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PubMed1988

Calcium as a modulator of the immediate-early gene cascade in neurons.

Increases in intraneuronal free calcium result in the rapid, transient, induction of the fos and jun proto-oncogenes. In PC12 cells, induction may be elicited either by membrane depolarization or by direct activation of voltage-dependent calcium channels with BAY K 8644 both of which provoke an influx of calcium. The calmodulin pathway appears to link the elevated intracellular calcium to gene induction. In the brain, c-fos and c-jun may be induced by elevated neuronal activity such as occurs during pentylenetetrazole (PTZ) seizures. The N-methyl-D-aspartate (NMDA) form of the glutamate receptor, which can directly gate calcium, plays a role in the induction of c-fos expression in PTZ seizures. In addition, NMDA can directly stimulate c-fos in the brain. Fos and Jun form a noncovalent nucleoprotein complex that binds to the consensus recognition sequence of the AP-1 transcription factor. Thus in a larger picture we envisage Fos and Jun as members of a concerted stimulus-transcription coupling pathway that links alterations in external stimuli to long term adaptive responses. In this context Fos, Jun and the other immediate-early genes should be viewed as third messengers which are regulated by second messengers such as intracellular calcium.

Animals↗

PubMed1986

Synthesis of simian rotavirus SA11 double-stranded RNA in a cell-free system.

A cell-free system was developed to study the replication of simian rotavirus SA11. The components of the system included (i) subviral particles prepared from infected cells to template the synthesis of viral RNA and (ii) an mRNA-dependent rabbit reticulocyte lysate to support protein synthesis. Based upon nuclease-sensitivity, approximately 20% of the RNA made in vitro was double-stranded (dsRNA) and 80% single-stranded (ssRNA). Electrophoretic analysis of the RNA products on polyacrylamide and low pH agarose gels showed that the system supported the synthesis of 11 dsRNAs and 11 positive-sense ssRNAs that corresponded in size to authentic viral RNAs. The synthesis of dsRNA in vitro was determined to be an asymmetrical process in which a nuclease-sensitive positive-strand RNA acted as a template for the synthesis of negative-strand RNA. The system also supported the initiation of negative-strand RNA using exogenous viral positive-strand RNA as a template. Finally, analysis of subviral particles recovered from reactions suggested that viral proteins made in vitro assembled into nucleoprotein complexes which were similar to those present in infected cells. Together, these results indicate that the cell-free system supported rotavirus RNA replication, transcription and the assembly of subviral particles.

Animals↗

PubMed1992

DNA inversions in phages and bacteria.

In certain phages and bacteria, there is a recombination system that specifically promotes the inversion of a DNA fragment. These inversion events appear to act as genetic switches allowing the alternate expression of different sets of genes which in general code for surface proteins. The mechanism of inversion in one class of inversion systems (Gin/Hin) has been studied in detail. It involves the formation of a highly specific nucleoprotein complex in which not only the two recombination sites and the DNA invertase participate but also a recombinational enhancer to which the DNA-bending protein Fis is bound.

Bacteria↗

PubMed1987

Isolation and characterization of DNA from the plasma of cancer patients.

UNLABELLED: Ten out of 37 patients with advanced malignant diseases were found to have extractable amounts of DNA in their plasma whereas no DNA could be detected in 50 normal controls. After its purification from the original nucleoprotein complex, DNA plasma levels ranging from 0.15 to 12 micrograms/ml were measured, the lowest concentration detectable with our method being 0.1 microgram/ml. Knowing from recovery experiments performed with 32P-DNA that the loss of DNA during the extraction procedure is about 65%, the real concentration of DNA in the plasma corresponds to about 3 times the given figures. The purified DNA was shown to be double-stranded and composed of fractions ranging from 21 kb to less than 0.5 kb, as determined by agarose gel electrophoresis. All these fractions hybridized with a 32P-labelled human DNA probe indicating the human origin of the bulk of the circulating DNA. IN CONCLUSION: the finding of extractable amounts of DNA in the plasma of 27% of the investigated cancer patients, and its absence from the controls, suggests some correlation with malignancy.

DNA, Neoplasm↗

PubMed1996

Electric birefringence of polytetrafluoroethylene particles in agarose gels.

