SEARCH · PubMed Health

Results for “nucleoprotein complex”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 631 records · Page 35Linked to original sources

PubMed2001

Effect of mutations in the Mu-host junction region on transpososome assembly.

Mu transposition occurs through a series of higher-order nucleoprotein complexes called transpososomes. The region where the Mu DNA joins the host DNA plays an integral role in the assembly of these transpososomes. We have created a series of point mutations at the Mu-host junction and characterized their effect on the Mu in vitro strand transfer reaction. Analysis of these mutant constructs revealed an inhibition in transpososome assembly at the point in the reaction pathway when the junction region is engaged by the transposase active site (i.e. the transition from LER to type 0). We found that the degree of inhibition was dependent upon the particular base-pair change at each position and whether the substitution occurred at the left or right transposon end. The MuB transposition protein, an allosteric effector of MuA, was shown to suppress all of the inhibitory Mu-host junction mutants. Most of the mutant constructs were also suppressed, to varying degrees, by the substitution of Mg(2+) with Mn(2+). Analysis of the mutant constructs has revealed hierarchical nucleotide preferences at positions -1 through +3 for transpososome assembly and suggests the possibility that specific metal ion-DNA base interactions are involved in DNA recognition and transpososome assembly.

Attachment Sites, Microbiological↗

PubMed1997

Chromatin studies by DNA-protein cross-linking.

Our current level of understanding of chromatin structure was to a large extent achieved with the help of DNA-protein cross-linking. The versatile inventory of cross-linking techniques allows the identification of the contacts between DNA and proteins with a single nucleotide-single amino acid precision, to detect minor components of the complex nucleoprotein systems, to reveal the interactions of the flexible protein domains with DNA, and to assay for conformational changes in the nucleosomes.

Animals↗

PubMed1998

Unwinding of the Escherichia coli origin of replication (oriC) can occur in the absence of initiation proteins but is stabilized by DnaA and histone-like proteins IHF or HU.

The unwinding of the origin of replication (oriC) is a critical step in initiation of DNA replication in Escherichia coli. Previous observations indicate that efficient unwinding of supercoiled plasmid templates containing oriC sequences requires the DnaA initiation protein and one or more accessory factors. The precise contribution of each protein to this process is unknown. Here, we demonstrate that unwinding can occur under physiological conditions at the same bases in oriC, in either the presence or the absence of initiation proteins, as detected by a single-strand specific nuclease, P1. This suggests that oriC unwinding is a spontaneous event determined solely by DNA sequence. DnaA and IHF, as part of a large nucleoprotein complex, may function to stabilize the DNA strand opening prior to initiation of DNA replication.

Adenine↗

PubMed2003

Biochemical characterization of bacteriophage lambda genome packaging in vitro.

Bacteriophage lambda has been extensively studied, and the abundance of genetic and biochemical information available makes this an ideal model system to study virus DNA packaging at the molecular level. Limited in vitro packaging efficiency has hampered progress toward this end, however. It has been suggested that limited packaging efficiency is related to poor activity of purified procapsids. We describe the construction of a vector that expresses lambda procapsids with a yield that is 40-fold greater than existing systems. Consistent with previous studies, packaging of a mature lambda genome is very inefficient in vitro, with only 4% of the input procapsids utilized. Concatemeric DNA is the preferred packaging substrate in vivo, and procapsids interact with a nucleoprotein complex known as complex I to initiate genome packaging. When complex I is used as a packaging substrate in vitro, capsid utilization is extremely efficient, and 40% of the input DNA is packaged. Finally, we provide evidence for a packaging-stimulated ATPase activity, and kinetically characterize this reaction quantifying the energetic cost of DNA packaging in bacteriophage lambda.

Adenosine Triphosphatases↗

PubMed2005

The role of the cytoskeleton during viral infection.

