Deoxyribonucleoprotein Complex Research Articles

Comparative Analysis of Molecular Functions and Biological Role of Proteins from Cell-Free DNA-Protein Complexes Circulating in Plasma of Healthy Females and Breast Cancer Patients.

Cell-free DNA (cfDNA) circulates in the bloodstream packed in membrane-coated structures (such as apoptotic bodies) or bound to proteins. To identify proteins involved in the formation of deoxyribonucleoprotein complexes circulating in the blood, native complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies from plasma of healthy females (HFs) and breast cancer patients (BCPs). It was found that the nucleoprotein complexes (NPCs) from HF plasma samples contained shorter DNA fragments (~180 bp) than BCP NPCs. However, the share of DNA in the NPCs from cfDNA in blood plasma in HFs and BCPs did not differ significantly, as well as the share of NPC protein from blood plasma total protein. Proteins were separated by SDS-PAGE and identified by MALDI-TOF mass spectrometry. Bioinformatic analysis showed that in the presence of a malignant tumor, the proportion of proteins involved in ion channels, protein binding, transport, and signal transduction increased in the composition of blood-circulating NPCs. Moreover, 58 (35%) proteins are differentially expressed in a number of malignant neoplasms in the NPCs of BCPs. Identified NPC proteins from BCP blood can be recommended for further testing as breast cancer diagnostic/prognostic biomarkers or as being useful in developing gene-targeted therapy approaches.

The Influence of Proteins on Fate and Biological Role of Circulating DNA.

Circulating DNA has already proven itself as a valuable tool in translational medicine. However, one of the overlooked areas of circulating DNA research is its association with different proteins, despite considerable evidence that this association might impact DNA’s fate in circulation and its biological role. In this review, we attempt to shed light on current ideas about circulating DNA origins and forms of circulation, known biological effects, and the clinical potential of circulating tumor deoxyribonucleoprotein complexes.

Protein Content of Circulating Nucleoprotein Complexes.

In the current study we have investigated the protein content of blood plasma deoxyribonucleoprotein complexes. The complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies. Proteins were separated by SDS PAAGE and identified by MALDI-TOF mass-spectrometry. 111 and 56 proteins (excluding histones), respectively, were identified with a good score in deoxyribonucleoprotein complexes of healthy females and breast cancer patients. However, only four of these proteins were found in 30 % of all samples. Fourteen proteins previously described as tumor specific proteins were found in cancer patients whereas not one of them was found in healthy individuals. The data obtained demonstrate the involvement of different cellular and extracellular proteins in circulating cell-free DNA.

Bovine sperm chromatin is not protected from the effects of ultrasmall gold nanoparticles

The response of ejaculated bovine spermatozoa to gold nanoparticles was studied by the standard method of nuclear chromatin decondensation in vitro. After the treatment of semen samples with a hydrosol containing gold nanoparticles with an average diameter of 3.0 nm and a concentration of 1 x 10(15) particles/mL, the ability of sperm nuclei to decondense in the presence of sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) dramatically changed compared to the control. The frequencies of gametes with nondecondensed ("intact"), partially decondensed, and completely decondensed nuclei correlated as 40 : 32 : 28% and 0 : 36 : 64% in the experiment and the control, respectively. Moreover, the appearance of a sufficiently large number of gametes with destructed and almost completely destroyed nuclei was noticed in the spermatozoa treated with gold nanoparticles. This article suggests the putative mechanisms of action of ultrasmall gold nanoparticles on the structural and functional integrity of the deoxyribonucleoprotein (DNP) complex of mature male gametes.

Effect of catalin on radical production in aqueous solutions and cross-linking in deoxyribonucleoprotein complex

The effect of catalin on cross-linking in the DNA-protein system is studied after illumination with visible and UV light. Catalin is shown to stimulate this process. The use of a hydroxylamine trap reveals that catalin (10–100 μM) induces the accumulation of free radicals in aqueous solutions as soon as 10 minutes after irradiation. A considerable amount of O2- is fixed in the medium during illumination of catalin aqueous solution. Superoxide dismutase added to the incubation medium considerably reduced the number of recorded radicals.

Linker histone-DNA complexes: enhanced stability in the presence of aluminum lactate and implications for Alzheimer's disease

The binding of human brain linker histone proteins to a radiolabelled human Alu repetitive element was examined by mobility shift assay.. Analysis of the complexes formed from protein extracts of whole neocortical nuclei, under physiological conditions in vitro revealed that linker histone H1° has the highest affinity for the Alu DNA sequence. The linker histone-DNA complexes assembled in the presence of aluminum lactate were more resistant to sodium chloride-induced dissociation than those formed in the presence of sodium lactate. The enhanced stability of deoxyribonucleoprotein (DNP) complexes in the presence of the aluminum cation may be of significance in neurodegenerative conditions such as Alzheimer's disease where aluminum preferentially associates with DNA containing structures of the nucleus.

