Isopycnic banding of chromatin in chloral hydrate gradients

Chromatin from Ehrlich ascites tumor cells bands in chloral hydrate gradients at densities ranging from 1.40 to 1.60 g/cm³. The total chromatin after recovery from the gradient has a composition similar to that of native chromatin. Chloral hydrate does not dissociate the chromatin into its nucleic acid and protein components. The chloral hydrate is inert toward both the DNA and the histone, as indicated by the lack of influence either on the melting curves of the DNA, or on the electrophoretic patterns of the histones, when these substances are recovered from chromatin treated with chloral hydrate. The resolution of chromatin on the gradients reflects different buoyant densities of the chromatin, and this in turn, different protein to DNA ratios. Extraction of the histones from chromatin obtained from different regions of the gradients reveals differences in prevalence of at least two of the histone species, and provides evidence suggesting that different stretches of DNA in the genome may associate with different histones.     

Chromatin regions,released by endogenous nucleases,are enriched in immunogenic tissue-specific proteins

Localization of immunogenic tissue-specific proteins in chromatin regions, hypersensitive to endogenous nucleases, has been studied using rabbit antibodies against rat thymus chromatin. It is shown that the first 1–2,5% of the chromatin (calculating on DNA), released by Mg²⁺-, Mn²⁺-, and Ca²⁺/Mg²⁺-dependent nuclear endonucleases are drastically enriched in tissue-specific antigenic determinants. The released chromatin fractions are found to contain a heterogeneous set of nonhistone proteins and are deficient in histones. The cleavage of nuclear DNA by endogenous acidic nuclease, independent on bivalent ions, resulted in a significantly less enrichment of the released fractions with immunogenic proteins.     

Binding of progesterone by rat uterus in vitro

Circular dichroism (CD) spectra have been determined for chromatin fractions obtained by ECTHAM-cellulose chromatography. The molecular ellipticity at the positive long wavelength maximum is about 3000 deg cm²/dmol for early-eluted chromatin fractions, thought to be relatively repressed $\text{in vivo}$, and 5000–6000 deg cm²/dmol for late-eluted chromatin fractions, those thought to be preferentially transcribable $\text{in vivo}$. CD bands in the peptide bond spectral region also differ for the two chromatin fractions, early-eluted chromatin having a more helical conformation for proteins. In addition to previously known differences in protein content, the biological activity of a native chromatin fraction can now be correlated with the conformation of its DNA.     

Circular dichroism of native whole SV40 chromatin.

Circular dichroism properties of SV40 virions, isolated minichromosomes from virions, and SV40 Form I (supercoiled) DNA were studied in a buffer of low ionic strength. The isolated minichromosomes are compact as judged by sedimentation and electron microscopy. The molar ellipticity at 284 nm of the virion, which may be regarded as a minichromosome in its native state, is about 1500 deg cm²/dmol phosphate; this value is in the same range as that reported for core particles (1300–2000) isolated from different sources. When the viral capsid is removed, there is a small increase in the molar ellipticity to about 2000. However, both of these values are much lower than that found for SV40 supercoiled DNA (about 8200). The results strongly suggest that the linker DNA of the native whole chromatin contributes in a similar fashion to the circular dichroic ellipticity as the core DNA.     

Laser Raman identification of an interaction site on DNA for arginine containing histones in chromatin.

Comparisons of the Raman spectra of DNA, chromatin, and complexes of DNA with poly-L-arginine and N-α-acetylarginine have been made. Both in native chromatin and in complexes of DNA with the arginine derivatives there is a marked decreased in the Raman intensity of the 1490±2 cm^?1 band due to guanine. Considerable evidence is presented to show that a decrease in the intensity of the 1490 cm^?1 Raman band of quanine in DNA is strong indication of a hydrogen bond being attached to the N-7 position of quanine. A specific model is presented for the interaction of the arginine residues with the guanine residues in the major groove of DNA. The Raman frequency of the histone Amide 1 band indicates that these protein molecules have a high α-helical content while the phosphate diester stretch frequency of the DNA shows the DNA to be in the B-family.     

Studies on erythrocruorin. V. Nuclear magnetic resonance quadrupole relaxation study of sodium, calcium and chloride binding.

