Analysis of chromatin reconstitutiion.

The ability of high molecular weight chicken erythrocyte chromatin to spontaneously self-assemble into native-like material, after dissociation by high ionic strength and reassociation by salt gradient dialysis, was critically examined. The native conformational state of the reassembled nucleoprotein complex was regenerated to the extent reflected by circular dichroism spectra and thermally induced helix--coil transition of the nucleoprotein DNA. However, internucleosomal packing of approximately 205 base pairs of DNA per repeating unit, as probed by digestion with micrococcal nuclease, was not regenerated upon reassembly and was replaced by a packing of approximately 160 base pairs per repeating unit. Thus, high molecular weight chromatin containing only lysine-rich histones (H1 and H5) and core histones (H2A, H2B, H3, and H4) is not a true self-assembling system in vitro using the salt gradient dialysis system used herein. Circular dichroism and thermal denaturation studies on core chromatin (lysine-rich histones removed) showed that core histones alone are not capable of reassembling high molecular weight DNA into native-like core particles at low temperature (4 degree C). Reassembly at 21 degree C restored the circular dichroism but not the thermal denaturation properties to those characteristic of undissociated core chromatin. Nonetheless, micrococcal nuclease digestions of both reassembled core chromatin products were identical with undissociated native core chromatin. Ressembly in the presence of the complete complement of histones, followed by removal of the lysine-rich histones, did regenerate the thermal denaturation properties of undissociated native core particles. These results indicated multiple functions of the lysine-rich histones in the in vitro assembly of high molecular weight chromatin.     

Thermal denaturation of nucleic acids in polyacrylamide gels

A system is described for measuring thermal denaturation of nucleic acid fractions directly in polyacrylamide gels. Total nucleic acids were fractionated by disc gel electrophoresis. The buffer within the gel was then exchanged for one commonly used in denaturation studies. Thermal denaturation profiles of DNA and ribosomal RNA in the gel were determined using a specially constructed Gel Carriage to position the appropriate fraction during spectrophotometric measurements. These profiles were compared with denaturation patterns obtained by classical methods in free solution; the two methods yielded similar patterns.Thermal denaturation profiles were also obtained for chloroplast light ribosomal RNA resolved by gel electrophoresis of total plant nucleic acids. Thus, denaturation patterns of individual, minor components present in complex nucleic acid mixtures can be directly measured in gels.     

Ionic strength-dependent conformational transitions of chromatin. Circular dichroism and thermal denaturation studies

High-molecular-weight chicken erythrocyte chromatin was prepared by mild digestion of nuclei with micrococcal nuclease. Samples of chromatin containing both core (H3, H4, H2A, H2B) and lysine-rich (H1, H5) histone proteins (whole chromatin) or only core histone proteins (core chromatin) were examined by CD and thermal denaturation as a function of ionic strength between 0.75 and 7.0 × 10^?3M Na⁺. CD studies at 21°C revealed a conformational transition over this range of ionic strengths in core chromatin, which indicated a partial unfolding of a segment of the core particle DNA at the lowest ionic strength studied. This transition is prevented by the presence of the lysine-rich histones in whole chromatin. Thermal-denaturation profiles of both whole and core chromatins, recorded by hyperchromicity at 260 nm, reproducibly and systematically varied with the ionic strength of the medium. Both materials displayed three resolvable thermal transitions, which represented the total DNA hyperchromicity on denaturation. The fractions of the total DNA which melted in each of these transitions were extremely sensitive to ionic strength. These effects are considered to result from intra- and/or internucleosomal electrostatic repulsions in chromatin studied at very low ionic strengths. Comparison of the whole and core chromatin melting profiles indicated substantial stabilization of the core-particle DNA by binding sites between the H1/H5 histones and the 140-base-pair core particle.     

Effects of shearing on chromatin structure

The effects of mechanical shearing on chromatin structure were investigated by using thermal denaturation and circular dichroism (CD) spectroscopy. Under ordinary conditions of mechanical shearing used for preparation of soluble chromatin, we observed only minor changes (less than 10%) of chromatin properties with respect to (a) absorption melting curves, (b) CD spectra, (c) CD melting curves and (d) histone transfer from chromatin to exogenous DNA. Such small pertubation of structural properties could be due to the generation of free ends when a large chromatin was cut into smaller fragments and by weakening the binding of histones to DNA near these free ends. In addition to mechanical shearing, sonication was used to shear some samples of chromatin. The effect of sonication on chromatin structure was investigated by the same physical methods used for mechanically sheared chromatin. The results indicate that sonication only slightly changes the chromatin properties with respect to CD spectra, similar to the results obtained by mechanical shearing, but sonication at high settings has a greater effect on the thermal denaturation property of chromatin as contrasted to our results from mechanically sheared chromatin.     

