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PubMed1981

Z-DNA conformation of N-2-acetylaminofluorene modified poly(dG-dC).poly(dG-dC) determined by reactivity with anti cytidine antibodies and minimized potential energy calculations.

The conformation of poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), and calf thymus DNA modified with N-acetoxy-N-2-acetylaminofluorene (N-acetoxy-AAF) was examined by extent of reaction with anti cytidine antibodies. In contrast to modified poly(dG).poly(dC0 and DNA, modified poly(dG-dC).poly (dG-dC) failed to react with the antibodies indicating that the base pairing in this polymer is intact. This in consistent with induction of the Z-DNA conformation in AAF modified poly(dG-dC).poly(dG-dC). Using minimized potential energy calculations on the dCpdG-AAF dimer as a model for the modified polymer, it is shown that the proposed Z-DNA conformation is energetically stable. A model is proposed for an AAF modified tetramer, dGpdCpdGpdC, in which the AAF is external to the Z-DNA duplex.

2-Acetylaminofluorene↗
PubMed1981

Conformational changes of poly(dG-dC) . poly(dG-dC) modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene.

Poly (dG-dC) . poly(dG-dC) was modified by the reaction with N-acetoxy-N-acetyl-2-aminofluorene. The conformations of poly(dG-dC) . poly(dG-dC) and of poly d(G-C)AAF were studied by circular dichroism under various experimental conditions. In 95% ethanol, the two polynucleotides adopt the A-form. In 3.9 M LiCl, the transition B-form-C-form is observed with poly(dG-dC) . poly (dG-dC) but not with poly d(G-C)AAF. In 1 mM phosphate buffer, poly d(G-C)AAF behaves as a mixture of B- and Z-form, the relative percentages depending upon the amounts of modified bases. The percentage of Z-form is decreased by addition of EDTA and is increased by addition of Mg++. Spermine favors the Z-form in modified and unmodified polynucleotides. No defect in the double helix of poly d(G-C)AAF is detected by SI endonuclease.

2-Acetylaminofluorene↗
PubMed2005

In vitro synthesis of uniform poly(dG)-poly(dC) by Klenow exo- fragment of polymerase I.

In this paper, we describe a production procedure of the one-to-one double helical complex of poly(dG)-poly(dC), characterized by a well-defined length (up to 10 kb) and narrow size distribution of molecules. Direct evidence of strands slippage during poly(dG)-poly(dC) synthesis by Klenow exo(-) fragment of polymerase I is obtained by fluorescence resonance energy transfer (FRET). We show that the polymer extension results in an increase in the separation distance between fluorescent dyes attached to 5' ends of the strands in time and, as a result, losing communication between the dyes via FRET. Analysis of the products of the early steps of the synthesis by high-performance liquid chromatography and mass spectroscopy suggest that only one nucleotide is added to each of the strand composing poly(dG)-poly(dC) in the elementary step of the polymer extension. We show that proper pairing of a base at the 3' end of the primer strand with a base in sequence of the template strand is required for initiation of the synthesis. If the 3' end nucleotide in either poly(dG) or poly(dC) strand is substituted for A, the polymer does not grow. Introduction of the T-nucleotide into the complementary strand to permit pairing with A-nucleotide results in the restoration of the synthesis. The data reported here correspond with a slippage model of replication, which includes the formation of loops on the 3' ends of both strands composing poly(dG)-poly(dC) and their migration over long-molecular distances (microm) to 5' ends of the strands.

Base Sequence↗
PubMed1994

Purification and characterization of the poly(hydroxyalkanoic acid) synthase from Chromatium vinosum and localization of the enzyme at the surface of poly(hydroxyalkanoic acid) granules.

