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At least 937 records · Page 52Linked to original sources
PubMed1980

Subnuclear fractionation by mild micrococcal-nuclease treatment of nuclei of different transcriptional activities causes a partition of expressed and non-expressed genes.

Extremely mild treatment with micrococcal nuclease of isolated nuclei yields subnuclear fractions in which the majority of RNA polymerase II transcriptional complexes formed in vivo are segregated [Tata & Baker (1978) J. Mol. Biol. 118, 249-272]. We now describe different approaches followed to established whether or not the nuclei are thus resolved into transcribed and non-transcribed DNA. First, we have compared the sensitivity to deoxyribonuclease I, which is known to digest preferably expressed genes as present in nuclei or chromatin, of three micrococcal-nuclease-derived fractions from nuclei of different transcriptional activities. In transcriptionally active nuclei (rat liver, hen liver and oviduct, and Xenopus liver), the DNA in a polynucleosomal fraction comprising 6-15% of DNA and the majority of template-engaged RNA polymerase II (fraction P2) was 10-50 times as sensitive to deoxyribonuclease I as the DNA in the other two fractions (fractions P1 and S, comprising 78-88% of total nuclear DNA as large polynucleosomal aggregates and 2-6% of DNA mostly as mononucleosomes, respectively). In transcriptionally inactive nuclei obtained from hen erythrocytes, micrococcal nuclease did not separate DNA into fractions exhibiting such differential sensitivities. Second, we have monitored the partition of an expressed gene. Hybridization of complementary DNA to Xenopus albumin mRNA revealed a 5-10-fold enrichment of the albumin (but not the globin) gene in the P2 fraction of nuclei from Xenopus liver in which this gene is fully expressed. Third, a large part of the nascent rapidly labelled RNA synthesized in vivo in rat liver nuclei was recovered in the micrococcal-nuclease-derived fraction that is more susceptible to digestion with deoxyribonuclease I. It is concluded that mild micrococcal-nuclease treatment of nuclei causes their separation into transcribed and non-transcribed DNA as determined by a number of very different criteria.

Animals↗
PubMed1994

Localization and alternative splicing of agrin mRNA in adult rat brain: transcripts encoding isoforms that aggregate acetylcholine receptors are not restricted to cholinergic regions.

Agrin is a protein implicated in the formation and maintenance of the neuromuscular junction. In addition to motor neurons, agrin mRNA has been detected in the brains of embryonic rat and chick and adult marine ray, suggesting that this molecule may also be involved in the formation of synapses between neurons. As a step toward understanding agrin's role in the CNS, we utilized Northern blot and in situ hybridization techniques to analyze the regional distribution and cellular localization of agrin mRNA in the spinal cord and brain of adult rats. The results of these studies indicate that the agrin mRNA is expressed predominantly by neurons broadly distributed throughout the adult CNS. Moreover, expression of agrin mRNA is not restricted to cholinergic structures or regions of the brain receiving cholinergic input. Recently, RNA isolated from rat embryonic spinal cord was shown to contain four alternatively spliced agrin mRNAs, referred to as agrin0, agrin8, agrin11, and agrin19, each of which encodes agrin proteins that are active in acetylcholine receptor aggregating assays (Ferns et al., 1992). Using the polymerase chain reaction we demonstrate that all four of these agrin transcripts are expressed within the adult CNS. Agrin0, agrin8, and agrin19 were present in all regions analyzed. In contrast, agrin11 was detected only in forebrain. Results of these studies indicate that both the level of expression and pattern of alternative splicing of agrin mRNA are differentially regulated in the brain. The broad and predominantly neuronal distribution of agrin mRNA in the adult brain suggests that, in addition to its role at the neuromuscular junction, agrin may play a role in formation and maintenance of synapses between neurons in the CNS.

Agrin↗
PubMed2006

In vitro synthesis of long DNA products in reactions with HIV-RT and nucleocapsid protein.

