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Results for “Poly(A) selection”

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At least 127 records · Page 7Linked to original sources

PubMed2004

Solvent-induced microphase separation in diblock copolymer thin films with reversibly switchable morphology.

We have studied the surface morphology of symmetric poly(styrene)-block-poly(methyl methacrylate) diblock copolymer thin films after solvent vapor treatment selective for poly(methyl methacrylate). Highly ordered nanoscale depressions or striped morphologies are obtained by varying the solvent annealing time. The resulting nanostructured films turn out to be sensitive to the surrounding medium, that is, their morphologies and surface properties can be reversibly switchable upon exposure to different block-selective solvents.

Journal Article↗

PubMed2001

Effect of pendant chain lengths and backbone functionalities on the chemical selectivity of sulfonated amphiphilic copolymers as pseudo-stationary phases in electrokinetic chromatography.

Amphiphilic copolymers of AMPS (2-acrylamido-2-methyl-1-propanesulfonic acid) and hydrophobic monomers with various chemical structures were synthesized, characterized and used as novel electrokinetic chromatography polymeric pseudo-stationary phases, showing significant chemical selectivity differences from that of the conventional monomeric pseudo-stationary phase, sodium lauryl sulphate. Copolymers of AMPS and methacrylates with different pendant chain lengths (C8, C12 and C18) were investigated and no significant difference in chemical selectivity was observed among them. However, the spacer bonding chemistry was shown to contribute to significant chemical selectivity difference, e.g. poly(AMPS-lauryl methacrylate) showed different chemical selectivity from poly(AMPS-lauryl methacrylamide). Linear solvation energy relationship analysis of 20 solutes by eight different polymeric pseudo-stationary phases was employed to investigate the solute molecule structural contributions to the retention. Hydrogen-bonding properties (described by system constants b and a) of poly(AMPS-alkyl methacrylamide) were found stronger than those of poly(AMPS-alkyl methacrylate).

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2025

RNA Sequencing Protocols for Short-Read Sequencing.

RNA sequencing (RNA-seq) methodologies allow the discovery of novel variants and transcripts. These comprise three general steps: (1) capture of RNA species of interest, (2) conversion of RNA to complementary DNA (cDNA), and (3) modification of cDNA to fit the sequencing platform. Here we describe four different library preparation protocols for short-read sequencing: cDNA synthesis with poly(A) selection, library preparation with ribosomal depletion, and cDNA synthesis with SMART® (Switching Mechanism at 5' end of RNA Template) technology for low and Pico inputs.

Gene Library↗

PubMed1996

Induction of murine erythroleukemia cell differentiation is associated with methylation and differential stability of poly(A)+ RNA transcripts.

Murine erythroleukemia (MEL) cells exposed to DMSO were assessed for their ability to methylate poly(A)+ RNA and accumulate RNA transcripts of globin and nonglobin genes (c-myc, beta-actin and MER5). Cells were pulse-labeled with L-[methyl-3H]methionine, cytoplasmic RNA was isolated, selected for poly(A)+ RNA and analyzed by HPLC chromatography for methylated nucleosides. When MEL cells were exposed to inhibitors of RNA methylation (neplanocin A, 3-deazaneplanocin A and cycloleucine) and assessed for their ability to differentiate by DMSO, accumulate RNA transcripts, produce hemoglobin, methylate poly(A)+ and poly(A)- RNA and synthesize S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), we observed the following: (a) MEL cells treated with DMSO underwent hypermethylation in poly(A)+ RNA that preferentially occurred at the 5'-cap structures (7-methylguanosine and 2'-O-methylcytidine and 2'-O-methyluridine); (b) inducer-treated MEL cells exhibited a decrease in the intracellular level of SAH that led to a lower ratio of SAH/SAM, an event that favors methylation; and (c) treatment of MEL cells with inhibitors of RNA methylation suppressed methylation of poly(A)- and poly(A)+ RNA, reversed the ratio SAH/SAM seen in differentiated MEL cells and prevented differentiation to occur. Moreover, we observed that treatment of MEL cells with selective inhibitors of RNA methylation caused fragmentation of beta major globin and c-myc mRNAs, two RNA transcripts coded by developmentally regulated genes, while had no detectable effect on the structural integrity of poly(A)+ RNA transcripts transcribed by two housekeeping genes (beta-actin and MER5). These data indicate that induction of erythroid cell differentiation of MEL cells is associated with changes in methylation of poly(A)+ RNA and selective differential stability of RNA transcripts, two events that might be related to each other.

Actins↗

PubMed2004

Stability of the inner polyaniline solid contact layer in all-solid-state K+-selective electrodes based on plasticized poly(vinyl chloride).