A recombinant strain of Escherichia coli, which overexpressed phaC and phaE from Chromatium vinosum, was used to isolate poly(3-hydroxyalkanoic acid) synthase. The isolation was performed by a two-step procedure including chromatography on DEAE-Sephacel and Procion Blue H-ERD. The poly(3-hydroxyalkanoic acid) synthase consisted of two different kinds of subunit (PhaC, M(r) 39,500 and PhaE, M(r) 40.500). PhaC was separated from the poly(3-hydroxyalkanoic acid) synthase complex by chromatography on phenyl-Sepharose: PhaE was enriched by solubilization of protein inclusion bodies. The stoichiometry of PhaC and PhaE in the enzyme complex was not determined. The poly(3-hydroxyalkanoic acid) synthase (PhaEC) exhibited a native relative molecular mass of M(r) 400,000 and most probably consists of ten subunits. The Km value of the enzyme for D(-)-3-hydroxybutyryl-CoA was 0.063 mM. The enzyme synthesized poly(3-hydroxybutyric acid) in vitro from D(-)-3-hydroxybutyryl-CoA or, together with propionyl-CoA transferase in a coupled enzyme reaction, synthesized the same product from acetyl-CoA plus D(-)-3-hydroxybutyric acid. Antibodies were raised against both subunits of the poly(3-hydroxyalkanoic acid) synthase. By immunoelectron microscopy, the poly(3-hydroxyalkanoic acid) synthase was localized within the cytoplasm in cells of C. vinosum grown under non-storage conditions. In cells grown under poly(3-hydroxybutyric acid) storage conditions, the enzyme was observed to be located at the surface of the poly(3-hydroxybutyric acid) granules. Immunoblots with anti-PhaC, anti-PhaE IgG and crude extract proteins indicated that poly(3-hydroxyalkanoic acid) synthases with partial sequence similarities are widespread among purple sulphur bacteria.

Acyltransferases↗
PubMed1984

Enzyme-linked immunosorbent assays for antibodies to poly(A), poly dAT and histones: possibly useful tools for the evaluation of prognosis and disease activity in systemic lupus erythematosus.

In a prospective study 222 sera from 56 patients with systemic lupus erythematosus (SLE) were tested for antibodies to poly(A), poly dAT and histones using enzyme-linked immunosorbent assay (ELISA). Patients with active disease had significantly more often antibodies to poly(A), poly dAT and histones than patients with inactive disease. There was a positive correlation between the activity score of the disease and the levels of poly(A)-antibodies of IgG and IgM class, poly dAT-antibodies and antibodies to histones. Patients with SLE-nephritis had a higher level of poly dAT and IgG class poly(A) antibodies than patients without nephritis. Interestingly, patients with an SLE-nephritis had lower levels of IgM-poly(A)-antibodies than those without nephritis. Attempts to use the ELISAs in predicting the SLE exacerbations were unsuccessful. However, the assays can be used as parameters in the estimation of the disease activity.

Adolescent↗
PubMed1990

Detection of poly(I):poly(C12U), mismatched double-stranded RNA, by rapid solution hybridization: blood values after intravenous infusion.

A rapid solution hybridization technique has been developed for estimating blood concentrations of poly(I):poly(C12U), a high molecular weight bioactive double-stranded RNA. Samples were prepared by mixing 100 microL of blood with, 165 microL of 6 M guanidine thiocyanate (GuSCN) and 0.16 M EDTA pH 8.0, and freezing. Hybridizations were carried out with a [3H]poly(C) probe in 3 M GuSCN for 10 min at 37 degrees C. Ribonuclease-resistant hybrids were collected by precipitation with trichloroacetic acid and filtration. Validation studies demonstrated minor interassay variance; the assay was accurate in the range 0.1 to 10 ng poly(I):poly(C12U). Thirty-one blood samples from 15 patients were collected and prepared before and immediately after an average 35 min intravenous infusion of 40-500 mg poly(I):poly(C12U). Postadministration values averaged 48% (s.d. 23%) of the theoretical maximum (range 20-102%). These results confirm previous observations of rapid elimination kinetics of poly(I):poly(C12U) in patients.

Adult↗
PubMed1989

The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro.

Using an in vitro mRNA decay system, we investigated how poly(A) and its associated poly(A)-binding protein (PABP) affect mRNA stability. Cell extracts used in the decay reactions were depleted of functional PABP either by adding excess poly(A) competitor or by passing the extracts over a poly(A)-Sepharose column. Polyadenylated mRNAs for beta-globin, chloramphenicol acetyltransferase, and simian virus 40 virion proteins were degraded 3 to 10 times faster in reactions lacking PABP than in those containing excess PABP. The addition of purified Saccharomyces cerevisiae or human cytoplasmic PABP to PABP-depleted reactions stabilized the polyadenylated mRNAs. In contrast, the decay rates of nonpolyadenylated mRNAs were unaffected by PABP, indicating that both the poly(A) and its binding protein were required for maintaining mRNA stability. A nonspecific single-stranded binding protein from Escherichia coli did not restore stability to polyadenylated mRNA, and the stabilizing effect of PABP was inhibited by anti-PABP antibody. The poly(A) tract was the first mRNA segment to be degraded in PABP-depleted reactions, confirming that the poly(A)-PABP complex was protecting the 3' region from nucleolytic attack. These results indicate that an important function of poly(A), in conjunction with its binding protein, is to protect polyadenylated mRNAs from indiscriminate destruction by cellular nucleases. A model is proposed to explain how the stability of an mRNA could be affected by the stability of its poly(A)-PABP complex.