In vitro reaction conditions using HIV reverse transcriptase (RT) and nucleocapsid protein (NC) that allowed efficient synthesis of single-stranded DNA products over a thousand nucleotides in length from genomic HIV RNA were characterized. Consistent with previous reports, the reactions required high concentrations of NC and RT. Long products were produced as a result of frequent strand transfer between RNA templates, averaging at least one transfer per 300 nucleotides synthesized. No change in RT processivity was observed in the reactions in the presence versus absence of NC. Synthesis of long products required formation of a high molecular mass aggregate between NC and nucleic acids. The aggregate formed rapidly and pelleted with low speed centrifugation. The aggregate was accessible to RT as pre-formed aggregates synthesized long products when RT was added. NC finger mutants lacking either finger one or two or with the finger positions switched were all effective in promoting long products. This suggests that the aggregation/condensation but not helix-destabilizing activity of NC was required. We propose that these high molecular mass aggregates promote synthesis of long reverse transcription products in vitro by concentrating nucleic acids, RT enzyme and NC to close proximity, thereby mimicking the role of the capsid environment within the host cell.

DNA Primers↗
PubMed1993

Inhibition of c-Ha-ras gene expression by hammerhead ribozymes containing a stable C(UUCG)G hairpin loop.

Catalytic RNAs recognize specific sequences of RNA and cleave at a specific site. In this study, we designed hammerhead ribozymes with a thermodynamically stable loop of the sequence 5'C(UUCG)G3' to prevent the aggregation of ribozymes with hammerhead structures. The cleavage activities of these ribozymes were examined using a synthetic pentadecamer with the sequence for the c-Ha-ras mRNA mutated at codon 12 (GGU-->GUU). For in vivo studies, we constructed a plasmid which expressed a highly active ribozyme targeted against the mutated c-Ha-ras mRNA. When this ribozyme-encoding gene and the activated c-Ha-ras gene were cotransfected into NIH3T3 cells, morphologically normal cells were obtained. We also determined that the expression of the c-Ha-ras gene was inhibited in these cells. These results show that ribozymes containing this stable hairpin loop are useful for the regulation of specific gene expression in vivo.

3T3 Cells↗
PubMed1982

Luminescence of the solid complexes of acridine orange with RNA.

The products of interaction between acridine orange (AO) and natural RNA, or the synthetic RNA homopolymers are precipitates insoluble over a wide range of ionic strength. These complexes have a composition of 1 AO molecule per 1 phosphate. The reaction is highly cooperative and the complex exhibits metachromatic luminescence. Significant differences in the luminescence spectra, related to base composition of RNA, characterize the insoluble complexes. This observation suggests that dye-base interactions take place in the AO-RNA complexes. During titration of poly(rA) with AO and simultaneous measurement of the luminescence and light scatter a two-step formation of the particles could be detected. The cooperative binding of the ligand at D/P less than 0.6 coincides with formation of small particles (molecular aggregates or micelles) and is followed at D/P greater than 0.6 by a cooperative agglomeration; the product of this agglomeration is particles of the size 0.35 micrometer and larger. Evidence is presented that suggests that the long wave length luminescence (approximately 650 nm) is a consequence of the solute-solid state transition of the AO-RNA interaction product rather than the classic dye-dye interactions previously visualized in the stacking model. Since these novel observations cannot be fully explained by the previously postulated molecular mechanisms of AO binding, an alternative model is advanced. Its implications in quantitative cytochemistry of nucleic acid as applied to flow cytometry are discussed.

Acridine Orange↗
PubMed1996

Molecular polymorphism and phenotypic variation in Aspergillus carbonarius.

