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PubMed1983

Isolation and characterization of polyoma nucleoprotein complexes.

A method for the isolation of polyoma nucleoprotein complexes has been developed using neuraminidase treatment of infected cell lysates. At least three distinct forms of polyoma virion intermediates were identified by their [3H]thymidine labeling kinetics and sedimentation coefficients: a rapidly labeled 95 S "replicating complex" which chases to a 75 S minichromosome and then to a 240 S virion structure. The general properties of these distinct intermediates were similar to those found for SV40. In contrast to SV40, however, a continuum of labeled polyoma viral DNA sedimented between 240 S and 95 S. These complexes were characterized by their release from cell debris with neuraminidase, precipitation with antivirion antibody, complete disruption in 1 M NaCl, and association with hemagglutinating (HA) activity. These intermediates may represent incremental capsid protein additions to the 75 S minichromosome, hypothesized in the current models for SV40 assembly. The ability to isolate a complete complement of polyoma subviral complexes provides a basis for studying the growth defect of polyoma host-range mutants, and the properties of neuraminidase release, hemagglutination, and specific immunoprecipitation suggest purification steps for further characterization of these virion assembly intermediates.

Animals↗

PubMed1973

Properties of nucleoprotein complexes containing replicating polyoma DNA.

Short-lived nucleoprotein complexes (r-py complex) containing replicating polyoma DNA were isolated from infected cells after lysis with Triton X-100. The Triton lysing procedure of Green, Miller, and Hendler (1971) releases most complexes containing supercoiled viral DNA (py complex) from nuclei, but liberates only a portion of r-py complexes. r-py Complexes are associated more strongly with nuclear sites but can be extracted by prolonged incubation of nuclei in lysing solution. Complexes containing replicating polyoma DNA appear to be precursors to stable complexes containing supercoiled DNA. Sedimentation and buoyant density studies indicate that protein is bound to both r-py complexes and py complexes at a ratio of protein to DNA of about 1 to 2/1. Both types of complexes sediment as if the viral DNA is more compact than free DNA and both undergo major reversible configurational changes with increased salt concentration. Changes resulting from enzymatic and chemical treatment indicate that there may be two or more protein components in both r-py complex and py complex. One component is digested by Pronase and trypsin while another is resistant to the enzymes but released by deoxycholate. The abundance and similarity in chemical and physical properties of protein bound to all forms of polyoma DNA suggest that part of the protein molecules may serve in a structural capacity.

Animals↗

PubMedDate not supplied

[Affinity chromatography of nucleoprotein complexes].

Affinity sorbent containing cellular membranes of the cerebral cortex was obtained by means of an inclusion into porous matrix of polyacrylonitrile. Sorption of model and natural nucleoprotein complexes on the obtained sorbent was studied by the method of affinity chromatography. Distribution coefficients were determined. Natural nucleoprotein complexes isolated from brain possess a greater selectivity of binding to the cellular membranes of the cerebral cortex than that from liver.

Acrylic Resins↗

PubMed1984

A comparison of methods for the extraction of nucleoprotein complexes from SV40-infected cells.

A comparison was made between two alternative methods for the nuclear extraction of Simian virus 40 (SV40) virions and nucleoprotein complexes (NPCs) from SV40-infected TC7 cells. The low-salt hypotonic method of Su and DePamphilis (1976) was compared with the detergent method of Garber et al. (1978), since other methods had been shown to result in virion breakdown. There was no disruption of mature SV40 virions with either of these extraction procedures. There was, however, considerably more effective extraction of SV40 NPCs, known to contain large tumor (T) antigen, using the low-salt hypotonic method as opposed to the detergent method. Thus, the low-salt hypotonic method for extraction should be the method of choice when studying SV40 DNA replication or the function of SV40 T antigen in SV40 nucleoprotein complexes.

Antigens, Viral, Tumor↗

PubMed1987

Changes in the DNase I sensitivity of DNA sequences within the yeast 2 micron plasmid nucleoprotein complex effected by plasmid-encoded products.

We examined the effect of plasmid-encoded gene products on two DNase-I-sensitive regions of DNA in the yeast 2 micron plasmid nucleoprotein complex. For these studies, each sensitive region was cloned into an appropriate vector, and the chimeric plasmids were transformed into yeast. Nucleoprotein complexes of the chimeric plasmids were partially purified and tested for sensitivity to DNase I digestion. One sensitive region is between the 3' end of the 2 micron plasmid coding region D and the plasmid REP3 locus. This region was more sensitive and exhibited a different cleavage pattern when purified from a yeast strain containing endogenous 2 micron plasmid copies than when purified from a yeast strain lacking plasmid copies. Examination of the effect of individual gene products and combinations of the various gene products revealed that the plasmid's REP1, REP2 and D loci were all necessary to restore the pattern to that found in the preparation containing endogenous 2 micron plasmid copies. The other sensitive region studied brackets the binding site of the plasmid-encoded FLP protein, which catalyzes site-specific recombination between the 2 micron plasmid's inverted repeated sequences. In contrast to the first sensitive region examined, the sensitive region in the inverted repeat was less sensitive in chimeric plasmids isolated from a yeast strain containing endogenous 2 micron plasmid copies than from one lacking endogenous copies. Presumably, this protection results from the binding of the FLP protein.