Binding Sites↗
PubMed1992

Effect of E. coli ribosomal protein S1 on the fidelity of the translational elongation step: reading and misreading of poly(U) and poly(dT).

Ribosomal protein S1 was selectively removed from E. coli ribosomes by affinity chromatography and the effect of added S1 on the translation of poly(dT) [which is read as poly(U) in the presence of neomycin] and on the misreading of poly(U) and poly(dT) were examined. S1 enhances the translation of poly(dT) at low template concentration, which is similar to the effect of S1 on poly(U) translation. The misreading of poly(dT) by E. coli ribosomes is at a lower level than is the case with poly(U). This low misreading is the same for "S1-dependent" and "S1-independent" modes of translation. On the other hand, the misreading of poly(U) is significantly reduced when S1 is present. These results thus indicate that S1 not only facilitates the binding of mRNA to the ribosome as already known, but also plays a role in the correct codon-dependent selection of aminoacyl-tRNA.

Bacterial Proteins↗
PubMed1981

Poly(ADP-ribose) synthetase, a main acceptor of poly(ADP-ribose) in isolated nuclei.

Poly(ADP-ribose) synthetase was identified as the main acceptor of this polymer produced in isolated nuclei of rat liver. When the nuclei were incubated with [32P]NAD at a limited concentration (2.4 microM) and for a brief period (10 s), a protein with Mr = 110,000 was predominantly poly(ADP-ribosyl)ated, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The modification of this protein increased upon longer incubations or at higher NAD concentrations, and induced a marked increase in the apparent molecular weight. A comparison with poly(ADP-ribose) synthetase (Mr = 110,000) of rat liver under various conditions suggested that the increase in the molecular weight of the acceptor resembled that of the synthetase undergoing multiple auto-poly(ADP-ribosyl)ation. This interpretation was further supported by the following observations: 1) [32P]poly(ADP-ribose) attached to the acceptor co-eluted with the synthetase activity from a hydroxyapatite column; 2) the [32P]poly(ADP-ribose).acceptor complex isolated on the column was converted to a very large complex by further incubation with NAD; and 3) a group of large poly(ADP-ribose).acceptor complexes were reduced to a single molecular species with Mr = 110,000 by extensive digestion with poly(ADP-ribose) glycohydrolase. These findings altogether suggested that poly(ADP-ribose) synthesized in isolated nuclei was principally bound to the synthetase itself.

Animals↗
PubMedDate not supplied

[Biological activity of the poly(G).poly(C) complex, modified by divalent platinum compounds].

Modification of poly(G).poly(C) with cys-diaminodichloroplatinum (cys-DDP) at the level of rb = 0.02 increased the in vivo antiviral and interferon-inducing activity of the complex, in contrast to the data reported for complex poly(G).poly(C). Antiinfluenza activity in this case depends on the method of modification and increases more intensively when a ready complex is treated with cys-DDP, as against treatment of poly(G) alone before the formation of a complex with poly(C). If rb is increased, the activity reduces again. Modification with trans-DDP at rb = 0.02 also leads to an increase of antiinfluenza activity of poly(G).poly(C), but mainly after pretreatment of poly(G).

Animals↗
PubMed1994

Enrichment of human DNAs that flank poly(dA).poly(dT) tracts by triplex DNA formation.

Human DNA fragments which contain poly(dA).poly(dT) tracts and their immediate flanking regions were enriched by means of triplex DNA formation. Human DNA fragments were mixed with biotinylated (dT)34 in the presence of Mg2+ and the triplex DNA [(dT)34.poly(dA).poly(dT)] was adsorbed onto streptavidin-coated magnetic beads and the DNA fragments which formed triplexes were eluted from the beads with a buffer containing EDTA. A control experiment using a plasmid with a poly(dA).poly(dT) tract indicated that DNA fragments with the tract could be enriched over 60-fold after one cycle of the treatment. After PCR amplification, the sample was subjected to the next cycle of the affinity enrichment. After four cycles, we obtained a human genomic DNA library of clones with inserts ranging from 500 to 1000 bp among which 86% had at least one poly(dA).poly(dT) tract. No less than 60% of the clones were close or distant members of the Alu family. Sequence determination of 25 clones revealed that the length of the poly(dA).poly(dT) tracts was 14 to 37 (average 28) bp and that they were located close to either end of the fragments. While 15 clones were identified as Alu family homologues and one as a human L1 family member, nine clones were of unknown origin. None of these nine clones were a highly repeated sequence nor a part of transcriptionally active regions.