Thirteen collection strains and field isolates of Aspergillus carbonarius were examined by using various genotypic and phenotypic approaches. Restriction fragment length polymorphism analysis of the ribosomal RNA gene cluster and the mitochondrial DNA of the strains revealed only slight variations, except for one field isolate (IN7), which exhibited completely different ribosomal RNA gene cluster and mitochondrial DNA patterns. The mitochondrial DNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. Strain-specific characters could be detected by the random amplified polymorphic DNA technique. Isoenzyme analysis and examination of carbon source utilisation patterns of the strains also revealed some intraspecific variability, though much smaller than that observed by using DNA-based techniques. The dendrograms constructed based on genotypic and phenotypic data suggest that strain IN7 might represent a new subspecies of A. carbonarius.

Acid Phosphatase↗
PubMed2007

RNA interference-mediated knockdown of alpha-synuclein protects human dopaminergic neuroblastoma cells from MPP(+) toxicity and reduces dopamine transport.

The critical observation in the pathology of Parkinson's disease (PD) is that neurodegeneration is largely restricted to dopaminergic neurons that develop cytoplasmic inclusions called Lewy bodies. These aggregations contain the protein alpha-synuclein. Furthermore, it is becoming apparent that alpha-synuclein expression levels are a major factor in PD pathogenesis. Patients with additional copies of the alpha-synuclein gene develop PD with a severity proportional to levels of alpha-synuclein overexpression. Similarly, overexpression of alpha-synuclein in in vitro and in vivo models has been shown to be toxic. However, little is known about the effects of reducing alpha-synuclein expression in human neurons. To investigate this, we have developed a system in which levels of alpha-synuclein can be acutely suppressed by using RNA interference (RNAi) in a physiologically relevant human dopaminergic cellular model. By using small interfering RNA (siRNA) molecules targeted to endogenous alpha-synuclein, we achieved 80% protein knockdown. We show that alpha-synuclein knockdown has no effect on cellular survival either under normal growth conditions over 5 days or in the presence of the mitochondrial inhibitor rotenone. Knockdown does, however, confer resistance to the dopamine transporter (DAT)-dependent neurotoxin N-methyl-4-phenylpyridinium (MPP(+)). We then demonstrate for the first time that alpha-synuclein suppression decreases dopamine transport in human cells, reducing the maximal uptake velocity (V(max)) of dopamine and the surface density of its transporter by up to 50%. These results show that RNAi-mediated alpha-synuclein knockdown alters cellular dopamine homeostasis in human cells and may suggest a mechanism for the increased survival in the presence of MPP(+), a toxin used extensively to model Parkinson's disease.

1-Methyl-4-phenylpyridinium↗
PubMed1997

Agrin gene expression in mouse somatosensory cortical neurons during development in vivo and in cell culture.

Agrin is an extracellular matrix protein involved in the formation of the postsynaptic apparatus of the neuromuscular junction. In addition to spinal motor neurons, agrin is expressed by many other neuronal populations throughout the nervous system. Agrin's role outside of the neuromuscular junction, however, is poorly understood. Here we use the polymerase chain reaction to examine expression and alternative splicing of agrin in mouse somatosensory cortex during early postnatal development in vivo and in dissociated cell culture. Peak levels of agrin gene expression in developing cortex coincide with ingrowth of thalamic afferent fibres and formation of thalamocortical and intracortical synapses. Analysis of alternatively spliced agrin messenger RNA variants shows that greater than 95% of all agrin in developing and adult somatosensory cortex originates in neurons, including isoforms that have little or no activity in acetylcholine receptor aggregation assays. The levels of expression of "active" and "inactive" isoforms, however, are regulated during development. A similar pattern of agrin gene expression is also observed during a period when new synapses are being formed between somatosensory neurons growing in dissociated cell culture. Changes in agrin gene expression, observed both in vivo and in vitro, are consistent with a role for agrin in synapse formation in the central nervous system.

2-Amino-5-phosphonovalerate↗
PubMed1996

Vaginal and rectal infection of cats with feline immunodeficiency virus.