Upon infection, virions or subviral nucleoprotein complexes are transported from the cell surface to the site of viral transcription and replication. During viral egress, particles containing viral proteins and nucleic acids again move from the site of their synthesis to that of virus assembly and further to the plasma membrane. Because free diffusion of molecules larger than 500 kDa is restricted in the cytoplasm, viruses as well as cellular organelles employ active, energy-consuming enzymes for directed transport. This is particularly evident in the case of neurotropic viruses that travel long distances in the axon during retrograde or anterograde transport. Viruses use two strategies for intracellular transport: Viral components either hijack the cytoplasmic membrane traffic or they interact directly with the cytoskeletal transport machinery. In this review we describe how viruses--particularly members of the Herpesviridae, Adenoviridae, Parvoviridae, Poxviridae, and Baculoviridae--make use of the microtubule and the actin cytoskeleton. Analysing the underlying principles of viral cytosolic transport will be helpful in the design of viral vectors to be used in research as well as human gene therapy, and in the identification of new antiviral target molecules.

Animals↗

PubMed2006

Strategies to identify potential therapeutic target sites in RNA.

Antisense agents are powerful tools to inhibit gene expression in a sequence-specific manner. They are used for functional genomics, as diagnostic tools and for therapeutic purposes. Three classes of antisense agents can be distinguished by their mode of action: single-stranded antisense oligodeoxynucleotides; catalytic active RNA/DNA such as ribozymes, DNA- or locked nucleic acid (LNA)zymes; and small interfering RNA molecules known as siRNA. The selection of target sites in highly structured RNA molecules is crucial for their successful application. This is a difficult task, since RNA is assembled into nucleoprotein complexes and forms stable secondary structures in vivo, rendering most of the molecule inaccessible to intermolecular base pairing with complementary nucleic acids. In this review, we discuss several selection strategies to identify potential target sites in RNA molecules. In particular, we focus on combinatorial library approaches that allow high throughput screening of sequences for the design of antisense agents.

Animals↗

PubMed1997

Nucleic acid transport in plant-pathogen interactions.

Inter- and intracellular transport of nucleic acids during plant-pathogen interaction is described on the examples of cell-to-cell movement of plant viruses and nuclear import of Agrobacterium T-DNA. In both cases, the transport process is mediated by specialized proteins produced by the pathogen. Plant virus movement occurs through the intercellular connections, plasmodesmata. In this process, the viral genomic nucleic acid is bound by virus-encoded movement protein. The nucleoprotein complex is then targeted to plasmodesmata, potentially via interaction with the host cell cytoskeleton. Prior to translocation, the plasmodesmal channel is dilated by the movement of protein. Nuclear import of Agrobacterium T-DNA is also mediated by bacterial proteins associated with the transported nucleic acid molecule. Specifically, the VirD2 and VirE2 proteins complex with the transferred DNA, providing it with the nuclear localization signals (NLSs). The VirD2 NLS is an evolutionarily conserved signal, active both in plant and animal cells. In contrast, the VirE2 NLS is plant-specific. Both VirD2 and VirE2 NLSs most likely interact with the plant cell nuclear import machinery to initiate the transport process.

Bacterial Proteins↗

PubMed1994

A genetic approach to the identification of functional amino acids in protein p6 of Bacillus subtilis phage phi 29.

Protein p6 of the Bacillus subtilis phage phi 29 is essential for in vivo viral DNA replication. This protein activates the initiation of phi 29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. We report on the development of an in vivo functional assay for protein p6. This assay is based on the ability of protein p6-producing B. subtilis non-suppressor (su) cells to support growth of a phi 29 sus6 mutant phage. We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region. The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein. These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6. Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.

Bacillus Phages↗

PubMed1980

Organization of chromatin during spermiogenesis: beaded fibers, partly beaded fibers, and loss of nucleosomal structure.

The aggregation of chromatin during spermiogenesis in the house cricket and many other animals is an orderly process involving the formation of a series of long, thick, well defined structures. The differentiation of chromatin preliminary to the development of such unusual structures is given attention here. Examination of nuclei after lysis and spreading indicated that fibers with closely spaced nucleosomes, like the fibers of somatic chromatin, make up the chromatin in all stages of early spermiogenesis and most of middle spermiogenesis. The thick structures of late spermatids cannot be formed by aggregation of fibers of this somatic type, however; just before thick structures form, chromatin fibers lose the nucleosomal structure. During the process, fibers with nucleosomes spaced at irregular intervals and with long stretches of smooth thin fiber are found, as if nucleosomes at one site on a fiber are broken down independently of those at adjacent sites. Since prior studies of cricket proteins have indicated that somatic histones persist during the stages when nucleosome structure disappears, the observations imply that the histones which are organized in nucleosomes during early stages must become incorporated into different kinds of nucleoprotein complexes during succeeding stages of spermiogenesis.