Bacterial Proteins↗
PubMed1988

Cytoplasmic poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase in AEV-transformed chicken erythroblasts.

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase activities were both investigated in chicken erythroblasts transformed by Avian Erythroblastosis Virus. Respectively 21% and 58% of these activities were found to be present in the post-mitochondrial supernatant (PMS). Fractionation of the PMS on sucrose gradients and poly(A+) mRNA detection by hybridization to [3H] poly(U) show that cytoplasmic poly(ADP-ribose) polymerase is exclusively localized in free mRNP. The glycohydrolase activity sedimented mostly in the 6 S region but 1/3 of the activity was in the free mRNP zone. Seven poly(ADP-ribose) protein acceptors were identified in the PMS in the Mr 21,000-120,000 range. The Mr 120,000 protein corresponds to automodified poly(ADP-ribose) polymerase. A Mr 21,000 protein acceptor is abundant in PMS and a Mr 34,000 is exclusively associated with ribosomes and ribosomal subunits. The existence of both poly(ADP-ribose) polymerase and glycohydrolase activities in free mRNP argues in favour of a role of poly(ADP-ribosylation) in mRNP metabolism. A possible involvement of this post translational modification in the mechanisms of repression-derepression of mRNA is discussed.

Animals↗
PubMed1978

The relationship between hnRNA+-poly(A) and mRNA+-poly (A) in non-dividing human lymphocytes. Evidence for distinct synthetic pathways for mRNA precursor- and nonprecursor-hnRNA.

The processing of hnRNA+-poly(A) to mRNA+-poly(A) has been studied in resting lymphocytes from human peripheral blood. In pulse-chase experiments, two types of hnRNA+-poly(A) have been distinguished: the first is labeled predominantly with exogenous radioactive precursors supplied during the pulse, and the second incorporates primarily scavenged labeled precursors made available during a chase incubation. When the disappearance of both types of hnRNA+-poly(A) was quantitatively compared with the appearance of stable and labile mRNA+-poly(A), only 10% of the anticipated cytoplasmic material was actually obtained. Statistically, 90% of the poly(A)-bearing hnRNA molecules processed were degraded. The two types of hnRNA+-poly(A) were found to be functionally different. Pulse-labeled material was processed to poly(A)-bearing mRNA; "chase-labeled" molecules did not leave the nucleus and never served as precursors for cytoplasmic mRNA. The data fit a model in which there are distinct pathways for mRNA precursor- and nonprecursor-hnRNA+-poly(A).

Cell Nucleus↗
PubMed1993

Production of a monoclonal antibody reactive with poly(A) and its application in the isolation of poly(A)+ RNA.

Hybridoma cells producing antibody reactive with poly(A) were isolated following fusion of spleen cells from a BALB/c mouse immunized with poly(A).poly(dT) and myeloma cells. The antibody was purified from ascitic fluid by the formation of immune complexes with poly(A). The antibody reacted with poly(A) and some homopolymers (poly(dT), poly(I) and poly(dC)) which was most probably due to its reaction with the ribose-phosphate part of the molecules, but not with cellular RNAs and DNA. Poly(A) tailed 5 S rRNA (about 20 nucleotides) and tobacco mosaic virus RNA (1060 nucleotides) were precipitated quantitatively by the antibody. Dot hybridization and in vitro translation confirmed that the RNA precipitated by the antibody from mouse spleen total RNA contained biologically active mRNA.

Antibodies, Monoclonal↗
PubMed1978

Steps in the processing of Ad2 mRNA: poly(A)+ nuclear sequences are conserved and poly(A) addition precedes splicing.