Metabolically labeled non-histone chromosomal proteins of high specific activity were fractionated on the basis of their sequential extractability from Krebs II chromatin with urea/salt solutions according to Bekhor et al. (1974a). The binding of each of these NHCP² classes to protein-free DNA and histone-DNA complexes (nucleohistone) was measured and compared to the binding to DNA substituted with 5-bromo-2′-deoxyuridine. After reconstitution of the interacting components, the binding of NHCP and histones was measured according to Scatchard formalism by titration of fixed amounts of DNA with increasing inputs of protein ligands under stringent conditions of 0.25 ionic strength, pH 8.0. Histone binding to either native DNA or BrUrd-substituted DNA was found to be essentially the same. In the presence of histones, the binding for all NHCP classes, except for medium 3 NHCP, was enhanced by an order of magnitude over the binding values for NHCP to DNA in the absence of histones. The binding of NHCP to DNA was thus strongly influenced by histones bound to DNA. A general and significant decrease in histone content in the complexes relative to increased NHCP binding was also apparent, with medium 3 NHCP having the greatest activity to weaken histone interaction with DNA and medium 0 the least. Enhancement in NHCP binding to BrUd-substituted DNA in the presence of histones was decreased to about 50% of the binding to control DNA. The distribution and quantity of DNA binding and non-DNA binding NHCP was also estimated by photochemical attachment to 33% BrUrd-substituted DNA in tryptophan-labeled chromatin and by direct binding assays. We have obtained 30% crosslinking for either histones or NHCP to DNA in stringently formed complexes. In histone-NHCP-DNA complexes, histone crosslinking remained unchanged, while that of NHCP increased to 70%. This is further evidence for a modification in the binding of NHCP to DNA in the presence of histones. The percentage of NHCP crosslinked to DNA in native chromatin ranged from 24% for medium 0 NHCP to 50% for medium 1 and 3 NHCP with an average of 35% for total NHCP. These results plus the direct binding assays indicate that NHCP, in addition to high affinity DNA binding, also interacts non-specifically to DNA and to proteins in chromatin. A mechanism is also being proposed to account for the observed BrUrd effects in chromatin.     

DNA Polymerase Activity Associated with the Membrane Fraction of E. coli

Spheroplasts were disrupted with 0.2% Brij 58 and the separation of intact cells, spheroplasts, disrupted spheroplasts, fragmented membrane, and supernatant was performed on a linear 40~55% sucrose gradient. About half an amount of nucleic acid components was distributed in disrupted spheroplast fractions, while only a small amount of protein components was found in these fractions.

DNA polymerase in the fragmented membrane fraction incorporated ³H-TTP more rapidly than that in the supernatant fraction for the first 5 to 6 min, and then the incorporation rate decreased, while DNA polymerase in the supernatant fraction incorporated ³H-TTP linearly up to 20 min when native DNA was used as a primer. The former required native DNA as a primer and showed little activity towards denatured DNA, while the latter incorporated ³H-TTP at a similar rate to both the primer DNA’s.

DNA polymerase of the fragmented membrane fraction synthesized various sizes of DNA from short to a size of primer when native DNA was used as a primer, while when denatured DNA was used, products were only short. DNA polymerase of the supernatant fraction synthesized various sizes of DNA when both native and denatured DNA’s were used as primers.     

Nucleosomes as antigens. Characterization of determinants and cross-reactivity

Antibodies raised against chicken erythrocyte nucleosomes were characterized in terms of their binding to individual chromatin components and tested for cross-reactivity with chromatin from other species. Most of the proteins present in the immunogen elicited antibodies. Of the histones, H5 elicited the strongest response, followed by H2B, H2A, H3 and H4. In addition, antibodies specific for several non-histone chromosomal proteins (NHCPs) were present, especially those NHCPs which remain bound to DNA in 5.0 M urea, 2.5 M NaCl, at pH 5.0. No antibodies to native DNA could be detected. Strong cross-reactions were observed between the antinucleosome sera, and nuclei, chromosomes, or nucleosomes from other vertebrate (calf, African green monkey), insect (Drosophila melanogaster) and higher plant (Haemanthus katherinae) species. In the case of Drosophila the cross-reaction was shown to be mainly due to the C-terminal (63–128) portion of H2B with smaller contributions from H3 and H4. These results are discussed in relation to exposed antigenic sites on nucleosomes, and the extent to which they have been conserved during evolution. Also, parallels between the immunological response to nucleosomes and the anti-nuclear antibodies characteristics of systemic lupus erythematosus are considered.     