Animals↗

PubMed1996

Features of autoantigens.

The major cellular antigens recognized by autoantibodies in SLE and other systemic autoimmune diseases have been identified and characterized over the past 25 years. The pioneering studies of Eng Tan demonstrate the importance of autoantibodies as diagnostic markers. However, why certain autoantibodies, such as anti-Sm, are pathognomonic of SLE, while others are markers of other autoimmune disease subsets, remains unanswered. This central question continues to drive much current research into the pathogenesis of SLE. Features of the autoantigens recognized by autoantibodies may provide important clues to the causes of lupus. Most autoantigens in systemic autoimmunity are multicomponent nucleoprotein complexes. These particles are encountered by the immune system as units, resulting in the tandem production of autoantibodies recognizing several components of the same complex. However, the intermolecular-intrastructural spreading of autoimmunity is regulated by mechanisms that at present are defined poorly. Also unexplained is the observation that the antigenic determinants recognized by autoantibodies are restricted and frequently correspond to active sites or functional domains. Analysis of experimental models of autoimmunity suggests that altering the structure of autoantigens, due to abnormal protein-protein interactions, hapten binding, altered degradation, or other mechanisms, could help to explain both the restricted patterns of autoantibody spreading and the selective targeting of antigenic sites. This may be a worthwhile area for further investigation of the pathogenesis of systemic autoimmune diseases.

Animals↗

PubMed1986

Nuclear differentiation and ultimate fate during epidermal keratinization. Two-wavelength and cytofluorometric DNA investigations completed by computerized scanning image analysis.

Quantitative DNA cytophotometric investigations were performed to clarify some aspects of the differentiation and fate of nuclei in bovine snout and human epidermis representing various sites and different degrees of keratinization. We elaborated optimal conditions for hydrolysis and Feulgen staining. Diverse cytophotometric techniques, including computerized scanning cytophotometry and image analysis were applied. This approach provided the first quantitative data concerning changes of nucleotype during soft keratinization. Cytophotometric DNA measurements provide evidence for a continuous decline of nuclear DNA content from immediately beyond the basal layer to the transition zone. The overall loss of DNA is an orderly process that intensifies gradually and culminates in the stratum granulosum. Gradual nuclear degeneration, however, is not a general phenomenon, and a significant number of nuclei retains a DNA content within the diploid limits throughout the entire stratum spinosum and part of the stratum granulosum. At any level of differentiation or decay, residual nucleoprotein complexes remain intact, as judged from their resistance to acid hydrolysis. Karyological features change completely during keratinization. Basal cell nuclei are rather compact, ellipsoid and heterochromatic. Beyond the basal layer, nuclei enlarge, round up and obviously evolve to an extremely euchromatic state, with preferential localization of the dispersed heterochromatic clumps at the more peripheral sites. In the upper stratum spinosum, nuclei undergo even more drastic changes: nuclear area and volume shrink, nuclei partially regain the ellipsoid shape and revert to heterochromasia. Nevertheless, euchromatin remains the major constituent of decaying nuclei. Terminal differentiation stages, except in human sole, are marked by heterochromatin clumping. In human sole, persistence or even progression of heterochromatin dispersion is observed. Heterochromatic dots are situated along the nuclear membrane in human terminal keratinocytes, but are almost randomly distributed in bovine stratum granulosum nuclei. Finally, nuclear contrast analysis partially reveals statistically significant changes throughout keratinization.

Animals↗

PubMed1979

A model of translational control involving mRNA-associated proteins in chick embryonic muscles.