Electric birefringence studies of strongly elongated, rod-like particles of polytetrafluoroethylene (PTFE) in agarose gels show that the negative effect observed by semi-diluted aqueous suspensions at low frequencies and at low electric field strengths (the so called "anomaly') disappears. The absolute value of the low frequency effect increases 3-4 times and the amplitude of modulation decreases faster compared to that of the suspensions. This together with decreased decay relaxation times in gels make the possibilty that the PTFE particles orientation in gels is not due to dipolar but to electrophoretic orientation mechanism quite probable. Similar change in the orientation mechanism could be expected also for suspensions of higher concentrations. The further elucidation of the orientation mechanism using fractions with lower polydispersity, broader ranges of experimental conditions (particle concentration, ionic strength and composition, electric field strengths, frequencies, etc.) could be interest for several fields: colloid electro-optics and especially that of concentrated colloids, pulsed field gel electrophoresis of DNA (and especially its sinusoidal biased field variant) and of nucleoprotein complexes and for the gel research.

Journal Article↗

PubMedDate not supplied

The control of puffing by ions - the Kroeger hypothesis: a critical review.

We have re-examined the several papers which appear to us to represent the principal lines of evidence for what we call the Kroeger hypothesis. To do this we have stated this hypothesis in its simplest, most concrete form, a form that has been repeatedly and forceably enunciated in the literature (Kroeger, 1963a, 1965, 1966, 1967, 1968; Kroeger and Lezzi 1966; Lezzi and Frigg, 1971). The evidence suggests to us that ecdysone's effect on puffing is probably not mediated by the [K+]/[Na+]. While such a model cannot, even now, be excluded, we see little reason to believe in it. We take the general issues raised by Kroeger's ideas very seriously. Nucleoprotein complexes are exquisitely sensitive to changes in salt concentration and ionic selectivity is a well-known property of proteins (and of ion-exchangers in general, see, for example, Diamond and Wright, 1969). Thus it might not be shocking if cells utilized this specificity in some general control over chromsosome structure, perhaps a second-layer of control superimposed upon other transcriptional controls. Therefore it is our feeling that Kroeger's data merits very careful and critical study, the more so because the experiments involved are intrinsically difficult. It is in this vein that we have tried to review Kroeger's data.

Animals↗

PubMed1995

The role of estrogen receptor in modulation of chromatin conformation in the 5' flanking region of the rat prolactin gene.

To determine whether the estrogen receptor (ER) has a role in the modification of chromatin structure, we developed cell lines to model discrete stages in the estrogen response. Each cell line carries a population of stably expressed papillomavirus-based minichromosomes containing the 5' flanking region of the rat prolactin gene. We examined ER effects at the distal enhancer domain of the rat prolactin promoter, using DNaseI to probe for alterations of the nucleoprotein complex. Within 1 h after the start of estrogen treatment, modifications in the chromatin state of the distal enhancer region were detected in a pituitary-derived, permissive cell line (GH3G1J). In rat-1 fibroblast cell lines that maintain the same stably expressed papillomavirus-based minichromosomes in the absence of ER or pituitary-specific transcription factors (Rat-1.2A2; non-permissive), no estrogen-induced modifications in the chromatin state were detected at 1 or 24 h. In rat-1 fibroblast cell lines that also contained ectopically expressed, functional ER (Rat-1 + ER.8A1), no estrogen-induced modifications in the chromatin state were detected at 1 h, but a 24 h a specific modification in the local structure was induced. These data support a model in which the ER interacts with chromatin to modify local structure in such a way as to induce a permissive state for interactions of transcription factors necessary for hormone-induced activation of gene transcription.

Animals↗

PubMed1993

Tyr26 and Phe73 are essential for full biological activity of the Fd gene 5 protein.

Using site-saturation mutagenesis, we have established all possible amino acid substitutions at Tyr26 and Phe73 that are compatible with biological activity of the gene 5 protein in vivo. No substitutions were found at either site that gave rise to a fully functional gene 5 protein, indicating that these two amino acid residues are crucial. However, partial activity was found if either residue was replaced by another aromatic amino acid (Y26F, Y26W, F73Y, F73W). The results suggest that both Tyr26 and Phe73 are important for base stacking in the nucleoprotein complex. The functional consequences of the removal of the hydroxyl group from Tyr26 argue that this residue may, in addition, be involved in hydrogen bond formation to confer greater stability on the complex. In contrast, the addition of such a group to Phe73 reduces activity.