Primary structure of the chromosomal protein MC1 from the archaebacterium Methanosarcina sp. CHTI 55

The DNA of the thermophilic archaebacterium Methanosarcina sp. CHTI 55 has been shown to be associated with two proteins called MC1 and MC2, of molecular mass 11 kDa and 17 kDa (Chartier et al. (1988) Biochim. Biophys. Acta 951, 149–156). The most abundant of these proteins, protein MC1, can protect DNA against thermal denaturation. In the present paper we report the covalent structure of protein MC1 and its effect on transcription of DNA in vitro. The covalent structure was determined from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at aspartic acid and arginine residues. The amino-acid sequence of protein MC1 from Methanosarcina sp. CHTI 55 is closely related to that of the protein MC1 (previously called HMb) isolated from Methanosarcina barkeri strain MS: among the nine substitutions observed between the two proteins seven are conservative. Transcription of DNA in vitro is stimulated by protein MC1 at low protein-to-DNA ratio but is inhibited at a ratio higher than 0.1 (w/w), which is the one determined in the bacterial deoxyribonucleoprotein complex.

Characterization of the chromosomal protein MC1 from the thermophilic archaebacterium Methanosarcina sp. CHTI 55 and its effect on the thermal stability of DNA

In the deoxyribonucleoprotein complex of Methanosarcina sp. CHTI 55, DNA is associated with two proteins, named MC1 (methanogen chromosomal protein 1) ( M r 10760) and MC2 ( M r 17000). Protein MC1, the most abundant of these proteins, is closely related to the Methanosarcina barkeri MS protein MC1. The effect of Methanosarcina sp. CHTI 55 protein MC1 on the thermal stability of DNA has been studied in native deoxyribonucleoprotein complex, as well as in reconstituted complexes, and it has been compared to the effect of E. coli DNA-binding protein II. Both proteins are able to protect DNA against thermal denaturation, but the differences observed in the melting profiles suggest that they interact by different mechanisms. Moreover, our studies indicate that one molecule of protein MC1 protects eight base pairs of DNA.

The deoxyribonucleoprotein complex from the archaebacterium Methanosarcina barkeri: characterization and ultrastructural localization of the chromosomal protein MC1

The Methanosarcina barkeri protein, methanogen chromosomal protein 1 (MC1), a basic polypeptide of 93 amino acid residues, has been localized in the DNA-rich areas on immunolabelled bacterial cryosections revealed with the protein A – gold method. This localization strongly supports the previous studies concerning the association of the protein MC1 with the DNA in vivo. In the M. barkeri deoxyribonucleoprotein complex, the protein MC1 accounts for about 90% of the total proteins, and the protein MC1 so DNA ratio (by weight) is equal to 0.11. Staphylococcal nuclease digestion data and microscopic observations have failed to reveal the presence of repeating structural units and suggest that the protein MC1 is unevenly distributed along the DNA.

Electron spectroscopic imaging of DNA

Electron spectroscopic imaging (ESI) was carried out using a fixed beam electron microscope equipped with a parallel electron energy filter to form micrographs of purified plasmid DNA without the use of heavy metal stains and shadows. Inelastically scattered electrons that have ionized the phosphorus L II,III shell electrons were used to form phosphorus distribution maps of DNA and deoxyribonucleoprotein complexes. Signal-to-noise values of the net phosphorus content over single DNA molecules compared to two and four interwound DNA strands directly reflect the known stoichiometric levels of phosphorus content, illustrating that ESI can be used to determine the relative levels of nucleic acid in nucleoprotein complexes. An initial attempt to characterize nucleosomal and transcriptionally active chromatin from Saccharomyces cerevisiae with this technique reveals three distinct ultrastructural classes of the basic chromatin fiber.

Histamine receptor-mediated changes in nuclear chromatin of cultured endothelial and smooth muscle cells.

Histamine, acting via membrane receptors, modulates a number of potentially important processes within the vascular wall. Histamine receptor activation affects cellular activities such as contractility, movement, growth, and metabolism. The present study entails a characterization of endothelial (EC) and smooth muscle cell (SMC) histamine receptors with respect to cytochemical indices of DNA content and chromatin dispersion.Both EC and SMC exhibited functionally active H1 and H2 receptors. Activation of either receptor type resulted in a substantial ploidy shift in EC but not in SMC cultures. The physico-chemical state of chromatin as assessed by Feulgen reactivity was altered in both cell types. H2 receptor activation elevated Feulgen reactivity in 2C nuclei, while in 4C nuclei both H1 and H2 receptors were active in this regard. Elevation of Feulgen reactivity of EC and SMC nuclear chromatin in response to histamine receptor agonists presumably represents a “chromatin activation reaction” reflecting alterations in the conformation of the deoxyribonucleoprotein (DNP) complex and accompanying changes in template activity. Disparity between agonist effects on 2C and 4C populations may reflect the cell cycle regulation of histamine receptors.