A simple and powerful method based on UV-visible spectroscopy is presented for studying the stability of the inner electrically conducting polyaniline (PANI) solid contact (SC) layer in all-solid-state ion-selective electrodes (ISE). The influence of the plasticized poly(vinyl chloride) (PVC) membrane (ISM) composition and the pH of the sample solution on the stability of the solid contact is reported. PANI is used as a model compound in this study, but the method presented is universal and can be applied to different types and combinations of SCs and ISMs. It provides a tool for finding the best combination of conducting polymer and ISM for solid contact ISEs. PANI is deposited electrochemically either on glassy carbon or quartz glass covered with a thin layer of tin oxide, and a K+-selective ISM is deposited on top of the PANI layer. The short-term stability of the inner PANI layer is good for all membrane types in buffer solutions with pH 2, 6, and 9, indicating that the outer plasticized PVC membrane hinders the emeraldine salt-emeraldine base transition of the highly pH sensitive PANI layer. The solid contact K+-selective electrodes studied showed a Nernstian response of 58.2 +/- 0.1 mV/log aK. Significant differences are observed in the long-term stability of the inner PANI layer between the different membrane types. This indicates that water uptake of the PVC membrane and its permeability to OH- ions are critical parameters affecting the stability of the PANI layer. The solid contact electrodes based on PANI may require a composition of the PVC membrane different from those typically used in conventional ISEs with an inner solution.

Journal Article↗

PubMed2001

Physiology, regulation, and limits of the synthesis of poly(3HB).

The properties of poly(3-hydroxybutyrate) combined with the fact that it can be produced easily by numerous prokaryotes from renewable resources and even from potentially toxic waste products using well-known fermentation processes have generated keen interest in this biopolyester as a substitute for chemo-synthetic petroleum-derived polymers in many applications. However, the high price of poly(3HB) compared with the conventional synthetic materials currently in use has restricted its availability in a wide range of applications. If the economic viability of poly(3HB) production and its competitiveness are to be improved, more must be found out about the phenotypic optimization and the upper limits of bacterial systems as the factory of poly(3HB). In this chapter, two aspects of poly(3HB) are reviewed--poly(3HB) formation as a physiological response to external limitations and overcoming internal bottlenecks, and poly(3HB) as a commercially attractive polyester. From a physiological viewpoint, the ability to synthesize and degrade poly(3HB) is considered an investment in the future and provides organisms with a selective advantage. Poly(3HB) is presented as a strategic survival polymer, and it is shown that growth-associated synthesis is not as rare as reported. The influence of the efficiency and velocity of cell multiplication and product formation, of poly(3HB) content and of productivity on the overall yield, and finally on the economics of the whole process are discussed and evaluated from the technological or consumer's point of view. The specific production rate and poly(3HB) content appear to be more important than the yield coefficients.

Chemical Engineering↗

PubMed1997

Sequence and structure specific antibodies from phage display libraries.

A large combinatorial phage display library was panned against five nucleic acid antigens, calf thymus DNA, poly[d(GC)], poly[d(AT)], poly(dA) x poly(dT) and poly(rA) x poly(dT). After the third and fourth rounds of panning, many positive clones were selected against poly[d(GC)], poly(dA) x poly(dT) and poly(rA) x poly(dT). The specificity of these antibodies was tested by both direct and competitive solid phase radioimmune assays. All the clones derived from panning with poly[d(GC)] were non-specific and bound to all nucleic acids. The poly(rA) x poly(dT) derived clones were specific for single-stranded nucleic acids, with some sequence preferences, and the poly(dA) x poly(dT) derived clones showed considerable specificity for this antigen. The sequences of these phage-derived antibodies showed no similarities with DNA-binding antibodies from other sources. Even after six rounds of panning no positive clones were detected which bound to poly[d(AT)] and after seven rounds only two were derived from panning with calf thymus DNA. Therefore, sequence- and structure specific antibodies can be recovered from phage display libraries but not all sequences may be represented in the repertoire.

Amino Acid Sequence↗

PubMed1991

Occlusion of the HIV poly(A) site.