ATP as an alternative inhibitor of bacterial and endogenous nucleases and its effect on native chromatin compaction

The studies reported here demonstrate that ATP may be used in lieu of EDTA to inhibit nuclease digestion of DNA and chromatin. Because ATP is a milder chelator than EDTA and is a biochemical common to the cellular microenvironment in vivo, critical studies of cellular processes that require native structure to be maintained are more feasible without the presence of strong chelators. During the digestion of chromatin into its components by nuclease treatment, ATP assures the retention of nucleoprotein compaction, particularly for large to intermediate-sized oligosomes (2400bp–1000bp in length). ATP used at a concentration of 3.3 mM appears to be somewhat better than EDTA, 1.0 mM, for minimizing degradation of nuclease-treated chromatin. However, termination of nuclease digestion of chromatin and minimization of further degradation by the addition of ATP to a concentration of 1.0 mM was almost equivalent to the addition of EDTA to a concentration of 1.0 mM. Slightly more degradation was observed for the latter condition. In addition, ATP can be used to inhibit endogenous nuclease activity when specific restriction enzymes are needed. Standard low ionic strength DNP, deoxyribonucleoprotein, and DNA electrophoresis of proteinized and deproteinized chromatin oligomers, respectively, indicated that ATP effectively inhibits staphylococcal nuclease. Low ionic strength nucleoprotein electrophoresis to resolve staphylococcal nuclease-digested chromatin indicates that as little as 10^–4 M EDTA can promote structural unfolding resulting in changes in apparent mobilities for chromatin oligomers 250 and 600 by in length. Comparative digestion of chromatin with staphlococcal nuclease followed by reaction termination by ATP or EDTA showed that this observation was not merely the result of degradation due to inefficiency of ATP enzyme inhibition.     

An analysis of the bovine genome by Cs2SO4-Ag density gradient centrifugation

Calf DNA preparations having molecular weights of 5 to 7 × 10⁶ have been fractionated by preparative Cs₂SO₄—Ag⁺ density gradient centrifugation into a number of components. These may be divided into three groups: (1) the main DNA component (1.697 g/cm³; all densities quoted are those determined in CsCl density gradients), the 1.704 and 1.709 g/cm³ components form about 50, 25 and 10% of the genome, respectively; they are characterized by having symmetrical CsCl bands and melting curves, both of which have standard deviations close to those of bacterial DNAs of comparable molecular weight, and by their G + C contents being equal to 39, 48 and 54%, respectively; after heat-denaturation and reannealing, their buoyant densities in CsCl are greater than native DNA by 12, 10 and 3 mg/cm³, respectively. (2) The 1.705, 1.710, 1.714 and 1.723 g/cm³ components represent 4, 1.5, 7 and 1.5% of the DNA, respectively, and exhibit the properties of “satellite” DNAs; their CsCl bands and melting curves have standard deviations lower than those of bacterial DNAs; after heat-denaturation and reannealing, their buoyant densities are identical to native DNA, except for the 1.705 g/cm³ component, which remains heavier by 5 mg/cm³; in alkaline CsCl, only the 1.714 g/cm³ component shows a strand separation. (3) A number of minor components, forming 1% of the DNA, have been recognized, but they have not been investigated in detail; two of them (1.719 and 1.699 g/cm³) might correspond to ribosomal cistrons and mitochondrial DNA, respectively.     

Changes in the infrared microspectroscopic characteristics of DNA caused by cationic elements, different base richness and single-stranded form

Background

The infrared (IR) analysis of dried samples of DNA and DNA-polypeptide complexes is still scarce. Here we have studied the FT-IR profiles of these components to further the understanding of the FT-IR signatures of chromatin and cell nuclei.

