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PubMed1983

A morphologic and biochemical study of the transcriptional changes in rat liver during the acute inflammatory reaction.

Transcriptional activity in nuclei isolated from the liver of rats during the acute inflammatory reaction induced by a 24-hour treatment with turpentine was evaluated by biochemical and ultrastructural techniques. This experimental model was chosen because it is known to induce an increase in hepatic synthesis of several plasma proteins called acute phase reactants. Using an in vitro system for estimating the activity of RNA polymerases A and B as an approach to evaluate RNA synthesis, we have shown that the activity of enzyme A (responsible for pre-rRNA synthesis) was significantly enhanced, whereas the activity of enzyme B (responsible for giant heterogenous RNA synthesis) remained unchanged in treated rats compared with control rats. At the electron microscopic level, using the Miller technique to spread the nuclear contents, the non-nucleolar transcription units were similar in treated and control rats. In contrast to control rats, more nucleolar transcription units were vizualized in treated rats, although the ribonucleoprotein fibrils were less numerous; in addition, in the latter group 10 to 15 times more aggregates constituted of ribonucleoprotein fibrils and particles were observed, the morphology of which was in agreement with a nucleolar origin. These results suggest that, during the acute inflammatory reaction, there was no overall increase in giant heterogenous RNA synthesis, whereas an increase in rRNA synthesis might be observed.

Animals↗
PubMed1990

CD of the synthetic RNA duplexes poly[r(A-T)] and poly[r(A-U)] in salt and ethanolic solutions.

Synthetic RNA poly[r(A-T)] has been synthesized and its CD spectral properties compared to those of poly[r(A-U)], poly[d(A-T)], and poly[d(A-U)] in various salt and ethanolic solutions. The CD spectra of poly[r(A-T)] in an aqueous buffer and of poly[d(A-T)] in 70.8% v/v ethanol are very similar, suggesting that they both adopt the same A conformation. On the other hand, the CD spectra of poly[r(A-T)] and of poly[r(A-U)] differ in aqueous, and even more so in ethanolic, solutions. We have recently observed a two-state salt-induced isomerization of poly[r(A-U)] into chiral condensates, perhaps of Z-RNA [M. Vorlícková, J. Kypr, and T. M. Jovin, (1988) Biopolymers 27, 351-354]. It is shown here that poly[r(A-T)] does not undergo this isomerization. Both the changes in secondary structure and tendency to aggregation are different for poly[r(A-T)] and poly[r(A-U)] in aqueous salt solutions. In most cases, the CD spectrum of poly[r(A-U)] shows little modification of its CD spectrum unless the polymer denatures or aggregates, whereas poly[r(A-T)] displays noncooperative alterations in its CD spectrum and a reduced tendency to aggregation. At high NaCl concentrations, poly[r(A-T)] and poly[r(A-U)] condense into psi(-) and psi(+) structures, respectively, indicating that the type of aggregation is dictated by the polynucleotide chemical structure and the corresponding differences in conformational properties.

Ammonium Chloride↗
PubMed1996

Substrate-bound agrin induces expression of acetylcholine receptor epsilon-subunit gene in cultured mammalian muscle cells.

Expression of the epsilon-subunit gene of the acetylcholine receptor (AChR) by myonuclei located at the neuromuscular junction is precisely regulated during development. A key role in this regulation is played by the synaptic portion of the basal lamina, a structure that is also known to contain agrin, a component responsible for the formation of postsynaptic specializations. We tested whether agrin has a function in synaptic AChR gene expression. Synaptic basal lamina from native adult muscle and recombinant agrin bound to various substrates induced in cultured rat myotubes AChR clusters that were colocalized with epsilon-subunit mRNA. Estimation of transcript levels by Northern hybridization analysis of total RNA showed a significant increase when myotubes were grown on substrate impregnated with agrin, but were unchanged when agrin was applied in the medium. The effect was independent of the receptor aggregating activity of the agrin isoform used, and agrin acted, at least in part, at the level of epsilon-subunit gene transcription. These findings are consistent with a role of agrin in the regulation of AChR subunit gene expression at the neuromuscular junction, which would depend on its binding to the synaptic basal lamina.

