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At least 1,081 records · Page 60Linked to original sources
PubMed1996

Guanine-rich oligonucleotides targeted to a critical R . Y site located in the Ki-ras promoter. The effect of competing self-structures on triplex formation.

The promoter of the murine Ki-ras proto-oncogene contains a (C+G)-rich homopurine . homopyrimidine (R . Y) sequence that is essential for transcription activity. We have designed two G-rich oligonucleotides, d(TGGGTGGGTGGTTGGGTGGG) (20GT) and d(AGGGAGGGAGGAAGGGAGGG) (20AG), that have the potential to bind the critical Ki-ras sequence via triplex-helix formation. Band-shift experiments have shown that 20GT binds the Ki-ras R . Y duplex with a delta G value of -40 +/- 5 kJ/mol, while 20AG appeared to have a lower affinity under the experimental conditions adopted: 50 mM Tris/HCl, pH 7.4, 50 mM NaCl, 5 mM MgCl2, 25 degrees C. In the absence of Mg2+, 20GT did not bind to the Ki-ras R . Y target, while 20AG exhibited the same affinity observed in the magnesium-containing buffer. To gain insight into the solution properties of 20GT and 20AG, we have performed several experiments including polyacrylamide gel electrophoresis (PAGE), hydroxyapatite chromatography, ultraviolet absorption melting and circular dichroism (CD). We found that 20AG rapidly self-associates into presumably a duplex, even at low concentration (< 1 microM), while 20GT forms aggregates slowly, a process favoured by high oligonucleotide concentrations (> 25 microM). The critical Ki-ras sequence was inserted in Bluescript KS+, downstream from the T7 promoter, to investigate to what extent 20AG and 20GT, which are directed against the R . Y target, are able to inhibit T7 RNA polymerase transcription, under near-physiological conditions. Transcription experiments conducted in vitro at pH 7.4 have shown that oligonucleotide 20GT produced a remarkable repression of T7 RNA polymerase activity in the concentration range (10-25 microM), whereas 20AG had little effect on transcription. In conclusion, the results of this work together with other data reported in the literature [Olivas, W. M. & Maher, L. J. III (1995) Biochemistry 34, 278-284; Noonberg, S. B., François, J.-C., Garestier, T. & Hélène, C. (1995) Nucleic Acids Res. 23, 1956-1963], demonstrate that G-rich oligonucleotides, in particular (G,A)-sequences, may raise problems for in vivo application due to self-aggregation.

Absorption↗
PubMed1981

Conformational studies of Escherichia coli ribosomes with the use of acridine orange as a probe.

The interaction of E. coli vacant ribosomes with acridine orange (AO) was studied, to obtain conformational information about rRNAs in ribosomes. Acridine orange binds to an RNA in two different modes: cooperative outside binding with stacking of bound AO's and intercalation between nucleotide bases. Free 16S and 23S rRNAs have almost identical affinities to AO. At 1 mM Mg2+, AO can achieve stacking binding on about 40% of rRNA phosphate groups. The number of stacking binding sites falls to about 1/3 in the 30S subunit in comparison with free 16S rRNA. In the 50S subunit, the number of stacking binding sites is only 1/5 in comparison with free 23S rRNA. Mg2+ ions are more inhibitory for the binding of AO to ribosomes than to free rRNAs. The strength of stacking binding appears to be more markedly reduced by Mg2+ in active ribosomes than in rRNAs. "Tight couple" 70S particles are less accessible for stacking binding than free subunits. The 30S subunits that have irreversibly lost the capability for 70S formation under low Mg2+ conditions have an affinity to AO that is very different from that of active 30S but similar to that of free rRNA, though the number of stacking binding sites is little changed by the inactivation. 70S and 30S ribosomes with stacking bound AO's have normal sedimentation constants, but the 50S subunits reversibly form aggregates.

Acridine Orange↗
PubMed1978

Age-related changes in ribosomal profiles and in vitro protein synthesis in skeletal muscle during fasting and subsequent refeeding of rats.