Hydroxylapatite thermal fractionation of chromatin and DNA

Chromatin and DNA from developing muscle cultures were fractionated by hydroxylapatite thermal chromatography on the basis of differential thermal stability. A thermal chromatography system was developed in which protein mediated thermal stability of chromatin DNA was maximally expressed. The resulting chromatin and DNA elution profiles were similar to thermal denaturation profiles in low ionic strength solution. Additional studies showed this system was able to detect protein stabilization of DNA in in vitro nucleohistone preparations. Although some protein remained bound to hydroxylapatite during chromatin thermal elution, it did not affect the denaturation or elution behavior of free DNA on the same column. These studies show that fragments of chromatin or DNA can be segregated on the basis of differential thermal stability by hydroxylapatite chromatography.     

Interaction of Nucleic Acids with Lipid Membranes

Abstract

The thermodynamics of nucleic acids which were enclosed in reverse-phase evaporation vesicles was studied by thermal denaturation with optical recording. The denaturation curves were recorded with a dual wavelength spectrophotometer. The sum of the hypochromicity of the nucleic acid and of the change in turbidity of the vesicles was measured at 260 nm and was corrected for the change in turbidity at 320 nm. Cloned fragments of double-stranded DNA containing 180 base pairs and poly A:poly U were enclosed in REV with a yield up to every vesicle containing five nucleic acid molecules. Vesicles were prepared from egg- lecithin, and the surface charge of the vesicles was varied by addition of stearic acid, phosphatidyl-glycerol and phosphatidyl-serine. The helix-coil transition of the nucleic acid enclosed in the vesicle could be resolved from that of the free nucleic acid. Due to the enclosure into the egg-lecithin REV the transition is stabilized from 70.5° to 74°C, the transition is broadened from 0.7°C to 2.7°C. Varying the phosphatidyl-serine-lecithin-ratio from 0–100%, an optimum in the yield of enclosure at 20% was obtained, a further broadening of the transition to 5.5°C and a decrease of the stabilization down to a small destabilization at 100% phosphatidyl serine was observed. Qualitatively, similar effects were observed with poly A:poly U. Variation of the ionic strength led to the conclusion that the replacement of the counterions of the phosphate backbone by the surface charge of the membrane, as well as a direct contact between the nucleic acid and the membrane have to be assumed. At present, the biological relevance of the results may be more in the drastic decrease in cooperativity than in the slight modulation of the stability. From nearly 180 base pairs opening up cooperatively in free nucleic acid this number is lowered to less than 50, a size in the range of promotor regions.     

Formation of hybrid nucleosomes cantaining new and old histones.

5 mM hydroxyurea (HU) inhibits DNA synthesis in mouse P815 cells by 94-97% in less than 1 hr. Nevertheless, histone synthesis continues and newly-synthesised histones are incorporated into non-replicating chromatin at a rate of about 20% of that in control exponentially-growing cells. To study the organization of these histones in chromatin P815 cells were treated with 5 mM HU in medium containing dense (15N, 13C, 2H) - substituted amino acids. After inhibition of DNA synthesis, newly-synthesised histones were labelled with (3H)-arginine. The cells were harvested 90 min later, and mono- and oligonucleosomes were prepared and analysed on metrizamide-triethanolamine (MA-TEA density gradients. Analysis of the distribution of 3H-labelled histones in these gradients shows that they are incorporated into hybrid mononucleosomes containing both new and old histones. It is also shown that these hybrid nucleosomes are not randomly distributed, but show a certain tendency to be clustered in certain chromatin regions.     

Fractionation of chick oviduct chromatin. Nuclease-resistant deoxyribonucleic acid.

Chromatin isolated from several chick tissues was treated with micrococcal nuclease. A limited degree of tissue specificity of chromatin DNA resistance to nuclease digestion was observed. No difference in the extent of nuclease resistance of chromatin DNA was detected during oestrogen-induced oviduct differentiation. This suggested that the amount of non-histone chromosomal protein does not play an important role in the sensitivity of chromatin DNA to nuclease digestion. Studies of nuclease resistance of chromatin DNA after dissociation and reconstitution of chromatin proteins and ethanol extraction of chromatin indicate that the histones protect the DNA from nuclease attack. Slow thermal denaturation of nuclease-resistant DNA suggests that the protected DNA sequences may be (A+T)-rich, and the (G+C)-rich satellites present in total chick DNA are sensitive to nuclease.     