The objective of this study was to examine the potential of vaginal and rectal mucosal routes for feline immunodeficiency virus (FIV) uptake and infection, as a model of mucosal HIV infection, and to determine the fate of virus at these mucosal sites following transmission of infection. SPF cats were exposed to FIV isolates (PET, GL-8, T637), administered as either cell-associated or cell-free inocula, via the rectum or vagina. Establishment of infection was confirmed by isolation of infectious FIV from peripheral blood mononuclear cells (PBMC), and by presence of FIV proviral DNA in PBMC using a nested polymerase chain reaction. Fate of virus in tissue taken at necropsy from cats infected for 6-48 weeks was assessed by localizing FIV core and envelope proteins, p24 and gp41, using a biotin-streptavidin linked immunoperoxidase (IP) technique. Cells susceptible to infection were identified by an in situ hybridization technique for FIV viral DNA and RNA. Cell-free, as well as cell-associated, virus was infectious across intact vaginal and rectal mucosal surfaces. Transmission was most successful using cell-associated inocula, and via the rectal route. Cells infected with FIV were detected by IP staining in the colon of 6/9 rectally challenged cats and 1/5 vaginally challenged cats. Virus was predominantly localized within the epithelium at the base of the colonic crypts associated with lymphoid aggregates (follicle associated epithelium; FAE), and within the lymphoid follicle itself. Occasional infected cells were also noted within the lamina propria. The distribution of FIV DNA positive cells in the colon was similar to that for FIV antigen whilst FIV RNA positive cells were found more extensively, including within the lamina propria and lymphoid follicle. FIV infected cells were not detected within the vagina, or colonic and ileac lymph nodes. Similar patterns of infected cells were seen in all of the positive cats, indicating that colonic tissues remain persistently actively infected with FIV. We conclude that the FIV/cat model of rectal and vaginal mucosal infection should prove useful for characterizing the mechanism by which HIV infects mucosal surfaces and as a challenge system for the design of vaccines effective at preventing HIV infection via rectal and vaginal routes.

Animals↗
PubMed1968

The disorganization of hepatic cell nucleoli induced by ethionine and its reversal by adenine.

The structure of nuclei and nucleoli of hepatic cells after short-term ethionine administration was investigated with the electron microscope. By 1(1/2) hr after the injection, a distinct alteration occurred in the nucleoli which was characterized by the appearance of electron-opaque masses in the nucleolonema. After 6-8 hr, the nucleoli showed partial fragmentation into small, dense masses. Large aggregates of interchromatinic granules appeared in the nucleoplasm. Condensation of chromatin became prominent in the nucleoplasm particularly along the nuclear membrane. By 12 hr almost complete fragmentation of nucleoli had occurred. The administration of adenine or methionine at 4 hr prevented the development of nucleolar changes. Also, adenine administration at 8 hr after ethionine completely reversed the nucleolar lesion by 12 hr. After methionine administration at 8 hr, many nucleoli showed incomplete reconstruction with many twisted ropelike structures when viewed 4 hr later. Identical structures were found when adenine was given at 8 hr, and animals were sacrificed 2 hr later. On the basis of this observation, the simplified structures of nucleoli found 2 hr after adenine or 4 hr after methionine appeared to be precursors of the nucleolonema. It is suggested that nucleoli show at least two basic reaction patterns to inhibitors of RNA synthesis, one typified by actinomycin D and one by ethionine.

Adenine↗
PubMed2000

Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets.