Methodology/Principal Findings

Calf thymus and salmon testis DNA, and complexes of histone H1, protamine, poly-L-lysine and poly-L-arginine (histone-mimic macromolecules) with DNA were analyzed in an IR microspectroscope equipped with an attenuated total reflection diamond objective and Grams software. Conditions including polypeptides bound to the DNA, DNA base composition, and single-stranded form were found to differently affect the vibrational characteristics of the chemical groups (especially, PO₂ ⁻) in the nucleic acid. The antisymmetric stretching (ν_as) of the DNA PO₂ ⁻ was greater than the symmetric stretching (ν_s) of these groups and increased in the polypeptide-DNA complexes. A shift of the ν_as of the DNA PO₂ ⁻ to a lower frequency and an increased intensity of this vibration were induced especially by lysine-rich histones. Lysine richness additionally contributed to an increase in the vibrational stretching of the amide I group. Even in simple molecules such as inorganic phosphates, the vibrational characteristics of the phosphate anions were differently affected by different cations. As a result of the optimization of the DNA conformation by binding to arginine-rich polypeptides, enhancements of the vibrational characteristics in the FT-IR fingerprint could be detected. Although different profiles were obtained for the DNA with different base compositions, this situation was no longer verified in the polypeptide-DNA complexes and most likely in isolated chromatin or cell nuclei. However, the ν_as PO₂ ⁻/ν_s PO₂ ⁻ ratio could discriminate DNA with different base compositions and DNA in a single-stranded form.

Conclusions/Significance

FT-IR spectral profiles are a valuable tool for establishing the vibrational characteristics of individualized chromatin components, such as DNA and DNA-polypeptide complexes in dried samples.     

Developmental studies of sea urchin chromatin. Chromatin isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus

Chromatin was isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus. The isolated chromatin shows less absorptivity ratio of 230 nm : 260 nm and possesses less protein than does embryonic chromatin. The ratio of histone : DNA is 1.02; nonhistone : DNA 0.13; RNA : DNA 0.04. Sperm chromatin melts in two steps with T_ms 70°C and 84°C in 2.5 × 10⁻⁴, M EDTA in contrast to embryonic chromatin with a single T_m = 72°C. Disc electrophoresis of basic proteins of sperm revealed one minor component with extremely fast mobility and three major components. The one with the slowest mobility is characteristic of sperm. The embryo has in turn its characteristic histone which also migrated slowly in disc electrophoresis. Both of these unique histone fractions are selectively extracted from chromatin by 5% perchloric acid. Amino acid analyses of these chromatographically purified unique fractions show that both contain a large amount of lysine, while that from sperm, in addition, contains also a large amount of arginine. Minimal molecular weights of 33,000 for sperm and 16,200 for embryo unique histone were estimated from these analyses. Sperm chromatin supports a level of RNA synthesis in vitro with exogeneously supplied RNA polymerase about 2% that of the corresponding free DNA.     

DNA SYNTHESIS IN NEURONAL, GLIAL AND LIVER NUCLEI ISOLATED FROM THE ADULT GUINEA PIG

Abstract— [³H]Deoxythymidine-5′-triphosphate incorporation into P₅₁ (51% neuronal nuclei: 49% glial nuclei), P₃ (3% neuronal nuclei: 97% glial nuclei) and liver nuclear preparations, isolated from the adult guinea pig, was determined in the presence of the other three complementary deoxyribonucleo-tides. The enzymic characteristics of the DNA synthesis reaction were studied and DNA polymerase contents were estimated in neuronal, glial and liver nuclei. (1) Cerebral and liver nuclei exhibited similar enzymic properties for DNA synthesis activities with a few discrepancies. (2) P₅₁ nuclei synthesized DNA 2.4-fold more actively than P₃ nuclei. Liver nuclei carried out the most active DNA synthesis. The proportion of chromatin DNA available as template and primer was estimated by comparison with native calf thymus DNA. The available proportions found, in terms of the total chromatin DNA. were 2.39% for P₅₁ nuclei, 1.38% for P₃ nuclei and 37.6% for liver nuclei. (3) Exogenous native and heat-denatured calf thymus DNA were utilized as template and primer by DNA polymerase in nuclei in different ways depending on the nuclear species. The enzyme was saturated with native DNA by elevating the concentration and the activity reached a plateau. Denatured DNA inhibited the activity at the higher concentrations. (4) From the enzyme activities at a saturation concentration of exogenous DNA, DNA polymerase contents were estimated: P₅₁ nuclei, 39.2 ± 2.6 (s.e.m. ) units (fmol of TMP incorporated/30 min at 31°C)/μg of nuclear DNA; P₃ nuclei. 24.5 ± 1.6; and liver nuclei, 72.5 ± 8.1; the specific activity obtained on a protein basis was 1.55 times higher with P₃ nuclei than with P₅₁ nuclei. (5) Denatured DNA inhibited the nuclear DNA polymerase activity dependent on native DNA. The efficiency of inhibition was in the order: P₃ > P₅₁ > liver nuclei.     