Chromosomal proteins of conifers

During imbibition and germination of jack pine seeds, the composition of the total extractable chromatin varied. Relative to DNA, the histone levels decreased as the nonhistone chromosomal proteins (NHCP) increased. New chromosomal proteins were synthesized after 2 days of imbibition as judged by recovery of ¹⁴C-amino acids from the major protein fractions. Phosphorylation of histones from ³²P-phosphoric acid was detected before the incorporation of ¹⁴C-amino acids. In the seed the synthesis and relative changes of chromatin coincided with a fall in total soluble protein and free arginine N. By contrast, adenylate energy charge, free glutamine N and in vitro template activity of chromatin increased during chromatin protein synthesis. When seeds had germinated for 4 days after the start of imbibition more radioactivity, derived from free ¹⁴C-amino acids, was recovered from the NHCP than from the histones. The percentage amino acid composition of most histone fractions remained stable, whereas the composition of NHCP changed more with time. The phosphorylation of NHCP was 8- to 41-fold greater than that of the histones. Phosphorylation of histone H4 was not detected at any stage of germination. Correlations between recovery of radioactivity (³²P and ¹⁴C) from chromosomal proteins and higher adenylate energy charge were positive.     

Interaction of nucleic acids with lipid membranes

The thermodynamics of nucleic acids which were enclosed in reverse-phase evaporation vesicles was studied by thermal denaturation with optical recording. The denaturation curves were recorded with a dual wavelength spectrophotometer. The sum of the hypochromicity of the nucleic acid and of the change in turbidity of the vesicles was measured at 260 nm and was corrected for the change in turbidity at 320 nm. Cloned fragments of double-stranded DNA containing 180 base pairs and poly A:poly U were enclosed in REV with a yield up to every vesicle containing five nucleic acid molecules. Vesicles were prepared from egg-lecithin, and the surface charge of the vesicles was varied by addition of stearic acid, phosphatidyl-glycerol and phosphatidyl-serine. The helix-coil transition of the nucleic acid enclosed in the vesicle could be resolved from that of the free nucleic acid. Due to the enclosure into the egg-lecithin REV the transition is stabilized from 70.5 degrees to 74 degrees C, the transition is broadened from 0.7 degrees C to 2.7 degrees C. Varying the phosphatidyl-serine-lecithin-ratio from 0-100%, an optimum in the yield of enclosure at 20% was obtained, a further broadening of the transition to 5.5 degrees C and a decrease of the stabilization down to a small destabilization at 100% phosphatidyl serine was observed. Qualitatively, similar effects were observed with poly A:poly U. Variation of the ionic strength led to the conclusion that the replacement of the counterions of the phosphate backbone by the surface charge of the membrane, as well as a direct contact between the nucleic acid and the membrane have to be assumed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the structural organization of chromatin during spermatogenesis in the rat

In this study, histone H4 was shown to be extensively hyperacetylated in mid-spermatids of the rat during the time period when the entire complement of histones is replaced by basic spermatidal transition proteins. The degree of hyperacetylation of histone H4 was minimal in pachytene spermatocytes. Therefore, the hyperacetylation appears to be directly involved in the histone replacement process late in spermatogenesis in mid-spermatids. In order to investigate further the possible effects of histone H4 hyperacetylation and the other dramatic changes in the nuclear proteins on the structure of chromatin in germinal cells, we examined the thermal denaturation profiles of chromatin from various purified germinal cell types. Our analyses revealed that chromatins from pachytene spermatocytes and early spermatids have similar thermal denaturation profiles, with their major thermal transitions slightly lower than those for rat liver. However, the major thermal transitions for chromatin from mid-spermatids are much lower than those from pachytene spermatocytes and early-spermatids. We propose that the greatly lowered thermal stability of mid-spermatid chromatin represents a dramatic relaxation or decondensation of the chromatin in this cell type in preparation for the replacement of histone by the basic spermatidal transition proteins and that the decondensation is due in large part to the extensive histones hyperacetylation which occurs in these cells.     

Age-related decline in the activites of erythrocyte membrane adenylate cyclase and protein kinase.