Highly purified poly(A)-containing free and polysomal mRNP particles have been isolated by chromatography of subcellular fractions of chick embryonic muscles on oligo-dT-cellulose and elution with low salt buffer at 45 degrees. The free and polysomal mRNP represent two distinct classes of macromolecules, the free particles having a more complex nucleoprotein organization than the polysomal particles. Comparison of the protein moieties of three classes of poly(A)-containing cytoplasmic mRNP -- those released from nuclei after in vitro transcription and processing (transported mRNP), the free, and polysomal mRNP -- strongly suggests that the majority of the mRNA-associated proteins are exchanged in the cytoplasm during the various functional states of mRNA. A model of translational control involving the participation of mRNA-associated proteins in chick embryonic muscles and by analogy in other differentiated eukaryotic cells is proposed.

Animals↗

PubMed1993

The glucocorticoid receptor precludes the binding of a transcriptional repressor protein to the long terminal repeat of the mouse mammary tumor virus.

The long terminal repeat (LTR) of the mouse mammary tumor virus was used as a template to examine the dual binding parameters of the glucocorticoid-receptor (GR) and a repressor protein termed Inhibitory Factor 1 (IF1). The receptor binds specifically to the glucocorticoid response element and precludes the binding of IF1 to its juxtaposed binding site within the LTR. When the two DNA targets are separated by the insertion of an additional 52 base pairs, coincident binding of both proteins is observed. Gel retention assays reveal three distinct nucleoprotein complexes. The first complex consists of the receptor and the LTR, the second is comprised of IF1 and DNA and the third is a multiprotein-DNA complex consisting of the GR, IF1 and DNA, migrating at a higher molecular weight position. The inhibition of IF1 binding by the presence of prebound GR leads to the repression of transcription of juxtaposed genes. The GR may act to block access of a sequence, used by the cell to titrate repressor proteins and facilitate the onset of gene expression.

Animals↗

PubMed1996

Nuclear organization and transcriptional silencing in yeast.

Transcriptional repression at the yeast silent mating type loci requires the formation of a nucleoprotein complex at specific cis-acting elements called silencers, which in turn promotes the binding of a histone-associated Sir-protein complex to adjacent chromatin. A similar mechanism of long-range transcriptional repression appears to function near telomeric repeat sequences, where it has been demonstrated that Sir3p is a limiting factor for the propagation of silencing. A combined immunofluorescence/in situ hybridization method for budding yeast was developed that maintains the three-dimensional structure of the nucleus. In wild-type cells the immunostaining of Sir3p, Sir4p and Rap1 colocalizes with Y' subtelomeric sequences detected by in situ hybridization. All three antigens and the subtelomeric in situ hybridization signals are clustered in foci, which are often adjacent to, but not coincident with, nuclear pores. This colocalization of Rap1, Sir3p and Sir4p with telomeres is lost in sir mutants, and also when Sir4p is overexpressed. To test whether the natural positioning of the two HM loci, located roughly 10 and 25 kb from the ends of chromosome III, is important for silencer function, a reporter gene flanked by wild-type silencer elements was integrated at various internal sites on other yeast chromosomes. We find that integration at internal loci situated far from telomeres abrogates the ability of silencers to repress the reporter gene. Silencing can be restored by creation of a telomere at 13 kb from the reporter construct, or by insertion of 340 bp of yeast telomeric repeat sequence at this site without chromosomal truncation. Elevation of the internal nuclear pools of Sir1p, Sir3p and Sir4p can relieve the lack of repression at the LYS2 locus in an additive manner, suggesting that in wild-type cells silencer function is facilitated by its juxtaposition to a pool of highly concentrated Sir proteins, such as those created by telomere clustering.

Cell Nucleus↗

PubMed1994

Identification of multiple B-cell transcriptional repressor elements in S mu-C mu intron of mouse IgH chain locus.

