscopic, and chemical crosslinking studies on monomer chromatin subnits are reported as a function of ionic strength, pH, and temperature. In earlier studies, two salt-dependent conformational transitions were described (Gordon et al., Proceedings of the National Academy of Science, 75, 660, 1978). Transition one occurred between 0.7 and 2.0 mM ionic strength and transition two occurred between 5.0 and 11.0 mM ionic strength. Crosslinking at 11 mM ionic strength with formaldehyde suppressed both transitions. In this communication we report that the second transition was characterized by changes in the circular dichroism spectra in the 260--320 nm region as well as by changes in the hydrodynamic properties. As the ionic strength was increased from 5.0 to 11.0 mM, [theta]282 decreased from 2000 TO 1500 DEG CM2/DMOLE AND [THETA]295 decreased from 0 to -400 deg cm2/dmole. Both transitions occurred in the pH range from pH 6.0 to 9.2. At pH 5.0, the two ionic strength-dependent transitions were no longer observed and the characteristic changes in the circular dichroism spectra were suppressed. The spectra of the monomer subunits at pH 5.0 showed only small changes with ionic strength and resembled the spectra of the subunits at 11 mM ionic strength above pH 6.0. In order to characterize the transitions in thermodynamic terms an ionic strength near the midpoint of each transition was selected. Then, changes in s20,w and D20,w were measured as a function of temperature. These data allow an estimation to be made of the enthalpies and entropies of the transitions.

Reconstructed complexes of the inner histones (H2A, H2B, H3, H4) and a variety of DNAs were digested with micrococcal nuclease to yield very homogeneous populations of core nucleosomes (nu 1). Nucleosomes containing Micrococcus luteus DNA (72% G+C); chicken DNA (43% G+C), Clostridium perfringens DNA (29% G+C); or poly(A-dT.poly(dA-dT) have been examined by circular dichroism, thermaldetenaturation, electron microscopy, and DNAse I digestion. Circular dichroism spectra of all particles show a typically suppressed ellipticity at 260--280 nm and a prominent alpha-helix signal at 222 nm. All particles show biphasic melting except nu 1 (dA-dT), which show three prominent melting transitions at ionic strength less than or equal to 1 mM. DNAse I digestion of nu 1 (dA-dT) produces a ladder of DNA fragments fiffering in lengthy by one base residue. nu 1 (dA-dT) contain 146 base pairs of DNA and exhibit an average DNA helix pitch of 10.4-10.5 bases per turn. There appear to be two regions of different DNA pitch wihtin nu 1 (dA-dT). It is suggested that the two regions of DNA pitch might correspond to the two regions of the melting profiles.

Monomer nucleosomes (nu 1) from chicken erythrocyte nuclei were examined in aqueous buffers (8 greater than pH greater than 3) and in solvent mixtures (i.e., water and ethanol, ethylene glycol, dioxane, dimethyl sulfoxide, 2-methyl-2,4-pentanediol, polyethylene glycol, sucrose, or urea). Circular dichroism, laser Raman spectroscopy of nu 1, and the fluorescence of nu 1 labeled with N-(3-pyrene) maleimide on thiol groups of H3 histone were employed to detect conformational transitions in nu 1. The results of pH studies were as follows: 5.5 greater than pH greater than 4.8, suppression of DNA ellipticity and no change of histone alpha-helix; 4.6 greater than pH greater than 4.2 an irreversible increase in the B character of DNA, a slight loss of histone alpha-helix, and a parallel loss of pyrene excimer fluorescence; 4 greater than pH, aggregation of nu 1 and protonation of the DNA bases C and A. Results obtained in the studies of nu 1 in solvent mixtures included the following: sharp conformational transitions that variously involved an increase in the B character of DNA, a slight loss of histone alpha-helix, and a loss of pyrene excimer. Different solvents required different concentrations to effect these conformational changes.

The macronucleus of the ciliated protozoan Euplotes eurystomus contains about 10(6) copies of a single type of 5S ribosomal RNA gene. This 5S gene DNA is only 930 bp long, is flanked by telomeres, and contains a single coding region of 120 bp which serves as a template for transcription in vivo and in vitro. The 5S gene minichromatin possesses four positioned nucleosomes and hypersensitive cleavage sites in the telomeric regions.

Oxytricha nova is a hypotrichous ciliate containing a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Two-dimensional gel electrophoresis shows that macronuclei contain a normal complement of inner histones. However, despite extensive efforts, no classical H1-like protein has been detected. Micrococcal nuclease digestion indicates a nucleosome repeat length of approximately 220 bp for macronuclear chromatin. Thermal denaturation profiles of macronuclear chromatin in 0.2 mM EDTA display four transitions at about 46, 57, 64, and 79 degrees C. The lowest of these shifts to higher temperature as the ionic strength is raised to 3-5 mM Na phosphate. These results are consistent with the absence of H1 and a nucleosome repeat of 220-230 bp. Circular dichroism (CD) results agree with these findings. By contrast, micronuclear chromatin displays a much smaller premelt and a more suppressed DNA CD signal at 285 nm, consistent with a micronuclear chromatin repeat of 165-185 bp as determined by micrococcal nuclease digestion.