The circular dichroism and thermal denaturation properties of chromatin isolated from duck erythrocytes have been carefully examined as has the chromatography of sonicated erythrocyte chromatin on ECTHAM-cellulose. The circular dichroism spectrum and thermal denaturation profile resemble much more closely those of chromatin from liver than has been previously reported by other workers. The chromatography of erythrocyte chromatin on ECTHAM-cellulose gave results that differ dramatically from those obtained from chromatography of f1-containing chromatins on this weak anion exchanger, in that no variations in histone content, circular dichroism spectra or thermal denaturation profiles were observed in the eluted material. Coupled with our earlier finding of no variation in relative content of individual histones (Reeck, G. R. et al. (1974) Eur. J. Biochem. 49, 407–414), we interpret the results of ECTHAM-cellulose chromatography of erythrocyte chromatin to indicate that f2c-depleted regions analogous to the f1-depleted regions found in f1-containing chromatins do not exist in duck erythrocyte chromatin.     

Influence of lipid membranes on the conformational transitions of nucleic acids

The conformational transitions of nucleic acids which were enclosed in reverse phase evaporation vesicles (REV) were studied by thermal denaturation with optical recording. Cloned fragments of double-stranded DNA containing 179 base pairs and 187 base pairs, respectively, and polyA.polyU were enclosed in REV with a yield up to every vesicle containing 50 nucleic acid molecules. With the 179 base pairs DNA enclosed in the vesicle from egg lecithin two well resolved helix-coil transitions could be measured; one is very similar in the midpoint-temperature Tm and halfwidth delta T1/2 to the transition of the free nucleic acid, and the other transition occurs stabilized at a 3.5 degrees C higher Tm-value and with a broader delta T1/2, 2.7 degrees C instead of 0.6 degree C. Both transitions are from nucleic acids inside the vesicles. Varying the surface charge of the lipid membrane by adding the negatively charged phosphatidylserine or phosphatidylglycerol, an optimum in the yield of enclosure and a maximum in the increase in Tm (4.5 degrees C) and delta T1/2 (5.5 degrees C instead of 1.0 degrees C) was obtained at 20% phosphatidylserine or phosphatidylglycerol. In vesicles from pure negatively charged lipids no second population of nucleic acids was observed. Qualitatively, similar effects were observed with polyA.polyU. Stabilization and broadening of the second transition is higher for nucleic acids inside vesicles from lipids with unsaturated fatty acids, as dioleoyl-phosphatidylcholine, than with saturated fatty acids, dipalmitoyl-phosphatidylcholine. Stabilization and broadening decrease with increasing ionic strength, whereas the relative contributions of both transitions to the total hypochromicity remain unchanged; the second transition coincides with the first at 90 mM Na+. From the experimental results it was concluded that the interaction of nucleic acids and lipid membranes is mainly of electrostatic nature. The nucleic acids exist inside the vesicles in two populations, one behaving like nucleic acid free in solution and one influenced by the contact with the membrane. All results are in accordance with a model in which the interaction between the nucleic acid and the membrane is in competition with the dipole-dipole interaction inside the membrane surface.     

Thermal denaturation of nucleohistones--effects of formaldehyde reaction

Thermal denaturation of native or partially dehistonized nucleohistones shows two melting bands at 66 and 81° in 2.5 × 10^?4 M EDTA, pH 8.0. These correspond to the melting of DNA segments bound by the less basic and the more basic half-molecules of histones, respectively. These two melting bands combine into a broad melting band from around 70 to 85° when these nucleohistones are pre-treated with formaldehyde. A formaldehyde reaction which fixes histones on DNA by covalent bonds account for the effect. Formaldehyde fixation also increases the melting temperature of some free DNA segments from around 42 to around 55°. This is interpreted as a result of closed or rigid boundaries between free DNA and formaldehyde-reacted histone-bound DNA segments. MgCl₂ dissociates histones from DNA more effectively and leaves longer free DNA segments than does NaCl. Thermal denaturation of a formaldehyde-reacted nucleoprotein thus provides an effective tool for comparing the relative size of free DNA regions on nucleoproteins. The effect of reversible binding of ligands on helix-coil transition of DNA is descussed and found not adequate for thermal denaturation of nucleohistones.     

The use of propidium iodide in cytochemical studies of interphase chromatin structure

Different cytochemical conditions of using Propidium Iodide, a phenanthridinic fluorochrome specific for double-stranded nucleic acids, have been considered to study some structural aspects of the interphasic chromatin. Some molecular properties of the dye allow to define the structure of chromatin fibre (degree of condensation) by means of thermal denaturation of DNA, fluorochromization after extraction of different chromatin components and fluorochromization in condition of low dye-substrate molar ratio (relative unsaturation of DNA). Different biological situations such as cell specialization, neoplastic transformation and life cycles have been examined choosing some typical models (hepatocytes, nucleated erythrocytes, lymphocytes, endometrial cells).