Deoxyribonucleoprotein complexes and DNA synthesis of herpes simplex virus type 1

Two temperature-sensitive mutants of herpes simplex virus type 1 in complementation group 1-1 were analyzed to determine if the major DNA-binding protein they produced was thermolabile. Cells infected with these mutants were analyzed for deoxyribonucleo-protein complexes containing the DNA-binding protein. These complexes were found in cells infected at the permissive temperature but not at the nonpermissive temperature. In temperature shift-up experiments with mutant virus infected cells, the levels of the deoxyribonucleoprotein complexes decreased with time of incubation at the nonpermissive temperature. Viral DNA synthesis terminated in cells infected with these mutants after temperature shift-up. The kinetics of termination of viral DNA synthesis were similar to the kinetics of dissociation of the deoxyribonucleoprotein complexes. These results indicate that two mutants in complementation group 1-1 produce a thermolabile DNA-binding protein and that this protein is required for viral DNA synthesis. Furthermore, they suggest that the major DNA-binding protein of herpes simplex virus type 1 functions in viral DNA synthesis as a component of deoxyribonucleoprotein complexes.

Nuclear chromatin changes in hepatocytes of soman treated rats

Cytochemical and morphometric indices of DNA metabolism and nuclear transcriptional-translational capabilities were used to monitor liver responses in Sprague-Dawley × Wistar rats subcutaneously injected with 0.9 LD 50 soman, a highly toxic acetylcholinesterase-inhibiting organophosphate. Thirty minutes post-soman the following were evident: (1) increased polyploidization; (2) expansion of the nuclear envelope; and (3) elevated deoxyribonucleoprotein (DNP) complex lability to acid hydrolysis. Nuclear changes were accompanied by cytoplasmic correlates of impaired hepatocyte function, i.e., vacuolation and depressed basophilia. The overall data suggest that with acute soman toxication there are concomitant adaptive and maladaptive responses occurring within liver parenchymal elements. It is postulated that nuclear changes represent an early proliferative response and DNP activation to enhance detoxification capabilities, whereas cytoplasmic changes reflect the disruption of normal function and hepatotoxic injury.

Identification and characterization of deoxyribonucleoprotein complexes containing the major DNA-binding protein of herpes simplex virus type 1.

Nuclear extracts were prepared from cells infected with herpes simplex virus type 1 (HSV-1) and fractionated by sucrose gradient centrifugation to identify deoxyribonucleoprotein complexes involved in viral replication. Large amounts of an HSV-1 induced protein with a molecular weight of about 133,000 sedimented as a broad peak in the 25 S region of the gradient and cosedimented with 13 S DNA fragments. The sedimentation of both the protein and DNA decreased upon treatment of nuclear extracts with DNase. This result indicated that the protein and DNA were associated in deoxyribonucleoprotein complexes. The protein was identified as the HSV-encoded major DNA-binding protein ICP8 based on its molecular weight, its association with DNA in nuclear extracts, and its immunoprecipitation with monospecific antiserum and monoclonal antibody to ICP8. Deoxyribonucleoprotein complexes containing ICP8 could be immunoprecipitated from nuclear extracts. When DNA was extracted from these immunoprecipitates, fractionated by agarose gel electrophoresis, transferred to nitrocellulose paper, and hybridized to 32P-labeled HSV-1 or cell DNA, both HSV-1 and cell DNA sequences were identified. Cesium chloride gradient analysis of the immunoprecipitated DNA indicated that duplex DNA was present in the complexes. Thus, the major DNA-binding protein of HSV-1 is associated with both duplex HSV-1 and cell DNA in vivo.