The conservation of nuclear Ad2 sequences during nucleocytoplasmic transport has been estimated from the accumulation of 3H-uridine in nuclear and cytoplasmic Ad2-specific RNA from the major late transcription unit. From 10-28% is conserved of the total Ad2 nuclear RNA synthesized from each of five regions of the genome that specify groups of 3' co-terminal mRNAs. The sum of the conservation of all the regions was equivalent to 100%, signifying the conservation of at least a part of each transcript or all of about one fifth to one sixth of the transcripts. The conservation of poly(A)+ Ad2 nuclear RNA is about 4 times greater than of total Ad2 nuclear RNA, approaching 100% conservation of poly(A)+ nuclear sequences. Since each mRNA contains three "spliced" sequences that are probably encoded only once per transcript, these data on conservation of the Ad2 sequences suggest that each transcriptional event from the 16-99 transcription unit gives rise to one of a possible 13-14 mRNA molecules with destruction of the remainder of the transcribed RNA. The portion which is conserved resides next to the region to which which poly(A) is added. Three models for the choice of poly(A) sites were considered: termination at the poly(A) site, cleavage shortly after synthesis of one of the sites before transcription was complete, and cleavage after completion of transcription. The first model was ruled out by the demonstration of equimolar synthesis over the 16-99 region. The second model is strongly supported because 3H-uridine label appears equally rapidly in the time range 2-10 min in each of the five 3' poly(A) addition sites, whereas chain completion before cleavage would lead to a faster appearance of label in the most promoter-distal site. Furthermore, briefly labeled RNA molecules extending from 16 to each of several poly(A) addition sites were the first poly(A)- terminated 3H-uridine-labeled molecules detected, demonstrating that poly(A) addition precedes splicing. The choice of which mRNA emerges from each transcriptional event would appear to depend upon first choosing one of five 3' mRNA ends followed by a 5' splicing event.

Adenoviruses, Human↗
PubMed2000

Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I.

Current evidence suggests that the length of poly(A) tails of bacterial mRNAs result from a competition between poly(A) polymerase and exoribonucleases that attack the 3' ends of RNAs. Here, we show that host factor Hfq is also involved in poly(A) tail metabolism. Inactivation of the hfq gene reduces the length of poly(A) tails synthesized at the 3' end of the rpsO mRNA by poly(A) polymerase I in vivo. In vitro, Hfq stimulates synthesis of long tails by poly(A) polymerase I. The strong binding of Hfq to oligoadenylated RNA probably explains why it stimulates elongation of primers that already harbor tails of 20-35 A. Polyadenylation becomes processive in the presence of Hfq. The similar properties of Hfq and the PABPII poly(A) binding protein, which stimulates poly(A) tail elongation in mammals, indicates that similar mechanisms control poly(A) tail synthesis in prokaryotes and eukaryotes.

Carrier Proteins↗
PubMed1999

The poly(A)-limiting element is a conserved cis-acting sequence that regulates poly(A) tail length on nuclear pre-mRNAs.

Most vertebrate mRNAs exit the nucleus with a 200+-residue poly(A) tail and are deadenylated to yield heterogeneous polymers of 50-200 adenosine residues on any given mRNA. We previously reported that Xenopus albumin mRNA and pre-mRNA have an unusually short, discrete 17-residue poly(A) tail and showed that regulation of poly(A) length is controlled independently by two cis-acting poly(A)-limiting elements (PLE A and PLE B) located in the terminal exon. The present study sought to determine the generality of this regulatory mechanism. Transferrin mRNA also has a discrete <20-nt poly(A) tail, and deletion mapping experiments identified an element homologous to the albumin gene PLE B within the terminal exon of the transferrin gene that conferred poly(A) length regulation on a globin reporter mRNA. Based on this similarity the PLE B sequence was used in a database search to identify candidate mRNA targets for regulated polyadenylation. Of the several hundred sequences identified in this manner we focused on HIV-EP2/Schnurri-2, a member of a family of genes encoding related zinc finger transcription factors. A striking feature of the PLE-like element in these genes is its location 10-33 bp upstream of the translation stop codon. We demonstrate that HIV-EP2 mRNA has a <20-nt poly(A) tail, for which the identified PLE-like sequence is responsible. These results indicate that the presence of a PLE can predict mRNAs with <20-nt poly(A) tails, and that nuclear regulation of poly(A) tail length is a feature of many mRNAs.

Animals↗
PubMed1984

Antigenicity of mouse interferons induced in vitro and in vivo by poly(I).poly(C).

Various mouse IFNs induced by poly(I).poly(C) or poly(ICLC) were analyzed for the antigenic types by neutralization tests using anti-IFN-alpha and anti-IFN-beta antibodies. The IFN samples included IFNs produced by L cells, by spleen cells in vitro and ex vivo, and in plasma of mice injected with the inducers. L-cell IFN was estimated to consist of IFN-beta and IFN-alpha at a ratio of about 80:20. Spleen IFNs also consisted of both IFN species, but IFN-alpha was the major component. Blood IFN samples obtained 1-4 h after injection of poly(I).poly(C) or poly(ICLC) consisted of the two molecular species, whereas the samples 8 and 24 h after injection contained only IFN-alpha. High levels of IFN activity (IFN-alpha) persisted much longer in the circulation of mice injected with poly(ICLC) than in mice injected with poly(I).poly(C).

Animals↗