Amino Acid Sequence↗

PubMed1989

Poly(A), poly(A) binding protein and the regulation of mRNA stability.

This review has focused on the possibility that interactions between mRNA sequences and the poly(A)-nucleoprotein complex play important roles in mRNA turnover. It is important to stress that additional genetic and biochemical tests are necessary to characterize how PABP interacts with mRNA in cells and to determine whether the poly(A) protection hypothesis is accurate. Moreover, there may be a significant number of mRNAs whose half-lives are independent of polyadenylation. For example, the stabilities of poly(A)-containing and deadenylated alpha 2u-globulin and interferon mRNAs are similar in microinjected oocytes. Thus, an important challenge in this field will be to analyse the complex and interactive factors that determine the half-lives of specific mRNAs.

Carrier Proteins↗

PubMed1990

Bending and supercoiling of DNA at the attachment site of bacteriophage lambda.

Integration of the DNA of bacteriophage lambda into the chromosome of E. coli depends on the formation of a complex nucleoprotein array at a specific locus on the phage genome, the attachment site. Recent work shows how bending of this DNA (induced by a specific DNA-binding protein), and strain in this DNA (induced by supercoiling) contribute to the formation of the nucleoprotein structure. Further, there are new insights into the way this structure directs critical events during recombination.

Attachment Sites, Microbiological↗

PubMed2006

Chromatin structure and epigenetics.

In eukaryotic cells, the DNA molecule is found in the form of a nucleoprotein complex named chromatin. The basic unit of the chromatin is the nucleosome, which comprises 147 base pairs of DNA wrapped around an octamer of core histones (made of two molecules of each H2A, H2B, H3, and H4 histones). Each nucleosome is linked to the next by small segments of linker DNA. Most chromatin is further condensated by winding in a polynucleosome fibre, which may be stabilized through the binding of histone H1 to each nucleosome and to the linker DNA. The modulation of the structure of the chromatin fibre is critical for the regulation of gene expression since it determines the accessibility and the sequential recruitment of regulatory factors to the underlying DNA. Depending on the different transcriptional states, the structure of the chromatin may be altered in its constituents (e.g. the presence of repressors, activators, chromatin remodelling complexes, and/or incorporation of histone variants), and in covalent modifications of its constituents (such as DNA methylation at cytosine residues, and posttranslational modifications of histone tails). Here, we give an overview of the molecular mechanisms involved in chromatin regulation and the epigenetic transmission of its state, both in normal and pathological scenarios.

Animals↗

PubMed2004

The Bacillus subtilis DnaD protein: a putative link between DNA remodeling and initiation of DNA replication.

The Bacillus subtilis DnaD protein is an essential protein and a component of the oriC and PriA primosomal cascades, which are responsible for loading the main replicative ring helicase DnaC onto DNA. We present evidence that DnaD also has a global DNA architectural activity, assembling into large nucleoprotein complexes on a plasmid and counteracting plasmid compaction in a manner analogous to that recently seen for the histone-like Escherichia coli HU proteins. This DNA-remodeling role may be an essential function for initiation of DNA replication in the Gram +ve B. subtilis, thus highlighting DnaD as the link between bacterial nucleoid reorganization and initiation of DNA replication.

Bacillus subtilis↗

PubMed2003

DNA binding specificity of the replication initiator protein, DnaA from Helicobacter pylori.

The key protein in the initiation of Helicobacter pylori chromosome replication, DnaA, has been characterized. The amount of the DnaA protein was estimated to be approximately 3000 molecules per single cell; a large part of the protein was found in the inner membrane. The H.pylori DnaA protein has been analysed using in vitro (gel retardation assay and surface plasmon resonance (SPR)) as well as in silico (comparative computer modeling) studies. DnaA binds a single DnaA box as a monomer, while binding to the fragment containing several DnaA box motifs, the oriC region, leads to the formation of high molecular mass nucleoprotein complexes. In comparison with the Escherichia coli DnaA, the H.pylori DnaA protein exhibits lower DNA-binding specificity; however, it prefers oriC over non-box DNA fragments. As determined by gel retardation techniques, the H.pylori DnaA binds with a moderate level of affinity to its origin of replication (4nM). Comparative computer modelling showed that there are nine residues within the binding domain which are possible determinants of the reduced H.pylori DnaA specificity. Of these, the most interesting is probably the triad PTL; all three residues show significant divergence from the consensus, and Thr398 is the most divergent residue of all.