To investigate the selective use of poly(A) sites in the 3' long terminal repeat (LTR) but not the 5' LTR of retroviruses, we have studied the poly(A) site of the human immunodeficiency virus (HIV-1). Using hybrid HIV/alpha-globin gene constructs, we demonstrate that the HIV poly(A) site is inactive or occluded when adjacent to an active promoter, either the homologous HIV promoter or the alpha-globin gene promoter. Furthermore, this occlusion of the HIV poly(A) site occurs over a considerable distance of up to at least 500 bp. In contrast, two nonretroviral poly(A) sites [alpha-globin and a synthetic poly(A) site] are active when close to a promoter. We also show that a short fragment of approximately 60 nucleotides containing the HIV poly(A) site is fully active when placed at the 3' end of the human alpha-globin gene or within the rabbit beta-globin gene. This result rules out the requirement of more distant upstream elements for the activity of the HIV poly(A) site, as has been suggested for other viral poly(A) sites. Finally, we show that the GT-rich downstream region of the HIV poly(A) site confers poly(A) site occlusion properties on a synthetic poly(A) site. This result focuses attention on this more variable part of a poly(A) site in retroviruses as a possible general signal for poly(A) site occlusion.

Animals↗

PubMed1999

Selective assembly of cyclodextrins on poly(ethylene oxide)-poly(propylene oxide) block copolymers.

This paper presents a computational study on the formation of a molecular necklace formed by specific threading of cyclodextrins (CDs) on block copolymers. Structural as well as energetic principles for the selective complexation of alpha- and beta-cyclodextrin with poly(ethylene oxide)-poly(propylene oxide) block copolymers (PEO-PPO) are elucidated considering a diblock copolymer of equimolecular composition (PEO)4-(PPO)4 as guest. A non-statistical distribution of CDs, i.e. alpha-CDs primarily located on the PEO chain and beta-CDs on PPO blocks of the polymer, is based on a variety of structural features and energetic preferences considering both potential as well as solvation energies. This selectivity becomes already obvious considering 1:1 complexes between PEO and PPO monomers and the two CDs, but is increasingly evident when calculating higher order ensembles. Besides the host-guest interaction, docking between CDs themselves is an important, also non-statistical, prerequisite for the self-assembly of highly ordered tubes. The formation of intermolecular hydrogen bonds between adjacent CDs in a tubular aggregate gives an important contribution to the overall stability of the molecular necklace. The net effect, based on the preferential interaction between host and guest as well as between the host molecules themselves, results in the formation of a stable, highly ordered macromolecular, multicomponent aggregate.

Cyclodextrins↗

PubMed2005

Supercritical fluid crystallization of griseofulvin: crystal habit modification with a selective growth inhibitor.

Poly (sebacic anhydride) (PSA) was used as a growth inhibitor to selectively modify habit of griseofulvin crystals formed via the Precipitation with a compressed-fluid antisolvent (PCA) process. PSA and griseofulvin were coprecipitated within a PCA injector, which provided efficient mixing between the solution and compressed antisolvent process streams. Griseofulvin crystal habit was modified from acicular to bipyramidal when the mass ratio of PSA/griseofulvin in the solution feed stream was <or=1:1. The habit modification was attributed to the preferential adsorption of PSA to the fastest growing crystal face of the acicular crystal form, which inhibited growth. Scanning electron microscopy (SEM) was used to characterize the griseofulvin and PSA particles, and gave results consistent with a selective growth inhibition mechanism. SEM micrographs showed regions on griseofulvin crystals where PSA microparticles had preferentially adsorbed. X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) analysis of the griseofulvin crystals indicated no changes in the crystalline form after the habit modification. Powder compressibility decreased from 49 +/- 3% to 28 +/- 7% with the modification in crystal habit. No change in the physical stability of the processed powder was observed after being stored at 25 degrees C/60% RH and 40 degrees C/70% RH for 23 days. Despite the change in crystal habit, griseofulvin crystals achieved 100% dissolution within 60 min in a simulated gastric fluid.

Anhydrides↗

PubMed1977

Comparative studies on the '5'-cap' and in vitro translational activity of cytoplasmic and nuclear poly A(+) RNA1.

The translational activities of cytoplasmic poly A(+)RNA of normal rat liver and Novikoff hepatoma cells in the wheat germ cell free system were found to be approximately 15-20 times greater than tose of the corresponding nuclear poly A(+) RNA. The translationsl activities were 85 and 62 pmoles 3H-leucine incorporated/micron g cytoglasmic poly A(+) RNA for the liver and tumor respectively and 3-4 pmoles 3H-leucine incoporated/micron g nuclear poly A(+)RNA. Inasmuch as intergity of the '5'-cap' of mRNA is essential for its translational activity, quantitative comparisons were made of its content in these RNA fractions. Of the total 32P incorporated into the tumor cytoplasmic poly A(+) RNA, 0.41% was in the '5'-cap'; in nuclear poly A(+) RNA, the '5'-cap' contained 0.11%. After periodate oxidation and labeling with KB3H4, m7 guanosine, the 5'-terminal nucleoside in both liver and Novikoff hepatoma nuclear poly A(+) RNA contained approximately 20% as much isotope as in the cytoplasmic poly A(+) RNA. These results suggest the lower translational activity of nuclear poly A(+) RNA is partly related to its lower content of the '5'-cap'. Molecular selection of poly A(+) RNA for transport out of the nucleus or further cytoplasmic processing may account for the higher percentage of the '5-cap' and the greater translational activity of the cytoplasmic poly A(+) RNA. During these studies, it was also found that the m7 guanosine of the '5'-cap' was not removed during translation of the mRNA in the wheat germ system; this result suggests that the '5'-cap' may associate with allosteric binding sites of initiation factor(s).