Age-dependent changes in ribosomal profiles and in in vitro protein synthesis in skeletal muscle (gastrocnemius muscle) were studied in rats subjected to fasting and subsequent refeeding. Male rats of 4, 7, and 21 weeks of age were used. Incorporation of 14C-leucine into protein in vitro using skeletal muscle ribosomes from fed rats decreased with the age of the animals. Sucrose density gradient analyses of ribosomal profiles revealed that the changes in protein synthesis were accompanied by progressive declines in the concentrations of polyribosomes and total RNA in skeletal muscle. After fasting for 1 or 2 days, protein synthesis in muscle of young rats dropped markedly and this change could be attributed to the fall in the concentrations of polyribosomes. Also, the concentration and total RNA in the muscles of the young rats decreased. During refeeding of rats after fasting for 2 days, the incorporation of 14C-amino acids into protein recovered more efficiently using ribosomes of skeletal muscle of young animals compared with using those of older animals. This rapid increase in the protein synthesizing activity of skeletal muscle may be related to the efficient recovery of the ribosomes toward heavier aggregates. The results indicate that young animals adapt more efficiently to fasting and refeeding than do older animals.

Aging↗
PubMed1993

Synchronous expression of LINE-1 RNA and protein in mouse embryonal carcinoma cells.

L1, or LINE-1, is a repetitive DNA family found in all mammalian genomes that have been examined. At least a few individual members of the L1 family are functional transposable elements. Expression of these active elements leads to new insertions of L1 into the genomic DNA by the process of retrotransposition. We have detected coexpression of full-length, sense-strand L1 RNA transcripts and L1-encoded protein in mouse embryonal carcinoma cell lines. Both of these L1 expression products are candidates for intermediates in the retrotransposition process. L1 protein is found in what appear to be cytoplasmic aggregates and is not localized to any known cytoplasmic organelles. The six embryonal carcinoma cell lines tested were chosen to represent commitment to different developmental pathways in early mouse embryogenesis. The only two cell lines that express L1 are unique among the six in that they have a strong predilection to differentiate into extraembryonic endoderm. This observation is consistent with L1 expression and transposition in primordial germ cells of the mouse. An important implication of these studies is that L1 expression may provide a new marker for use in determining the origin of primordial germ cells during mouse embryogenesis.

Animals↗
PubMedDate not supplied

A heat-shock-like response with cytoskeletal disruption occurs following hydrostatic pressure in MG-63 osteosarcoma cells.

Human osteosarcoma cells, MG-63, were exposed to a hydrostatic pressure shock of 4.0 MPa for 20 min. Changes in subcellular distribution of the cytoskeletal elements and heat shock protein 70 (hsp70) were followed by indirect immunofluorescence and by avidin-biotin-peroxidase protocols. During recovery, total cellular RNA was determined and actin and aldolase mRNA content was followed using reverse transcription-polymerase chain reaction techniques. Hydrostatic pressure caused cell rounding (but not cell death), disruption of microtubules, collapse of intermediate filaments to a perinuclear location, collapse of actin stress fibers into globular aggregates in the cytoplasm, and the formation of several large elongated intranuclear actin inclusions. During recovery, the cells flattened, reorganized microtubules, and redistributed intermediate filaments prior to the reappearance of actin stress fibers. At 20 and 60 min following the initiation of hydrostatic pressure, there was increased anti-hsp 70 staining at the nuclear membrane and concentration of hsp70 in four to six granules in the nucleus. At 120 min following the hydrostatic pressure, hsp70 showed intense staining in the cytoplasm and hsp70-containing granules in the nucleus disappeared. Cellular RNA decreased during the first 120-min posthydrostatic pressure shock and then recovered to near prehydrostatic pressure treatment levels by 240 min. Actin mRNA abundance, in relation to aldolase mRNA abundance, showed the same temporal pattern of initial decrease, followed by increase as did total RNA. Review of the literature indicated that eukaryotic cells respond to heat shock and to hydrostatic pressure by disruption of the cytoskeletal elements and by similar modifications in genetic expression. In this study, the observed responses of MG-63 cells to a 4-MPa hydrostatic pressure shock was like the reported response of mammalian cells to a 43 degrees C heat shock.

Actins↗
PubMed1981

Modulation of hepatic transcription and translation during early stages of aflatoxin B1 carcinogenesis.