Induction of DNA synthesis by 2,4-dichlorophenoxyacetic acid during callus induction in Jerusalem artichoke tuber tissues

During the induction of DNA synthesis in Jerusalem artichoke (Helianthus tuberosus L.) tuber by 2,4-D, the 2-¹⁴C-2, 4-D from the agar medium rapidly incorporated into the ethanol soluble and insoluble fractions. Although the 2,4-D level in the ethanol soluble fraction decreased on transplantation of the tissue from the 2-¹⁴C-2,4-D medium to medium without the auxin, its level in the buffer-soluble and -insoluble macromolecular fractions increased. The purified, buffer-insoluble macromolecules were chromatin. The 2,4-D binding to chromatin particularly increased during DNA synthesis. The histone contents of chromatin decreased as DNA synthesis progressed. The polyacrylamide gel electrophoretic patterns of the histones showed a decrease in the moderately lysine-rich histone fraction as compared to other fractions. Thus, the decrease in the histone level caused by 2,4-D and the presence of the 2,4-D moderately lysine-rich histone complex may be closely related to the induction of DNA synthesis by 2,4-D in cells.     

Effect of chromatin associated factors on the activity of thyroid hormone receptors in rat liver and brain

Receptors for thyroid hormones were extracted by 0.4 M KCl from the nuclei of rat liver and brain, and their binding properties compared to the properties of these receptors in unextracted nuclear suspensions. The inhibititory effect of a non-iodinated thyroid hormone analogue, 3,5,dimethyl-3′-isopropyl-l-thyronine (DIMIT) on [¹²⁵I]-T₃ binding was observed in the nuclear suspension of brain, but absent when the solubilized receptors of the same organ were tested. The initial properties of the receptor could be restored in a system containing the receptor and the extracted chromatin. Moreover, when the liver solubilized receptor was supplemented with the brain chromatin extract, the hepatic receptor acquired the binding ability of the brain receptors. The data suggest that chromatin associated components may confer organ specificity in thyroid hormone effects, and play a role in the selectivity of the recognition of thyroid hormone analogues by the receptor.     

Synthesis of compositionally unique DNA by terminal deoxynucleotidyl transferase

Studies on the composition and characterization of DNA product(s) synthesized by calf thymus terminal deoxynucleotidyl transferase were performed using homopolymeric single-stranded, calf thymus double-stranded, and native DNA resident in calf thymus chromatin preparations as priming DNA species. Synthesis was carried out using equimolar concentrations of all four deoxynucleoside triphosphates as substrates and Mg²⁺ or Mn²⁺ as an effective divalent cation. Irrespective of the nature of the priming DNA or the divalent cation, the DNA product contained 60–70% dGMP residues, 10–15% each of the two pyrimidine residues, and 5–10% dAMP residues. The product synthesized using chromatin DNA as initiator was predominantly single-stranded and its synthesis was resistant to actinomycin D. The predilection of terminal deoxynucleotidyl transfease to synthesize dGMP-rich products on natural or homopolymeric DNA primers suggests that such products may represent biologically important recognition signal sequences.     

Chromosomal position effects determine transcriptional potential of integrated mammary tumor virus DNA

We have investigated two mammary tumor virus proviruses that are integrated at different chromosomal sites in the genomes of two clonal isolates of cultured rat hepatoma cells. One of these cell lines, J2.17, expresses MTV² RNA only in the presence of glucocorticoid hormones. In contrast, the proviral genes in the other line, J2.15, fail to be transcribed in the presence or absence of glucocorticoids, despite the fact that the viral genes and the cellular components that mediate hormone responses appear intact and normal. Low-level DNAase I digestion of chromosomes in isolated nuclei reveals that the J2.17 MTV DNA sequences are packaged in chromatin that is highly sensitive to DNAase I attack, whereas the chromatin of the J2.15 provirus is relatively resistant to DNAase I. These results demonstrate that the same genetic element located at two different chromosomal loci within a single cell line can differ in both chromatin structure and gene expression. Analysis of the chromatin structure of the appropriate DNA sequences in uninfected HTC cells suggests that the difference in the chromatin structure of the two proviruses may reflect a “spreading effect”, in which heterologous integrating DNA is packaged into chromatin that is similar in configuration to the surrounding chromatin. Thus, we propose that chromosomal position determines the folding pattern of the newly introduced DNA sequences, and that this pattern in turn determines whether the genes can subsequently be expressed in response to the hormonal inducing signal.