Agrin↗
PubMed1976

The isolation and properties of a DNA-directed RNA polymerase from yeast mitochondria.

A method is described for the rapid isolation of yeast mitochondrial DNA-directed RNA polymerase. The enzyme obtained had a specific activity of 1.56 nmol UMP incorporated per mg protein in 20 min at 37 degrees C, and is some 95% pure. This purified enzyme upon polyacrylamide gel elctrophoresis consists of a single polypeptide of 68 000 mol. wt. However, the enzyme forms aggregates easily which are affected by ionic strength, an increase decreasing the apparent molecular weight of the aggregates. This property also explains the presence of two peaks of activity upon DEAE-cellulose chromatography. The purified enzyme is still sensitive to rifamycin and to a number of rifamycin derivatives. The enzyme's sensitivity to rifamycin and rifamycin derivatives was compared with Escherichia coli and yeast nuclear RNA polymerases.

Cell Nucleus↗
PubMed1990

Distribution of msDNAs among serotypes of enteropathogenic Escherichia coli strains.

A genetic element, called a retron, is present in certain Escherichia coli strains. It consists of genes for the production of a covalently linked DNA-RNA compound and a reverse transcriptase. The presence of a retron can be detected by testing for a satellite DNA band by polyacrylamide gel electrophoresis. This DNA band consists of the DNA portion of the DNA-RNA compound and is called msDNA (multicopy single-stranded DNA). In a survey of intestinal E. coli isolates we detected msDNAs in classical enteropathogenic (EPEC) strains and in strains with aggregative adherence to tissue-culture cells (AA), but not in enteroinvasive (EIEC) and enterotoxigenic (ETEC) strains. Among 76 EPEC strains belonging to 14 different serotypes, msDNA was found to be present in 7 serotypes. In total, five different types of msDNA were found, although within each serotype, the msDNAs were the same. These results suggest that different retrons are clonally inherited.

DNA, Bacterial↗
PubMed1999

A role for RNA metabolism in inducing the heat shock response.

Yeast HSF is constitutively trimeric and DNA bound. Heat shock is thought to activate HSF by inducing a conformational change. We have developed an assay in which we can follow a conformational change of HSF that correlates with activity and thus appears to be the active conformation. This conformational change requires two HSF trimers bound cooperatively to DNA. The conformational change can be induced in whole cell extracts, and is thus amenable to biochemical analysis. We have purified a factor that triggers the conformational change. The factor is sensitive to dialysis, insensitive to NEM, and is not extractable by phenol. It is small, and apparently not a peptide. Mass spectroscopy identifies a novel guanine nucleotide that tracks with activity on columns. This novel nucleotide, purchased from Sigma, induces the conformational change (although this does not prove the identity of the activating factor unambiguously, because Sigma's preparation is contaminated with other compounds). What is the source of this nucleotide in cells? Activity can be generated by treating extracts with ribonuclease; this implicates RNA degradation as a source of HSF-activating activity. The heat shock response is primarily responsible for monitoring the levels of protein chaperones; how can RNA degradation be involved? Synthetic lethal interactions link HSF activity to ribosome biogenesis, suggesting a possible model. Ribosomal proteins are produced in large quantities, and in excess of rRNA; unassembled r-proteins are rapidly degraded (t1/2 approximately 3 min). Unassembled r-proteins aggregate readily. It is likely that unassembled r-proteins represent a major target of chaperones in vivo, and for proteasome-dependent degradation. Interference with rRNA processing (e.g., by heat shock) requires hsp70s to handle the aggregation-prone r-proteins, and proteasome proteins to help degrade the unassembled r-proteins before they aggregate. A nucleotide signal could be generated from the degradation products of the rRNA itself.

DNA↗
PubMed1999

Maternal function of a retroviral-type zinc-finger protein is essential for Drosophila development.