Isolated rat hepatocytes have been used to study the transcription and translation processes at varied time intervals after administration of a single dose (6 mg/kg) of hepatocarcinogen aflatoxin B1 (AFB1). The effects of AFB1 during the first 24 h of drug treatment show a characteristic inhibition followed by periods of rapid recovery and also a hyperactive state when both heterogeneous nuclear RNA (HnRNA) transcription and cytoplasmic translation processes reach about 200% of control. Analysis of [3H]orotic acid pulse-labeled HnRNA and cytoplasmic polyadenylic acid(poly (A)) containing mRNA on sucrose gradients under conditions which prevent aggregation show a stepwise reduction in the total isotopic incorporation as well as in the size distribution during the first 9 h of AFB1 treatment. Between 9 and 24 h after AFB1 treatment, there is a recovery in the total incorporation as well as in the size of HnRNA and poly (A) containing mRNA. Analysis of translation products by a two-dimensional procedure shows loss of some high molecular weight products in AFB1-treated cells. At 24 h after AFB1 treatment (hyperactive stage) there is a total recovery of activity. Also, several new translation products not detected in the control hepatocytes are synthesized at this stage. The hyperactive stage might represent gene-derepression or reprogramming of gene expression.

Aflatoxin B1↗
PubMed1976

Novel mechanism for translational control in regulation of ferritin synthesis by iron.

Poly(A)-containing RNA was isolated from the polyribosomal and post-ribosomal fractions of the livers of normal and iron-treated rats. These RNA fractions were then translated in a wheat germ system to provide a measure of the amount of ferritin mRNA present in each fraction. Following iron administration, there was a 2-fold increase in the amount of ferritin mRNA in the polyribosomal fraction. This increase was not inhibited by prior treatment of the rats with actinomycin D or cordycepin, suggesting a cytoplasmic control mechanism. In normal rats, the post-ribosomal fraction contained an amount of ferritin mRNA equal to that in the polyribosomes. When iron was administered, this untranslated ferritin mRNA became reduced to negligible quantities, thus accounting for the doubling of the ferritin mRNA content of the polyribosomal fraction. A scheme is proposed in which translation of the ferritin mRNA in the post-ribosomal fraction is prevented by adhering ferritin subunits. Iron administration removes this inhibition of the translation of ferritin mRNA by promoting aggregation of these subunits into ferritin.

Animals↗
PubMed1996

The identification of proteoglycan-associated mRNAs in human dental pulp cells.

Characterization of the dental pulp proteoglycans has been largely confined to the glycosaminoglycan component of the proteoglycan molecule, while the protein core has received little attention. This study was conducted to identify mRNAs of previously well-characterized proteoglycans-biglycan, decorin and versican-and link protein in dental pulp cells. Dermal fibroblasts were used as a positive control. Oligonucleotide probes were constructed based on published sequences for the four proteins from human tissues. Total RNA was isolated from cultured human pulp and dermal cells, separated according to size by formaldehyde gel electrophoresis and subsequently transferred to a nylon filter. Northern hybridizations using the oligonucleotide probes revealed the expression of biglycan, decorin, versican and link protein mRNAs. Biglycan and decorin are small proteoglycans that have a regulatory effect on collagen fibrillogenesis. Assuming expression of link protein and versican in vivo, the larger proteoglycans in the dental pulp are capable of forming large proteoglycan aggregates.

Biglycan↗
PubMed1984

Gene expression during development of Myxococcus xanthus. Analysis of the genes for protein S.

Protein S is an abundant spore coat protein produced during fruiting body formation (development) of the bacterium Myxococcus xanthus. We have cloned the DNA which codes for protein S and have found that this DNA hybridizes to three protein S RNA species from developmental cells but does not hybridize to RNA from vegetative cells. The half-life of protein S RNA was found to be unusually long, about 38 minutes, which, at least in part, accounts for the high level of protein S synthesis observed during development. Hybridization of restriction fragments from cloned M. xanthus DNA to the developmental RNAs enabled us to show that M. xanthus has two directly repeated genes for protein S (gene 1 and gene 2) which are separated by about 10(3) base-pairs on the bacterial chromosome. To study the expression of the protein S genes in M. xanthus, eight M. xanthus strains were isolated with Tn5 insertions at various positions in the DNA which codes for protein S. The strains which contained insertions in gene 1 or between gene 1 and gene 2 synthesized all three protein S RNA species and exhibited normal levels of protein S on spores. In contrast, M. xanthus strains exhibited normal levels of protein S on spores. In contrast, M. xanthus strains with insertions in gene 2 had no detectable protein S on spores and lacked protein S RNA. Thus, gene 2 is responsible for most if not all of the production of protein S during M. xanthus development. M. xanthus strains containing insertions in gene 1, gene 2 or both genes, were found to aggregate and sporulate normally even though strains bearing insertions in gene 2 contained no detectable protein S. We examined the expression of gene 1 in more detail by constructing a fusion between the lacZ gene of Escherichia coli and the N-terminal portion of protein S gene 1 of M. xanthus. The expression of beta-galactosidase activity in an M. xanthus strain containing the gene fusion was shown to be under developmental control. This result suggests that gene 1 is also expressed during development although apparently at a much lower level than gene 2.