Animals↗

PubMed2006

Hu proteins regulate polyadenylation by blocking sites containing U-rich sequences.

A recent genome-wide bioinformatic analysis indicated that 54% of human genes undergo alternative polyadenylation. Although it is clear that differential selection of poly(A) sites can alter gene expression, resulting in significant biological consequences, the mechanisms that regulate polyadenylation are poorly understood. Here we report that the neuron-specific members of a family of RNA-binding proteins, Hu proteins, known to regulate mRNA stability and translation in the cytoplasm, play an important role in polyadenylation regulation. Hu proteins are homologs of the Drosophila embryonic lethal abnormal visual protein and contain three RNA recognition motifs. Using an in vitro polyadenylation assay with HeLa cell nuclear extract and recombinant Hu proteins, we have shown that Hu proteins selectively block both cleavage and poly(A) addition at sites containing U-rich sequences. Hu proteins have no effect on poly(A) sites that do not contain U-rich sequences or sites in which the U-rich sequences are mutated. All three RNA recognition motifs of Hu proteins are required for this activity. Overexpression of HuR in HeLa cells also blocks polyadenylation at a poly(A) signal that contains U-rich sequences. Hu proteins block the interaction between the polyadenylation cleavage stimulation factor 64-kDa subunit and RNA most likely through direct interaction with poly(A) cleavage stimulation factor 64-kDa subunit and cleavage and polyadenylation specificity factor 160-kDa subunit. These studies identify a novel group of mammalian polyadenylation regulators. Furthermore, they define a previously unknown nuclear function of Hu proteins.

Animals↗

PubMed2004

Single nanocrystal arrays on patterned poly(ethylene glycol) copolymer microstructures using selective wetting and drying.

Single nanocrystal arrays were fabricated on sub-microwells of poly(ethylene glycol) (PEG) copolymer using selective wetting on the hydrophilic regions of the exposed substrate surface and subsequent drying. Templates were produced by molding a thin film of a PEG-based random copolymer on hydrophilic substrates such as glass or silicon dioxide. The polymeric microstructures provide a topographical barrier around the well, which makes it possible to create nanocrystal arrays with controlled geometrical features. The size of the nanocrystal was found to decrease with decreasing well size and also decrease with decreasing topological height. A simple empirical equation was derived to predict the size of the crystal as a function of the pattern size and height, which is in good agreement with the experimental data.

Journal Article↗

PubMed1995

Bop: a new T-cell-restricted gene located upstream of and opposite to mouse CD8b.

In the course of transient expression studies undertaken to determine the location of the mouse CD8b gene promoter, two additional promoter activities were detected within 600 nucleotides upstream of the gene. One activity directs transcription in the same direction as CD8b but fails to transcribe the CAT reporter gene due to an apparent transcription-blocking element lying between it and the gene. The second activity directs transcription opposite to that of the CD8b gene. Northern hybridization with a probe consisting of nucleotides -875 to -550 relative to the site of CD8b transcription initiation revealed hybridizing species of 4 kilobases (kb) and 1.8 kb in poly-A-selected RNA from mouse thymus but not from any other tissues. Similar RNA species were detected in poly-A+ RNA from concanavalin A-stimulated spleen cells and several long-term CTL lines but not from the EL4 or BW5147 T-cell lines or the J558L myeloma. The mRNA species were most abundant in cells of a secondary mixed leukocyte culture which were greater than 95% CD8(+). Northern hybridizations using single-stranded unidirectional probes indicated that these mRNAs represent transcription opposite to the CD8b gene. The tissue and cell type distribution of this newly-discovered gene (designated Bop for CD8b opposite) are consistent with T-cell-specific and possibly CD8-positive T-cell-specific expression. The head-to-head arrangement of the Bop and CD8b genes is reminiscent of the arrangement of the Tap1 and Lmp2 genes, and the expression of the Bop gene in CD8-positive cells raises the possibility that these genes are involved in the same functional pathway.