Platelets are known to contain platelet factor 4 and beta-thromboglobulin, alpha-chemokines containing the CXC motif, but recent studies extended the range to the beta-family characterized by the CC motif, including RANTES and Gro-alpha. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors. Polymerase chain reaction detected chemokine receptor messenger RNA in platelet RNA. CCR1, CCR3, and especially CCR4 gave strong signals; CXCR1 and CXCR4 were weakly positive. Flow cytometry with specific antibodies showed the presence of a clear signal for CXCR4 and weak signals for CCR1 and CCR3, whereas CXCR1, CXCR2, CXCR3, and CCR5 were all negative. Immunoprecipitation and Western blotting with polyclonal antibodies to cytoplasmic peptides clearly showed the presence of CCR1 and CCR4 in platelets in amounts comparable to monocytes and CCR4 transfected cells, respectively. Chemokines specific for these receptors, including monocyte chemotactic protein 1, macrophage inflammatory peptide 1alpha, eotaxin, RANTES, TARC, macrophage-derived chemokine, and stromal cell-derived factor 1, activate platelets to give Ca(++) signals, aggregation, and release of granule contents. Platelet aggregation was dependent on release of adenosine diphosphate (ADP) and its interaction with platelet ADP receptors. Part, but not all, of the Ca(++) signal was due to ADP release feeding back to its receptors. Platelet activation also involved heparan or chondroitin sulfate associated with the platelet surface and was inhibited by cleavage of these glycosaminoglycans or by heparin or low molecular weight heparin. These platelet receptors may be involved in inflammatory or allergic responses or in platelet activation in human immunodeficiency virus infection.

Adenosine Diphosphate↗
PubMed1991

Regulation of proenkephalin A gene expression in aggregating fetal rat brain cells.

1. Aggregating fetal rat brain cells express a significant amount of proenkephalin A (PENK) mRNA, a selective radioimmunoassay shows that this mRNA is also translated into enkephalins. 2. Depolarization with potassium chloride (KCl) or veratridine increases the expression of PENK mRNA in a time-dependent fashion, with a maximal increase of sixfold. It is interesting, however, that depolarization of the same cultures with KCl has no effect on the expression of prodynorphin mRNA. 3. An increase in PENK mRNA levels has been also observed in cultures treated with 8-Br-cAMP, phorbol 12-myristate-13-acetate (TPA), or dexamethasone. 4. However, incubation of the cultures with the opioid agonist etorphine or the antagonist naltrexone did not alter PENK gene expression, suggesting that there is not feedback control of opioids on PENK biosynthesis in these cells. 5. The increase in PENK mRNA in depolarized and in TPA-dexamethasone-, or 8-Br-cAMP-treated cultures was not accompanied by a significant increase in the amount of free immunoreactive met-enkephalin. Fetal brain cell cultures are therefore a useful neuronal model system for studying the mechanism that regulated the expression of PENK mRNA.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1977

Reverse transcriptase of foamy virus. Purification of the enzymes and immunological identification.

Reverse transcriptase from foamy virus, strain H4188 was estimated and purified. The enzyme has the following characteristics: 1. The reaction utilized preferentially oligo (dT) poly (rA) as a primer-template; however, the synthetic primer-template oligo (dT) poly (dA) could also be used to some extent. 2. The reaction utilized oligo (dG) poly (rC) as a primer-template with very low efficiency. 3. The crude virus preparation had a detectable endogenous reaction using the four deoxyribonucleotides for DNA polymerization. 4. The cation requirement for the enzyme reaction was much more biased for Mn++ than for Mg++ ions. 5. The molecular weight of the partially-purified enzyme was estimated to be about 80,000. Aggregates of 240,000 daltons were also seen. The activity of this enzyme was not inhibited by antisera against the reverse transcriptases of various type C RNA viruses, namely, feline endogenous leukemia virus, RD 114, Woolly simian sarcoma virus (SSV-1) and avian myeloblastosis virus (AMV). Antiserum against Rauscher leukemia virus (RLV) enzyme was marginally active against foamy virus enzyme, perhaps indicating a slight cross-reaction. The biochemical characteristics of foamy virus reverse transcriptase seemed to be very close to those of the type C RNA viruses, but the immunological reaction proved that the foamy virus reverse transcriptase was distinct from the others.

Epitopes↗
PubMed1988

Synthesis and fate of keratins 8 and 18 in nonepithelial cells transfected with cDNA.