Base Sequence↗

PubMed1991

Transcription of a subset of human class II major histocompatibility complex genes is regulated by a nucleoprotein complex that contains c-fos or an antigenically related protein.

Transcriptional regulation of the human major histocompatibility complex class II genes requires at least two upstream elements, the X and Y boxes, located in the -50- to -150-base-pair region of all class II promoters. The DRA and DPB promoters contain phorbol ester-responsive elements overlapping the 3' side of their X boxes. Mutation of this sequence down-regulates the efficiency of the DRA promoter, suggesting that a positive regulator(s) binds to this site. In this report, anti-sense c-fos RNA and an anti-c-fos antibody were used to show that the product of the protooncogene c-fos or an antigenically related protein is a component of a complex that binds to the X box and is required for maximal transcription from the DRA and DPB promoters. As c-fos (or its related proteins) cannot bind alone to DNA, these results suggest that it may dimerize with other members of the JUN/AP-1 family, such as hXBP1, to participate in the activation of a subset of class II major histocompatibility complex genes.

Amino Acid Sequence↗

PubMed1996

Distamycin-induced inhibition of formation of a nucleoprotein complex between the terminase small subunit G1P and the non-encapsidated end (pacL site) of Bacillus subtilis bacteriophage SPP1.

The small subunit of the Bacillus subtilis bacteriophage SPP1 terminase (G1P) forms a sequence-specific nucleoprotein complex with the SPP1 non-encapsidated end (pacL site) during initiation of DNA encapsidation. Gel mobility shift assay was used to study the G1P-pacL interaction. Distamycin, a minor groove binder that induces local distortion of the DNA, inhibits G1P-pacL complex formation. The competition of G1P with distamycin for DNA binding at the pacL site is independent of the order of addition of the reactants. Other minor groove binders, such as spermine or Hoechst 33258, which do not distort DNA, failed to compete with G1P for pacL DNA binding. Cationic metals, which generate a repertoire of DNA structures different from that caused by the minor groove binders, can partially reverse the distamycin-induced inhibition of G1P binding to pacL DNA. The major groove binder methyl green, which does not distort sequence-directed bending of pacL DNA, competes with G1P for binding at the pacL site. Our data suggest that the natural sequence-directed bend that exists within the pacL site is the architectural element that facilitates assembly of a nucleoprotein complex and hence initiation of DNA encapsidation by bacteriophage SPP1.

Anti-Bacterial Agents↗

PubMed1991

Formation of several specific nucleoprotein complexes on the human cytomegalovirus immediate early enhancer.

The major immediate early enhancer of the human cytomegalovirus (HCMV) is composed of unique and repeated sequence motifs, which interact with different nuclear proteins, thus forming a large nucleoprotein complex. Using DNAase I protection analysis, we determined at the nucleotide level the interactions of B cell and HeLa cell nuclear proteins with transcription factor binding sites in the enhancer/promoter. In agreement with in vivo activity, protein binding to the 18 bp repeats (kappa B element) was found predominantly with B cell extract. Competition for proteins with individual transcription factor binding sites allowed us to define boundaries of closely spaced and overlapping binding sites, and to group binding proteins into several classes. Using gel mobility shift assays, we could show that proteins, which bind to the 17 bp repeat, also bind to a classical NF1 site. In addition, several novel binding sites were identified. The presence of overlapping binding sites, together with differences in the occupation of the 18 bp repeats in the two cell types, suggest that the HCMV major IE enhancer has several possibilities of forming nucleoprotein complexes.

B-Lymphocytes↗

PubMedDate not supplied

[Nucleoprotein complexes of yeast and their biological effect on T-lymphocytes].

Complexes of nucleic acids and acid nuclear proteins that are active toward human T-lymphocytes were isolated from cells of bakers' yeast Saccharomyces cerevisiae. The conditions of isolation of nucleoprotein complexes by acid extraction followed by microfiltration for concentration of macromolecular components were optimized. Gel filtration and electrophoresis were used to study the composition and molecular weights of components of the preparations obtained. It was shown that nucleoprotein complex had a molecular weight of 1430 kDa. However, only one zone was determined by electrophoresis of the protein component with a molecular weight of 30 kDa.

Fungal Proteins↗

PubMed1972

Biosynthetic properties of a polyoma nucleoprotein complex: evidence for replication sites.