Polyamine-mediated phosphorylation of a nucleolar protein from Physarum polycephalum that stimulates rRNA synthesis

An acidic nucleolar phosphoprotein with a subunit M(r) of 70,000 was purified as an apparent dimer of 139,000 from isolated nuclei of the slime mold Physarum polycephalum. The protein was purified without the aid of strong dissociating agents after its selective phosphorylation in isolated nuclei by a polyamine-mediated reaction. Its amino acid composition resembled that of a nucleolar phosphoprotein from Novikoff hepatoma ascites cells. The phosphoprotein stimulated rRNA synthesis 5-fold by RNA polymerase I within a nucleolar, ribosomal deoxyribonucleoprotein complex isolated from nucleoli of P. polycephalum. It was also identified as a component of the complex. It bound with high affinity and specificity to the palindromic ribosomal DNA of 38 x 10(6)M(r) from P. polycephalum, which contained two coding sequences for 5.8S, 19S, and 26S rRNA. It also bound to three fragments of ribosomal DNA of M(r) 21.2 x 10(6), 17.1 x 10(6), and 8.1 x 10(6), prepared by cleavage with restriction endonucleases HindIII, PstI, and BamHI, respectively. All of these fragments included the symmetry axis of the palindromic ribosomal DNA. The phosphoprotein that had been treated with alkaline phosphataseagarose to hydrolyze the phosphate groups did not stimulate transcription and did not bind to ribosomal DNA or to the restriction fragments indicated. We have thus isolated a specific phosphoprotein with the capacity to stimulate transcription of a specific set of genes in a eukaryote. These findings suggest that this phosphoprotein may specifically regulate functions of ribosomal DNA in a manner dependent on its degree of phosphorylation.

Isolation of a minichromosome containing the ribosomal genes from Physarum polycephalum

The ribosomal genes of Physarum polycephalum are located on linear DNA molecules (rDNA) of 38 x 10(6) daltons present in each nucleolus in 100--200 copies. The rDNA is not part of the large chromosomal DNA. These molecules have now been solubilized in the form of transcriptionally active deoxyribonucleoprotein complexes from nucleoli purified through Percoll gradients. The ribosomal chromatin (rDNP) sediments in sucrose gradients as a homogeneous fraction with an approximate sedimentation coefficient of 100 S. It contains alpha-amanitin-resistant RNA polymerase I activity and rDNA as its major (or sole) DNA component. Buoyant density analysis indicates that ribosomal chromatin contains, besides RNA polymerase I, significant amounts of additional protein. RNA synthesized in vitro by the endogenous RNA polymerase is complementary to rDNA and is transcribed mainly from the sequences coding for 19S and 26S ribosomal RNA.

Analysis of structural changes of deoxyribonucleoprotein complex under the action of prospidin

Analysis of structural changes of deoxyribonucleoprotein complex under the action of prospidin

Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei

Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure.

Differential binding of tritiated actinomycin to the nuclei of mammalian spermatogenic cells in vivo.

Summary. Chinese hamsters, Cricetulus griseus, were given a single injection of 2·0 μg [3H]actinomycin D directly into the left testis. For 24 hr after injection, the distribution of the radioisotope in the reproductive organs, blood plasma and liver was followed, using liquid scintillation counting techniques. After 24 hr, nearly 50% of the injected dose remained in the left testis while insignificant amounts of label were associated with the right testis. The left epididymis gradually accumulated radioisotope probably by intratubular transport from the left testis. The right epididymis possessed negligible amounts of label after 24 hr. Levels of the antibiotic in blood plasma and liver steadily decreased during the experimental period. Localization of [3H]actinomycin D within the spermatogenic cells of the seminiferous epithelium was determined by autoradiography. The results indicated that the antibiotic was differentially bound by various types of spermatogenic cells. Of the spermatocytes, Stage VIII leptotene nuclei (31·6 grains/nucleus) were much more heavily labelled compared to early Stage V pachytene nuclei (6·2 grains/nucleus). Actinomycin D was lightly bound to nuclei of Step 5 and Step 8 round spermatids (3·6 and 3·8 grains/nucleus, respectively). Elongating spermatid nuclei (Steps 9 to 14) were lightly labelled at best, whereas elongated spermatid nuclei (Steps 15 to 18) were very heavily grained. The label intensity decreased in Step-19 spermatids (spermiation step). Labelling variations among spermatogenic cells may be attributable to the physicochemical state of deoxyribonucleoprotein complexes in these cells.

Acridine orange as a supravital fluorochrome indicating varying degrees of chromatin condensation

When acridine orange is used at 10−5 or 10−6M concentrations in supravital preparations of mouse or grasshopper testicular cells, it is evident that condensed chromatin in spermatogenic cells displays considerably higher levels of fluorescence than extended chromatin in vesicular nuclei. This result differs from that obtained traditionally in appropriately fixed and stained cytological preparations in which fluorescence is greatest in extended chromatin and lowest in condensed chromatin (Rigler, 1966, and others). While an explanation for this reversed staining pattern in unfixed cells is not immediately available, it is clear that acridine orange may offer novel opportunities for the experimental manipulation and examination of the chromosomes (for deoxyribonucleoprotein complex) of living cells.