To study the assembly of intermediate filaments in vivo we have transfected fibroblast cell lines with the cDNAs coding for keratins 8 and 18 under the control of the promoter of the SV40 early region and followed keratin expression by RNA hybridization, two-dimensional gel electrophoresis, and immunofluorescence analysis. When expressed individually, keratins 8 and 18 failed to polymerize into intermediate filaments but formed granular aggregates of variable size distributed throughout the cytoplasm as seen by staining with specific antibodies. The expression of one of these two keratins did not induce the synthesis of its partner or of any other keratin. Coexpression of the two keratins produced filamentous structures, frequently perinuclear, indicating that the two types of polypeptides were able to assemble into intermediate filaments but could not form the cytoskeleton characteristic of epithelial cells. These results demonstrate that assembly in heterocomplexes stabilizes keratins against cellular degradation, helping to explain why excess pools of simple keratins have never been detected.

Animals↗
PubMed1999

Co-translational folding.

Nascent proteins appear to fold co-translationally. The ribosome itself may function as a chaperone, providing a sheltered environment in which the nascent peptide is protected from aggregation and degradation, and in which folding into the tertiary structure is facilitated by interactions both with ribosomal proteins and with specific segments of the ribosomal RNA.

Amino Acid Sequence↗
PubMed1996

Mutagenesis and gene identification in Dictyostelium by shotgun antisense.

We have developed a mutagenesis technique that uses antisense cDNA to identify genes required for development in Dictyostelium discoideum. We transformed Dictyostelium cells with a cDNA library made from the mRNA of vegetative and developing cells. The cDNA was cloned in an antisense orientation immediately downstream of a vegetative promoter, so that in transformed cells the promoter will drive the synthesis of an antisense RNA transcript. We find that individual transformants typically contain one or occasionally two antisense cDNAs. Using this mutagenesis technique, we have generated mutants that fail to aggregate, aggregate but fail to form fruiting bodies, or aggregate but form abnormal fruiting bodies. The individual cDNA molecules from the mutants were identified and cloned using PCR. Initial sequence analysis of the PCR products from 35 mutants has identified six novel Dictyostelium genes, each from a transformant with one antisense cDNA. When the PCR-isolated antisense cDNAs were ligated into the antisense vector and the resulting constructs transformed into cells, the phenotypes of the transformed cells matched those of the original mutants from which each cDNA was obtained. We made homologous recombinant gene disruption transformants for three of the novel genes, in each case generating mutants with phenotypes indistinguishable from those of the original antisense transformants. Shotgun antisense thus is a rapid way to identify genes in Dictyostelium and possibly other organisms.

Animals↗
PubMed1995

Calf thymus Hsc70 protein protects and reactivates prokaryotic and eukaryotic enzymes.

The heat-shock 70 protein (Hsp70) chaperone family is very conserved and its prokaryotic homologue, the DnaK protein, is assumed to form one of the cellular systems for the prevention and restoration of heat-induced protein denaturation. By using anti-DnaK antibodies we purified the DnaK homologue heat-shock cognate protein (Hsc70) from calf thymus to apparent homogeneity. This protein was classified as an eukaryotic Hsc70, since (i) monoclonal antibodies against eukaryotic Hsc70 recognized it, (ii) its amino-terminal sequence showed strong homology to Hsp70s from eukaryotes and, (iii) it had an intrinsic weak ATPase activity that was stimulated by various peptide substrates. We show that this calf thymus Hsc70 protein protected calf thymus DNA polymerases alpha and epsilon as well as Escherichia coli DNA polymerase III and RNA polymerase from heat inactivation and could reactivate these heat-inactivated enzymes in an ATP-hydrolysis dependent manner, likely leading to the dissociation of aggregates formed during heat inactivation. In contrast to this, DnaK protein was exclusively able to protect and to reactivate the enzymes from E.coli but not from eukaryotic cells. Finally, the addition of calf thymus DnaJ co-chaperone homologue reduced the amount of Hsc70 required for reactivation at least 10-fold.

Adenosine Triphosphatases↗