The S mu-C mu intron of the IgH chain locus is conserved in rodents, but its biological function is unknown. It has been shown that switch recombination breakpoints are concentrated within the repetitive sequences in the S mu region in mitogen-activated normal B cells. In Ig-secreting hybridomas these breakpoints occur most frequently at the most 5' end, and immediately upstream of the S mu DNA. The S mu-C mu intron appears remarkably protected from recombination. Because the nucleoprotein complexes that drive transcription and recombination may overlap, the transcriptional characteristics of this fragment were studied. The cis-acting regulatory elements in the S mu-C mu intron were identified by ligating the entire intron, or a series of subfragments to the TK promoter and bacterial chloramphenicol acetyltransferase gene. Expression of these constructs was tested in activated B cells and the nonlymphoid cell lines HeLa and HepG2. The complete S mu-C mu intron (1 kb) had a negative effect on TK promoter activity in activated B cells only when placed upstream of the promoter, in both orientations. Segmentation of the S mu-C mu intron has revealed that this region contains multiple negative elements active in B cells. A subfragment located at the 3' end of the S mu-C mu intron contains a B-cell-specific negative element, while the subfragment located at the 5'end has cell-type-independent repressing activity.

Animals↗

PubMed1992

Structural and functional studies on phi 29 DNA polymerase.

The Bacillus subtilis phage phi 29 DNA polymerase, involved in protein-primed viral DNA replication, contains several amino acid consensus sequences common to other eukaryotic-type DNA polymerases. Using site-directed mutagenesis, we have studied the functional significance of a C-terminal conserved region, represented by the Lys-X-Tyr ("K-Y") motif. Single point mutants have been constructed and the corresponding proteins have been overproduced and characterized. Measurements of the activity of the mutant proteins indicated that the invariant Lys and Tyr residues play a critical role in DNA polymerization. Interestingly, substitution of the invariant Lys either by Arg or Thr, produced enzymes with an increased or a largely reduced, respectively, capability to use a protein as primer, an intrinsic property of TP-priming DNA polymerases. On the other hand, the viral protein p6, which stimulates initiation of phi 29 DNA replication by formation of a nucleoprotein complex at both DNA replication origins, increased (about 5-fold) the insertion fidelity of phi 29 DNA polymerase during the formation of the TP-dAMP initiation complex. We propose a model in which the special strategy to maintain the integrity of the phi 29 DNA ends, by means of a "sliding-back" mechanism, could also contribute to increase the fidelity of phi 29 DNA replication.

Amino Acid Sequence↗

PubMed2006

Regulation of cytokine transcription in the context of chromatin.

Understanding the transcriptional regulation of an important class of innate and adaptive immune system effector molecules, the cytokines, is increasingly important given the promise cytokine regulation holds for treating various autoimmune and inflammatory diseases. Studies defining the mechanisms regulating cytokine transcription initially focused on identifying the cis-acting elements and trans-acting factors that activate cytokine promoters and enhancers. In the past, these studies were largely completed in the absence of constraints instituted by cellular chromatin. Over the past decade it has become obvious that changes in chromatin accessibility critically control, rather than simply correlate with, the transcriptional activation of most genes, including cytokines. Hence candidate transcriptional activators are being re-evaluated for potency in the context of cellular chromatin. Several distinct mechanisms for manipulating the generally repressive context of chromatin have been identified for cytokine genes. Most recently, single nucleotide polymorphisms in cytokine transcriptional regulatory elements have been shown to play measurable roles in regulating cytokine levels in the context of naturally selected haplotypes. Overall, subtle differences in DNA sequence and nucleoprotein complex composition, including protein post-translational modification, come together in cell type-specific combinations to explain the normal variation in cytokine transcription throughout the human populace.

Animals↗

PubMed2005

The telomerase cycle: normal and pathological aspects.

Telomeres are nucleoprotein complexes that cap the end of eukaryotic chromosomes and are essential for their function and stability. Telomerase, a reverse transcriptase that extends the single-stranded G-rich 3' protruding ends of chromosomes, stabilizes telomere length in germ line cells and regenerative tissues as well as in tumor cells. In the absence of telomerase telomeres shorten with cell division, a process able to trigger cell growth arrest. When telomerase is present in the cell, its activity is tightly regulated at its site of action by factors specifically bound to the telomeric DNA. Recent data indicate that telomeres reorganize during the cell cycle. This review summarizes our current knowledge on how telomeres are dynamically organized and remodeled during cell cycle and stress response, pointing out the conservation and the difference between yeast and human. We then focus on the cellular consequences of telomere modifications in normal and cancer cells. This leads to a discussion of the different roles, seemingly contradictory, of telomeres and telomerase during the initiation and the progression of a cancer.

Animals↗