Under normal growth conditions, all of the newly synthesized polyoma deoxyribonucleic acid (py DNA) that could be extracted from infected mouse cell cultures by the Triton procedure of Green, Miller, and Hendler was in the form of a 55S nucleoprotein complex. Inhibition of protein synthesis by cycloheximide reduced the sedimentation rate of the polyoma complex synthesized during the first hour after addition of the drug to 25 to 35S. Since the 55S and the 25 to 35S complexes each contain closed circular 20S py DNA, it is suggested that the slower complex contains less protein per DNA molecule and that there is normally a small or unstable pool of protein available for binding to newly replicated py DNA. In the presence of cycloheximide, the newly formed 25 to 35S complex was not derived from preexisting 55S complex. Thus, some py DNA which was not solubilized by the Triton method served as a template for replication. Further evidence for the existence of polyoma replication sites is provided by the demonstration that, during the inhibition of protein synthesis, a class of newly replicated py DNA can be solubilized by the sodium dodecyl sulfate procedure of Hirt, but not by the Triton method. It is postulated that continuous protein synthesis is required to release py DNA from replication sites in the form of a Triton-extractable nucleoprotein complex.

Animals↗

PubMed1975

[Dyserythropoietic anemia, type I: ineffective erythropoiesis due to disorders in the DNA-nucleoprotein complex].

The relative DNA, RNA histone and hemoglobin contents and 3H-thymidine incorporation in vitro were determined sequentially in individual erythroblasts of type I dyserythropoietic anemia. The histone/DNA ratio was increased due to a rise in histone extinction, indicating a pathological DNA-nucleoprotein complex. In addition, the erythroblasts displayed an increase in DNA content exceeding tetraploid values, a loss of DNA-synthesis activity at immature stages with low hemoglobin content, and a reduction of RNA. These variations were assumed to be caused by the preceding and primary impairment of the DNA-nucleoprotein complex.

Adult↗

PubMed1998

Lac and lambda repressors relieve silencing of the Escherichia coli bgl promoter. Activation by alteration of a repressing nucleoprotein complex.

The Escherichia coli bgl promoter is kept in a repressed state by silencer sequences which flank the promoter and by the histone-like protein H-NS. Silencing of the bgl promoter is likely due to the formation of a repressing nucleoprotein complex of which H-NS is an essential component. Here, we show that silencing is abolished by the binding of Lac or lambda repressors to their respective operators that were inserted within the bgl upstream silencer. Efficient activation of bgl operon transcription by Lac and lambda repressors was independent of the position and phasing of the operators with respect to the promoter. Activation by Lac and lambda repressors as shown here is unprecedented. We conclude that the activation of bgl transcription by both repressors is achieved by a novel mechanism, that is by alteration of the repressing nucleoprotein complex rather than by protein-protein interactions with RNA polymerase and the catabolite activator protein, CAP.

Bacterial Proteins↗

PubMed1998

Photoaffinity labeling as an approach to study supramolecular nucleoprotein complexes.

The modern approaches for studying the detailed structure of nucleoprotein complexes involved in replication and transcription, based on the use of nucleic acids with photoreactive groups incorporated into definite positions of polynucleotide chain, are considered. Methods of preparation of photoreactive nucleic acids of this type are presented. Their use for positioning of RNA polymerase III and transcription factors as well as of the main participants of the replication machinery at the respective templates is described. A survey of the data concerning the amino acid residues modified in the course of photoaffinity labeling of proteins is also presented and some complications are discussed.

Affinity Labels↗

PubMed1971

Nucleoprotein complexes in simian virus 40-infected cells.

When African green monkey kidney cells (BSC-1) were infected with simian virus 40 (SV40) and extracted with 0.25% Triton X-100 after exposure to (3)H-thymidine, the (3)H-SV40 deoxyribonucleic acid (DNA) was present in a form which had a sedimentation coefficient in sucrose gradients of 44S. The change from the sedimentation coefficient of purified SV40 DNA (21S) was shown to result from the association of the SV40 DNA in the Triton extracts with protein by means of sensitivity to Pronase digestion and labeling with (14)C-amino acids. Short-term labeling experiments with (3)H-thymidine demonstrated that SV40 DNA molecules in the course of replication (25S) were also present as nucleoprotein complexes in Triton-extracted material. Labeled DNA extracted with Triton in the form of nucleoprotein complexes was obtained in amounts which were quantitatively equivalent to the amounts extracted with deoxycholate in parallel experiments. This indicated that the newly synthesized pools of SV40 DNA may not occur as free DNA in the infected cell.

Amino Acids↗

PubMed1989

A nucleoprotein complex mediates the integration of retroviral DNA.