Bacterial Proteins↗
PubMed2001

Escherichia coli RNA polymerase subunit omega and its N-terminal domain bind full-length beta' to facilitate incorporation into the alpha2beta subassembly.

The omega subunit of Escherichia coli RNA polymerase, consisting of 90 amino acids, is present in stoichiometric amounts per molecule of core RNA polymerase (alpha2betabeta'). The presence of omega is necessary to restore denatured RNA polymerase in vitro to its fully functional form, and, in an omega-less strain of E. coli, GroEL appears to substitute for omega in the maturation of RNA polymerase. The X-ray structure of Thermus aquaticus core RNA polymerase suggests that two regions of omega latch on to beta' at its N-terminus and C-terminus. We show here that omega binds only the intact beta' subunit and not the beta' N-terminal domain or beta' C-terminal domain, implying that omega binding requires both these regions of beta'. We further show that omega can prevent the aggregation of beta' during its renaturation in vitro and that a V8-protease-resistant 52-amino-acid-long N-terminal domain of omega is sufficient for binding and renaturation of beta'. CD and functional assays show that this N-terminal fragment retains the structure of native omega and is able to enhance the reconstitution of core RNA polymerase. Reconstitution of core RNA polymerase from its individual subunits proceeds according to the steps alpha + alpha --> alpha2 + beta --> alpha2beta + beta' --> alpha2betabeta'. It is shown here that omega participates during the last stage of enzyme assembly when beta' associates with the alpha2beta subassembly.

Amino Acid Sequence↗
PubMed1983

A rapid method for preparation of bacterial plasmids.

A method for isolating plasmids from Escherichia coli which requires less than 8 h from cell pellet to purified plasmid essentially free of protein, RNA, and chromosomal DNA is presented. By this procedure, amplified plasmid pBR322 was isolated from E. coli strain RR1. The final product had no detectable protein or RNA, and plasmid comprised approximately 99% of the total DNA. The procedure includes lysozyme treatment in hypertonic solution followed by lysis with a mild detergent in the presence of high salt and an RNase inhibitor--conditions which prevent unfolding of the bacterial nucleoid. After centrifuging out the nucleoid and cell debris, the nucleic acids are selectively precipitated with a neutral solution of sodium trichloroacetate and ethanol. RNA is degraded with RNase and the degradation products and RNase are eliminated through a second trichloroacetate/ethanol precipitation. Finally, the plasmid is resuspended and passed through a nitrocellulose filter to remove aggregates and any residual protein and single-stranded DNA--giving a plasmid preparation suitable for electrophoretic fractionation or cleavage with restriction nucleases.

Bacteria↗
PubMed1997

EBV-associated Kikuchi's histiocytic necrotizing lymphadenitis with cutaneous manifestations.

The clinical and pathologic findings of Kikuchi's histiocytic necrotizing lymphadenitis may mimic those of malignant lymphoma. We describe a 6-year-old boy with generalized lymphadenopathy, spiking fever, chills, myalgias, malaise, and erythematous, crusted papules. Although cutaneous manifestations have been noted in 16% to 40% of patients with histiocytic necrotizing lymphadenitis, only three publications described skin lesions. The skin lesions and affected lymph nodes revealed histiocytic aggregates, atypical lymphoid cells, karyorrhectic debris, and patchy necrosis. Spontaneous resolution occurred in 2 months. Results of serologic studies, Epstein-Barr virus (EBV) latent membrane protein immunoperoxidase staining, EBER-1 RNA in-situ hybridization, and EBV EBNA-1 DNA polymerase chain reaction implicate EBV as the causative agent.

Biopsy↗
PubMed1998

The cauliflower mosaic virus capsid protein: assembly and nucleic acid binding in vitro.

The capsid protein of the cauliflower mosaic virus (CaMV) was expressed in a bacterial system to study CaMV assembly. Bacterial lysates contained soluble particulate material and insoluble inclusion bodies that were both used for analysis. In vitro renaturation of pIV derivatives lead to the appearance of folded sheets or large tubular structures in electron microscopy. The region between amino acid positions 77 and 332 is sufficient for self-aggregation of pIV in vitro. C-terminal deletion to amino acid position 265 still allowed dimerization but prevented further aggregation. Nucleic acid binding assays of immobilized pIV derivatives demonstrated that a region located upstream of the retroviral "zinc finger-like" motif is involved in unspecific binding dsDNA, ssDNA and RNA.