Bacterial Proteins↗

PubMed2004

Interaction with capsid protein alters RNA structure and the pathway for in vitro assembly of cowpea chlorotic mottle virus.

Viruses use sophisticated mechanisms to allow the specific packaging of their genome over that of host nucleic acids. We examined the in vitro assembly of the Cowpea chlorotic mottle virus (CCMV) and observed that assembly with viral RNA follows two different mechanisms. Initially, CCMV capsid protein (CP) dimers bind RNA with low cooperativity and form virus-like particles of 90 CP dimers and one copy of RNA. Longer incubation reveals a different assembly path. At a stoichiometry of about ten CP dimers per RNA, the CP slowly folds the RNA into a compact structure that can be bound with high cooperativity by additional CP dimers. This folding process is exclusively a function of CP quaternary structure and is independent of RNA sequence. CP-induced folding is distinct from RNA folding that depends on base-pairing to stabilize tertiary structure. We hypothesize that specific encapsidation of viral RNA is a three-step process: specific binding by a few copies of CP, RNA folding, and then cooperative binding of CP to the "labeled" nucleoprotein complex. This mechanism, observed in a plant virus, may be applicable to other viruses that do not halt synthesis of host nucleic acid, including HIV.

Bromovirus↗

PubMed2004

Purine and pyrimidine-specific repression of the Escherichia coli carAB operon are functionally and structurally coupled.

Transcription of the carAB operon encoding the sole carbamoylphosphate synthetase of Escherichia coli proceeds from a tandem pair of promoters. P2, downstream, is repressed by arginine and the ArgR protein, whereas P1 is submitted to pyrimidine-specific regulation and as shown here to purine-specific control exerted by binding of the PurR protein to a PUR box sequence centered around nucleotide -128.5 with respect to the start of P1 transcription. In vivo analyses of the effects of trans and cis-acting mutations on the regulatory responses and single round in vitro transcription assays indicated that ligand-bound PurR is by itself unable to inhibit P1 promoter activity. To exert its effect PurR relies on the elaborated nucleoprotein complex that governs P1 activity in a pyrimidine-specific manner. Thus we reveal the existence of an unprecedented functional and structural coupling between the modulation of P1 activity by purine and pyrimidine residues that appears to result from the unique position of the PUR box in the carAB control region, far upstream of the promoter. Missing contact and premethylation binding interference studies revealed the importance of base-specific groups and of structural aspects of the PUR box sequence in complex formation. Permutation assays indicated that the overall PurR-induced bending of the carAB control region is slightly less pronounced than that of the purF operator. The PUR boxes of the carAB operon of E.coli and Salmonella typhimurium are unique in that they have a guanine residue at position eight. Interestingly, guanine at this position has been proposed to be extremely unfavorable on the basis of modeling and binding studies, as its exocyclic amino group would enter into a steric clash with the side-chain of lysine 55. To analyze the effect of guanine at position eight in the upstream half-site of the carAB operator we constructed the adenine derivative and assayed in vivo repressibility of P1 promoter activity and in vitroPurR binding to the mutant operator, and constructed a molecular model for the unusual lysine 55-guanine 8 interaction.

Bacterial Proteins↗

PubMed2005

Flexibility in repression and cooperativity by KorB of broad host range IncP-1 plasmid RK2.