Previous studies have shown that zygotic expression of the Drosophila lark gene, which encodes an RNA-binding protein, is essential for embryogenesis. We now show that lark mRNA is abundant in preblastoderm (0-2 h) embryos, prior to zygotic transcription, indicative of maternal inheritance. Lark can also be detected within the nurse cells of developing egg chambers, suggesting a function for the protein during oogenesis. To test the hypothesis that the maternal inheritance of lark is required for oogenesis or early development, we employed the "FLP-DFS" technique to eliminate the lark maternal component within the germ line. Unfertilized and fertilized eggs lacking the lark maternal component exhibit a "fragile" phenotype, indicating that the protein functions during oogenesis. Furthermore, differentiation of the fertilized eggs is abnormal-most embryos arrest prior to blastoderm formation and exhibit morphological phenotypes that might reflect underlying defects in syncytial nuclear cycling or cellularization. Mutational analysis of a retroviral-type zinc finger within the lark protein indicates that it is required for the maternal function of the protein: females are completely sterile when their only source of lark protein contains a zinc-finger mutation. The aggregate of our studies shows that the germ-line expression of lark is essential for development, and suggests that the retroviral-type zinc finger mediates important RNA-binding functions during oogenesis and/or early development.

Animals↗
PubMed1990

The chicken embryonic mesonephros synthesizes link protein, an extracellular matrix molecule usually found in cartilage.

Link protein is a macromolecule that is relatively abundant in the extracellular matrix of cartilage, where it acts as a stabilizing component in aggregates of the large chondroitin sulfate proteoglycan and hyaluronic acid. In the present study, link protein transcripts were demonstrated in the chicken embryonic mesonephros by RNA in situ hybridization using a cartilage link protein cDNA probe. The link protein transcripts of the mesonephros are of the same size as those seen in cartilage. In addition, mesonephroi contain a protein that is immunologically reactive with a link protein polyclonal antiserum and this protein is identical in size to link protein isolated from cartilage. No transcripts for cartilage proteoglycan core protein were detected in the mesonephros. Type II collagen and cartilage matrix protein transcripts were also not detectable in the mesonephros. From previous data on chondrogenesis in the developing limb bud, the transcription of link protein and the proteoglycan core protein genes appeared to be spatially and temporally regulated in a coordinated fashion. However, the presence of link protein transcripts in the mesonephros, independent of cartilage proteoglycan core protein gene expression, indicates that these genes can be regulated independently of each other.

Animals↗
PubMed1975

Intermolecular renaturation of hairpin loops leads to stable and specific aggregates of 28-S rRNA and precursor rRNAs from Xenopus laevis.

Under certain extraction conditions, 28-S rRNA of Xenopus laevis can form specific dimers by intermolecular renaturation of a particular hairpin loop near the B end of the molecules. This was demonstrated by electron microscopy of isolated aggregates under conditions where rRNA shows a reproducible pattern of secondary structure. In 50% formamide, 15 mM sodium chloride, 1.5 mM trisodium citrate, pH 7.0, the aggregates dissociate into their monomers at a temperature (61 degrees C) at which the most stable hairpin loops of the 28-S rRNA are melting. Precursor rRNAs containing the 28-S rRNA region also form aggregates with 28-S rRNA by the same linkage mechanism. Since such artificial aggregates yield molecular weights higher than expected for true precursors of Xenopus rRNA, they may interfere in precursor rRNA studies. Analysis of whole-cell RNA pulse-labeled with (Me-3H)methionine, or nucleolar RNA pulse-labeled with (3H)uridine in the presence of tubercidin (inhibitor of rRNA processing), as well as electron microscopy of high-molecular-weight RNA fractions confirm that the 40-S precursor rRNA (2.8 X 10(6)) is the primary transcript of the ribosomal genes in Xenopus laevis.

Animals↗
PubMed1981

Ultrastructural studies on chromatin digestion by micrococcal nuclease in the presence of polyamines.