The integration of viral DNA into the host genome is an essential step in the retrovirus life cycle. To understand this process better, we have examined the native state of viral DNA in cells acutely infected by murine leukemia virus (MLV), using both a physical assay for viral DNA and a functional assay for integration activity (Brown et al. 1987). The viral DNA and integration activity copurify during velocity sedimentation, gel filtration, and density equilibrium centrifugation, indicating that viral DNA is in a large (approximately 160S) nucleoprotein complex that includes all functions required for integration activity in vitro. Analysis by immunoprecipitation shows that the viral capsid protein is part of the active nucleoprotein complex, but recognition of the complex by only a subset of anti-capsid sera implies that the protein is constrained conformationally. The viral DNA within this structure is accessible to nucleases; the effects of nucleases on the integrity of the complex suggest that the integration-competent particle is derived from and similar to the core of extracellular virions.

Animals↗

PubMed2004

Polypeptide components of telomere nucleoprotein complex.

Chromosome telomeres of humans and many model organisms contain a structure called a t-loop, which is maintained by TERF, TINF2, Pot1, and other proteins. Increase in TERF1 concentration prevents telomere elongation by telomerase. Decrease in TERF2 concentration (preventing t-loop formation) is accompanied by blockade of proliferation and appearance of other signs of cellular senescence in experiments. Natural regulation of TERF1 involves tankyrase, ATM protein kinase, and fluctuations of the protein level across a cell cycle. The telomere nucleoprotein complex also interacts with various polypeptide macromolecules (e.g., Sir2, PinX1, Rap1, Ku, Rad50/Mre11/Nbs1) responsible for heterochromatin formation, modulation of telomerase activity, DNA repair, and signaling to other cell compartments about telomere state. Study of structure and functioning of telomere nucleoprotein complex may contribute to elucidation of poorly understood mechanisms of aging and processes of tumor transformation of cells.

Animals↗

PubMed1998

Formation of large nucleoprotein complexes upon binding of the high-mobility-group (HMG) box B-domain of HMG1 protein to supercoiled DNA.

High-mobility group (HMG) 1 is a relatively highly abundant chromosomal protein with structural- rather than sequence-specific preference for binding to DNA. HMG1 has two highly related, folded domains A and B (HMG boxes), attached by a short basic region to an acidic C-terminal domain. We have studied binding of the B-domain of HMG1 protein and its mutants to supercoiled DNA by a gel-retardation assay and electron microscopy. Using a gel-retardation assay, we have demonstrated that HMG1 or HMG1 lacking the acidic C-terminal domain [i.e. HMG1(A+B) bi-domain], but not the isolated B-domain, could preferentially bind supercoiled over-relaxed closed circular or linear DNA. Mutational analysis of the HMG1 B-domain revealed that replacement of Lys96 of the extended N-terminal segment (and much less the neighboring Arg97) and Lys128 of helix II to glutamic acid severely impaired binding of the HMG box domain to supercoiled DNA. The latter mutation within helix II significantly decreased the alpha-helical content of the B-domain as revealed by circular dichroism. We have also shown that mutation of several residues within helix I of the B-domain, in particular Arg110, resulted in a diminished binding to supercoiled DNA as revealed by intensive smearing and reduced retardation of the protein/DNA complexes. These findings indicated that the extended N-terminus, helix I and helix II of the HMG1 B-domain are likely in contact with DNA. Electron microscopy revealed that the B-domain could bind to supercoiled DNA at higher HMG/DNA molar ratios as oligomeric protein beads with subsequent association of the beads into large nucleoprotein complexes from which many looped DNA molecules emerged. Most of the introduced mutations within all three helices of the B-domain (involving mainly basic and aromatic residues) abolished formation of the large nucleoprotein complexes even though the binding of the HMG box to supercoiled DNA was retained as revealed by a gel-retardation assay. A model for the interaction of the B-domain of HMG 1 with supercoiled DNA is presented and discussed.

Amino Acid Sequence↗

PubMed1998

Involvement of HIV-I nucleocapsid protein in the recruitment of reverse transcriptase into nucleoprotein complexes formed in vitro.

Retroviral reverse transcription takes place within the virion core, where nucleocapsid (NC) protein (NCp) molecules cover the dimeric RNA genome. NCp thus has structural roles in the virion core but is also extensively involved in viral DNA synthesis and virion assembly. To further characterize the role of human immunodeficiency virus type 1 NCp7 during replication of the viral genome, we investigated the relationship between NCp7 and reverse transcriptase (RT) either directly or within nucleoprotein complexes in vitro. We show that NCp7 interacts directly with RT and enhances synthesis of full-length cDNA by increasing RT processivity. Using NCp7 mutants, we show that the complete amino acid sequence of NCp7 is required for functional interactions with RT. Our results suggest that NCp7 plays a role in recruitment of RT into stable and functional nucleoprotein complexes during viral DNA synthesis.

Amino Acid Sequence↗