KorB, encoded by plasmid RK2, belongs to the ParB family of active partitioning proteins. It binds to 12 operators on the RK2 genome and was previously known to repress promoters immediately adjacent to operators O(B)1, O(B)10 and O(B)12 (proximal) or up to 154 bp away (distal) from O(B)2, O(B)9 and O(B)11. To achieve strong repression, KorB requires a cooperative interaction with one of two other plasmid-encoded repressors, KorA or TrbA. Reporter gene assays were used in this study to test whether the additional KorB operators may influence transcription and to test how KorB acts at a distance. The distance between O(B)9 and trbBp could be increased to 1.6kb with little reduction in repression or cooperativity with TrbA. KorB was also able to repress the promoter and cooperate with TrbA when the O(B) site was placed downstream of trbBp. This suggested a potential regulatory role for O(B) sites located a long way from any known promoter on RK2. O(B)4, 1.9kb upstream of traGp, was shown to mediate TrbA-potentiated KorB repression of this promoter, but no effect on traJp upstream of O(B)4 was observed, which may be due to the roadblocking or topological influence of the nucleoprotein complex formed at the adjacent transfer origin, oriT. Repression and cooperativity were alleviated significantly when a lac operator was inserted between O(B)9 and trbBp in the context of a LacI+ host, a standard test for spreading of a DNA-binding protein. On the other hand, a standard test for DNA looping, movement of the operator to the opposite face of the DNA helix from the natural binding site, did not significantly affect KorB repression or cooperativity with TrbA and KorA over relatively short distances. While these results are more consistent with spreading as the mechanism by which KorB reaches its target, previous estimates of KorB molecules per cell are not consistent with there being enough to spread up to 1kb from each O(B). A plausible model is therefore that KorB can do both, spreading over relatively short distances and looping over longer distances.

Bacterial Proteins↗

PubMed2005

Cooperative and specific binding of Vif to the 5' region of HIV-1 genomic RNA.

The viral infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in vivo. Packaging of Vif into viral particles is mediated by an interaction with viral genomic RNA and association with viral nucleoprotein complexes. Despite recent findings on the RNA-binding properties of Vif suggesting that Vif could be involved in retroviral assembly, no RNA sequence or structure specificity has been determined so far. To gain further insight into the mechanisms by which Vif might regulate viral replication, we studied the interactions of Vif with HIV-1 genomic RNA in vitro. Using extensive biochemical analysis, we have measured the affinity of recombinant Vif proteins for synthetic RNAs corresponding to various regions of the HIV-1 genome. We found that recombinant Vif proteins bind specifically to HIV-1 viral RNA fragments corresponding to the 5'-untranslated region (5'-UTR), gag and the 5' part of pol (K(d) between 45 nM and 65 nM). RNA encompassing nucleotides 1-497 or 499-996 of the HIV-1 genomic RNA bind 9+/-2 and 21+/-3 Vif molecules, respectively, and at least some of these proteins bind in a cooperative manner (Hill constant alpha(H) = 2.3). In contrast, RNAs corresponding to other parts of the HIV-1 genome or heterologous RNAs showed poor binding capacity and weak cooperativity (K(d) > 200 nM). Moreover, RNase T1 footprinting revealed a hierarchical binding of Vif, pointing to TAR and the poly(A) stem-loop structures as primary strong affinity targets, and downstream structures as secondary sites with moderate affinity. Taken together, our findings suggest that Vif may assist other proteins to maintain a correct folding of the genomic RNA in order to facilitate its packaging and further steps such as reverse transcription. Interestingly, our results suggest also that Vif could bind the viral RNA in order to protect it from the action of the antiviral factor APOBEC-3G/3F.

5' Untranslated Regions↗

PubMed2006

Three-stage regulation of the amphibolic gal operon: from repressosome to GalR-free DNA.

The gal operon of Escherichia coli is negatively regulated by the Gal repressosome, a higher order nucleoprotein complex containing a DNA loop that encompasses two gal promoters. In the repressosome structure, Gal repressor (GalR) dimers are bound to the two operator sites, flanking the promoter region, thus generating a DNA loop. The DNA loop is stabilized by binding of the architectural HU protein to the apex of the loop, and negative supercoiling. The gal promoters are also regulated in opposite directions by GalR without DNA looping. The repressosome-mediated as well as looping-independent transcription regulation of the two promoters is lifted in the presence of the inducer D-galactose. We tested the effect of D-galactose on various DNA-protein and protein-protein interactions of different regulatory complexes and on transcription repression in vitro. We found that the inducer breaks up the repressosome with clear intermediates in a concentration-dependent manner. The sequential disassembly generates different stages of regulation of the gal operon. The D-galactose-dependent switch from one stage of regulation to another satisfies the amphibolic requirement of the gal operon.

Binding Sites↗