Nuclei purified from C57BL mouse submandibular salivary gland were treated with a range of micrococcal nuclease concentrations and times of treatment (from 0.5 unit for 2.5 min to 50 units for 30 min) in the presence of polyamines. About 50% of the chromatin was solubilized initially but with prolonged digestion this chromatin became insoluble again. Electron microscopy showed destruction of the finely dispersed chromatin with mild digestion, followed by aggregation of chromatin with more vigorous digestion. The early disappearance of finely dispersed chromatin filaments was not accompanied by preferential solubilization of chromatin associated with RNA polymerase II (euchromatin). These data suggest that the polyamines markedly reduce the susceptibility of euchromatin to micrococcal nuclease digestion.

Animals↗
PubMed1997

Differential roles of IRS-1 and SHC signaling pathways in breast cancer cells.

Several polypeptide growth factors stimulate breast cancer growth and may be involved in tumor progression. However, the relative importance of diverse growth factor signaling pathways in the development and maintenance of the neoplastic phenotype is largely unknown. The activation of such growth factor receptors as the insulin-like growth factor I receptor (IGF-I R), erbB-type receptors (erbB Rs) and FGF receptors (FGF Rs) controls the phenotype of a model breast cancer cell line MCF-7. To evaluate the function of 2 post-receptor signaling molecules, insulin receptor substrate-1 (IRS-1) (a major substrate of the IGF-IR) and SHC (a common substrate of tyrosine kinase receptors), we developed several MCF-7-derived cell clones in which the synthesis of either IRS-1 or SHC was blocked by antisense RNA. In MCF-7 cells, down-regulation of IRS-1 by 80-85% strongly suppressed anchorage-dependent and -independent growth and induced apoptotic cell death under growth factor- and estrogen-reduced conditions. The reduction of SHC levels by approximately 50% resulted in the inhibition of monolayer and anchorage-independent growth but did not decrease cell survival. Importantly, cell aggregation and the ability of cells to survive on the extracellular matrix were inhibited in MCF-7/anti-SHC clones, but not in MCF-7/anti-IRS-1 clones. Cell motility toward IGF was not attenuated in any of the tested cell lines, but motility toward EGF was decreased in MCF-7/anti-SHC clones. Our results suggest that in MCF-7 cells: 1) both IRS-1 and SHC are implicated in the control of monolayer and anchorage-independent growth; 2) IRS-1 is critical to support cell survival; 3) SHC is involved in EGF-dependent motility; and 4) normal levels of SHC, but not IRS-1, are necessary for the formation and maintenance of cell-cell interactions.

Adaptor Proteins, Signal Transducing↗
PubMed1990

Circular dichroism studies of the HIV-1 Rev protein and its specific RNA binding site.

The circular dichroism (CD) spectrum of the Rev protein from HIV-1 indicates that Rev contains about 50% alpha helix and 25% beta sheet at 5 degrees C in potassium phosphate buffer, pH 3, and 300 mM KF. The spectrum is independent of protein concentration over a 20-fold range. At neutral pH, Rev is relatively insoluble but can be brought into solution by binding to its specific RNA binding site, the Rev-responsive element (RRE), at a Rev:RNA ratio of about 3:1. Nonspecific binding to tRNA does not solubilize Rev. As judged by difference CD spectra, the conformation of Rev when bound to the RRE at neutral pH is similar to the conformation of unbound Rev at pH 3, although changes in the RNA may also contribute to the difference spectrum. Indeed, some difference is observed near 260 nm, consistent with a conformational change of the RRE upon Rev binding. Rev alone at pH 3 shows irreversible aggregation as the temperature is raised, while Rev bound to the RRE at neutral pH shows a reversible transition with a Tm of 68 degrees C.

Amino Acid Sequence↗
PubMed2001

Inducible model to study negative strand RNA synthesis and assembly of hepatitis C virus from a full-length cDNA clone.