Amino Acid Sequence↗
PubMed2005

Production of cyanophycin, a suitable source for the biodegradable polymer polyaspartate, in transgenic plants.

The production of biodegradable polymers in transgenic plants in order to replace petrochemical compounds is an important challenge for plant biotechnology. Polyaspartate, a biodegradable substitute for polycarboxylates, is the backbone of the cyanobacterial storage material cyanophycin. Cyanophycin, a copolymer of l-aspartic acid and l-arginine, is produced via non-ribosomal polypeptide biosynthesis by the enzyme cyanophycin synthetase. A gene from Thermosynechococcus elongatus BP-1 encoding cyanophycin synthetase has been expressed constitutively in tobacco and potato. The presence of the transgene-encoded messenger RNA (mRNA) correlated with changes in leaf morphology and decelerated growth. Such transgenic plants were found to produce up to 1.1% dry weight of a polymer with cyanophycin-like properties. Aggregated material, able to bind a specific cyanophycin antibody, was detected in the cytoplasm and the nucleus of the transgenic plants.

Journal Article↗
PubMed1994

Receptor tyrosine phosphatase beta is expressed in the form of proteoglycan and binds to the extracellular matrix protein tenascin.

The extracellular domain of receptor type protein tyrosine phosphatase beta (RPTP beta) exhibits striking sequence similarity with a soluble, rat brain chondroitin sulfate proteoglycan (3F8 PG). Immunoprecipitation experiments of cells transfected with RPTP beta expression vector and metabolically labeled with [35S]sulfate and [35S]methionine indicate that the transmembrane form of RPTP beta is indeed a chondroitin sulfate proteoglycan. The 3F8 PG is therefore a variant form composed of the entire extracellular domain of RPTP beta probably generated by alternative RNA splicing. Previous immunohistochemical studies indicated that both RPTP beta and the extracellular matrix protein tenascin are localized in similar regions of the central nervous system. We have performed co-aggregation assays with red and green Co-vaspheres coated with tenascin and 3F8 PG, respectively, showing that the extracellular domain of RPTP beta (3F8 PG) binds specifically to tenascin. The interaction between a receptor tyrosine phosphatase and an extracellular matrix protein may have a role in development of the mammalian central nervous system.

Alternative Splicing↗
PubMed1994

Differential scanning calorimetry study on the binding of nucleic acids to dimyristoylphosphatidylcholine-sphingosine liposomes.

Binding of DNA and RNA to sphingosine-containing dimyristoylphosphatidylcholine (DMPC) liposomes was characterized by differential scanning calorimetry. The thermal phase behaviour of neat DMPC liposomes was unaffected by the presence of the nucleic acids. However, significant alterations in the melting profiles of the DMPC/sphingosine composite membranes were produced by DNA and RNA, thus revealing their binding to the liposomes. For example, for 79:21 (molar ratio) DMPC/sphingosine liposomes a single endotherm at 29.1 degrees C with an enthalpy of 6.3 kcal/mol lipid was observed. In the presence of DNA at the nucleotide/sphingosine ratio of 0.6 this endotherm separated into three distinct peaks at 28.0, 31.4 and 35.1 degrees C, together with an approximately 22% reduction in the total enthalpy. Further increase in DNA concentration up to 1.5 nucleotides per sphingosine led to complete loss of the original heat absorption peak of the DMPC/sphingosine liposomes, while an endotherm at 34.3 degrees C with delta H of 2.7 kcal/mol developed. By visual inspection, rapid and extensive aggregation of the liposomes due to DNA was evident. Evidence for DNA-induced phase separation was also provided by compression isotherms of sphingosine containing DMPC monolayers recorded over an aqueous buffer both in the presence and absence of DNA. The effects of RNA on the thermal phase behaviour of the composite liposomes were qualitatively similar to those described above for DNA. Notably, the presence of eggPA abolished the nucleic acid induced heat capacity changes for DMPC/sphingosine liposomes probably because of neutralization of the positive charge of sphingosine. The binding of DNA to DMPC/sphingosine liposomes occurred both below and above the lipid phase transition temperature, as shown by fluorescence resonance energy transfer utilizing adriamycin-labelled DNA as a quencher and membrane incorporated pyrene-labelled phospholipid as a donor. However, the apparent binding to liquid crystalline liposomes was slightly more effective.

Animals↗