An inducible in vitro cell culture system was developed to assay HCV replication by direct biochemical means. A transcription plasmid containing a T7 promoter at the 5' end, full-length cDNA of the HCV genome, a ribozyme sequence from the antigenomic strand of hepatitis delta virus and a T7 terminator was prepared. To facilitate high-level transcription of HCV RNA, HepG2 cells were infected with replication deficient adenovirus containing the T7 RNA polymerase gene and later transfected with the transcription plasmid containing the full-length HCV genome. This transfection-based cell culture system expressed high levels of HCV structural (core, El and E2) and non-structural proteins (NS3 and NS5B) detectable by Western blot and immunofluorescence assays. Production of HCV RNA transcripts and presence of replicative negative strand of HCV was confirmed by ribonuclease protection assay indicating replication of HCV in the transfected HepG2 cell. The transfected HepG2 cells assembled 50-60 nm virus-like particles, which could be aggregated by anti-E2 antibodies. This model can be utilized for studying mechanisms of HCV replication, assembly of HCV particles and to test potential anti-HCV compounds.

Cloning, Molecular↗
PubMed2006

Deletion of the ubiquitin ligase CHIP leads to the accumulation, but not the aggregation, of both endogenous phospho- and caspase-3-cleaved tau species.

Accumulation of the microtubule-associated protein tau into neurofibrillary lesions is a pathological consequence of several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Hereditary mutations in the MAPT gene were shown to promote the formation of structurally distinct tau aggregates in patients that had a parkinsonian-like clinical presentation. Whether tau aggregates themselves or the soluble intermediate species that precede their aggregation are neurotoxic entities in these disorders has yet to be resolved; however, recent in vivo evidence supports the latter. We hypothesized that depletion of CHIP, a tau ubiquitin ligase, would lead to an increase in abnormal tau. Here, we show that deletion of CHIP in mice leads to the accumulation of non-aggregated, ubiquitin-negative, hyperphosphorylated tau species. CHIP-/- mice also have increased neuronal caspase-3 levels and activity, as well as caspase-cleaved tau immunoreactivity. Overexpression of mutant (P301L) human tau in CHIP-/- mice is insufficient to promote either argyrophilic or "pre-tangle" structures, despite marked phospho-tau accumulation throughout the brain. These observations are supported in post-developmental studies using RNA interference for CHIP (chn-1) in Caenorhabditis elegans and cell culture systems. Our results demonstrate that CHIP is a primary component in the ubiquitin-dependent degradation of tau. We also show that hyperphosphorylation and caspase-3 cleavage of tau both occur before aggregate formation. Based on these findings, we propose that polyubiquitination of tau by CHIP may facilitate the formation of insoluble filamentous tau lesions.

Animals↗
PubMed1986

Cyclic AMP and NH3/NH4+ both regulate cell-type-specific mRNA accumulation in the cellular slime mold, Dictyostelium discoideum.

Dictyostelium discoideum cells plated for development until aggregation stage, and then dissociated into media containing glucose, albumin, and cAMP will form into clumps and undergo prespore and prestalk differentiation. Differentiation in this in vitro system is dependent on three components: cAMP, multicellularity, and the acquisition of "differentiation competence" which the cells acquire in a period between interphase and aggregation stage when plated on Millipore filters. We have used this system to explore aspects of the multicellular environment which are involved in regulation the accumulation of the different prespore- and prestalk-specific messenger RNAs. Two classes of prespore messenger RNA, as well as a prestalk-specific messenger RNA, all require the acquisition of differentiation competence in order to be expressed in response to cAMP. Additionally, all of these messenger RNAs require agglomerate formation for maximal expression. The addition of 33 mM ammonium sulfate (NH4)2SO4, however, can entirely replace the requirement for agglomerate formation for expression of the prestalk-specific messenger RNA, and can partially substitute for agglomerate formation in inducing the expression of both classes of prespore-specific messenger RNAs. In this system, cAMP is essential for the initial induction of expression of all three classes of messenger RNAs. In this system, cAMP is essential for the initial induction of expression of all three classes of messenger RNAs while agglomerate formation or elevated NH3/NH+4 is essential only for the maintenance of the elevated levels of the messenger RNAs.

Albumins↗
PubMed1988

A chemoattractant receptor controls development in Dictyostelium discoideum.

During the early stages of its developmental program, Dictyostelium discoideum expresses cell surface cyclic adenosine monophosphate (cyclic AMP) receptors. It has been suggested that these receptors coordinate the aggregation of individual cells into a multicellular organism and regulate the expression of a large number of developmentally regulated genes. The complementary DNA (cDNA) for the cyclic AMP receptor has now been cloned from lambda gt-11 libraries by screening with specific antiserum. The 2-kilobase messenger RNA (mRNA) that encodes the receptor is undetectable in growing cells, rises to a maximum at 3 to 4 hours of development, and then declines. In vitro transcribed complementary RNA, when hybridized to cellular mRNA, specifically arrests in vitro translation of the receptor polypeptide. When the cDNA is expressed in Dictyostelium cells, the undifferentiated cells specifically bind cyclic AMP. Cell lines transformed with a vector that expresses complementary mRNA (antisense) do not express the cyclic AMP receptor protein. These cells fail to enter the aggregation stage of development during starvation, whereas control and wild-type cells aggregate and complete the developmental program within 24 hours. The phenotype of the antisense transformants suggests that the cyclic AMP receptor is essential for development. The deduced amino acid sequence of the receptor reveals a high percentage of hydrophobic residues grouped in seven domains, similar to the rhodopsins and other receptors believed to interact with G proteins. It shares amino acid sequence identity and is immunologically cross-reactive with bovine rhodopsin. A model is proposed in which the cyclic AMP receptor crosses the bilayer seven times with a serine-rich cytoplasmic carboxyl terminus, the proposed site of ligand-induced receptor phosphorylation.

Amino Acid Sequence↗
PubMed2006

Three-dimensional culture regulates Raf-1 expression to modulate fibronectin matrix assembly.

Oncogenic transformation has been associated with decreased fibronectin (FN) matrix assembly. For example, both the HT-1080 fibrosarcoma and MAT-LyLu cell lines fail to assemble a FN matrix when grown in monolayer culture (2-dimensional [2D] system). In this study, we show that these cells regain the ability to assemble a FN matrix when they are grown as aggregates (3-dimensional [3D] system). FN matrix assembly in 3D correlates with decreased Raf-1 protein expression compared with cells grown in monolayer culture. This effect is associated with reduced Raf-1 mRNA levels as determined by quantitative RT-PCR and not proteasome-mediated degradation of endogenous Raf-1. Interestingly, transient expression of a Raf-1 promoter-reporter construct demonstrates increased Raf-1 promoter activity in 3D, suggesting that the transition to 3D culture may modulate Raf-1 mRNA stability. Finally, to confirm that decreased Raf-1 expression results in increased FN matrix assembly, we used both pharmacological and small interfering RNA knockdown of Raf-1. This restored the ability of cells in 2D culture to assemble a FN matrix. Moreover, overexpression of Raf-1 prevented FN matrix assembly by cells cultured in 3D, resulting in decreased aggregate compaction. This work provides new insight into how the cell microenvironment may influence Raf-1 expression to modulate cell-FN interactions in 3D.

Animals↗
PubMed2002

Molecular visualization of immunoglobulin switch region RNA/DNA complex by atomic force microscope.

Immunoglobulin heavy-chain (IgH) class switch recombination (CSR) is initiated by DNA breakage in the switch (S) region featuring tandem repetitive nucleotide sequences. Various studies have demonstrated that S-region transcription and splicing proceed to genomic recombination and are indispensable for CSR in vivo, although the precise molecular mechanism is largely unknown. Here, we show the novel physical property of the in vitro transcribed S-region RNA by direct visualization using an atomic force microscope (AFM). The S-region sense RNA, but not the antisense RNA, forms a persistent hybrid with the template plasmid DNA and changes the plasmid conformation from supercoil to open circle in the presence of spermidine. In addition, the S-region transcripts generate globular forms and are assembled on the template DNA into a large aggregate that may stall replication and increase the recombinogenicity of the S-region DNA.

Immunoglobulin Class Switching↗