Animals↗

PubMed1996

Restoration of both structure and function to a defective poly(A) site by in vitro selection.

Efficient cleavage and polyadenylation at the human immunodeficiency virus type-1 (HIV-1) poly(A) site requires an upstream 3'-processing enhancer to overcome the suboptimal sequence context of the AAUAAA hexamer. The HIV-1 3'-processing enhancer functions to stabilize the association of the pre-mRNA with cleavage and polyadenylation specificity factor (CPSF), the factor responsible for recognition of the AAUAAA hexamer. Intriguingly, in the absence of the 3'-processing enhancer, CPSF binding and polyadenylation efficiency could be restored to near wild-type levels upon replacement of the 14-nucleotide region immediately 5' of the HIV-1 AAUAAA hexamer (the B segment) by the analogous sequences from the efficient adenovirus L3 poly(A) site. To further investigate the contributions of RNA sequence and structure to poly(A) site recognition, we have used an in vitro selection system to identify B segment sequences that enhance the polyadenylation efficiency of a pre-cleaved RNA lacking a 3'-processing enhancer. The final RNA selection pool was composed of two predominant classes of RNAs. Nuclease probing revealed that the selected sequences restored an RNA conformation that facilitates recognition of the AAUAAA hexamer by CPSF. These results indicate that both the sequence and structural context of the AAUAAA hexamer contribute to poly(A) site recognition by CPSF.

Base Sequence↗

PubMed1999

Cystic fibrosis transmembrane conductance regulator. Physical basis for lyotropic anion selectivity patterns.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel exhibits lyotropic anion selectivity. Anions that are more readily dehydrated than Cl exhibit permeability ratios (P(S)/P(Cl)) greater than unity and also bind more tightly in the channel. We compared the selectivity of CFTR to that of a synthetic anion-selective membrane [poly(vinyl chloride)-tridodecylmethylammonium chloride; PVC-TDMAC] for which the nature of the physical process that governs the anion-selective response is more readily apparent. The permeability and binding selectivity patterns of CFTR differed only by a multiplicative constant from that of the PVC-TDMAC membrane; and a continuum electrostatic model suggested that both patterns could be understood in terms of the differences in the relative stabilization of anions by water and the polarizable interior of the channel or synthetic membrane. The calculated energies of anion-channel interaction, derived from measurements of either permeability or binding, varied as a linear function of inverse ionic radius (1/r), as expected from a Born-type model of ion charging in a medium characterized by an effective dielectric constant of 19. The model predicts that large anions, like SCN, although they experience weaker interactions (relative to Cl) with water and also with the channel, are more permeant than Cl because anion-water energy is a steeper function of 1/r than is the anion-channel energy. These large anions also bind more tightly for the same reason: the reduced energy of hydration allows the net transfer energy (the well depth) to be more negative. This simple selectivity mechanism that governs permeability and binding acts to optimize the function of CFTR as a Cl filter. Anions that are smaller (more difficult to dehydrate) than Cl are energetically retarded from entering the channel, while the larger (more readily dehydrated) anions are retarded in their passage by "sticking" within the channel.

Algorithms↗

PubMed1992

Liver-specific RNA processing of the ubiquitously transcribed rat fibrinogen gamma-chain gene.

Fibrinogen gamma-chains differ in amino acid sequence at the carboxyterminus due to alternative 3' RNA processing. Previous studies reported differences between humans and rats in the mechanism of gamma-chain RNA processing and that it was a nonregulated event. To test the hypothesis that rat gamma-chain RNA processing involves both alternative splicing and poly(A) site selection and that it is regulated in a tissue-specific manner, we determined the tissue distribution of gamma-chain mRNA expression and the pattern of gamma-chain pre-mRNA processing. The results of in situ hybridization demonstrated that gamma A and gamma B transcripts were localized to and codistributed in liver hepatocytes, indicating that no subset of cells process gamma B mRNA. The ubiquitous expression of the fibrinogen gamma-chain promoter was demonstrated in marrow, lung, brain, and liver by RNase protection using a 5'-specific gamma-chain probe. RNase protection studies to map 3' RNA processing sites suggested that, in addition to the distal poly(A) signal previously identified, two alternative poly(A) signals within the last intron (ATTAAA and AATAAA) were used only in liver to produce gamma B transcripts. Approximately equal usage of the three poly(A) signals (27%, 37%, and 36%, respectively) to form the 3' end of mature gamma B transcripts suggested that poly(A) site selection is random. These results indicate that splicing of the last intron to produce gamma A mRNA is the ubiquitous and constitutive pattern of gamma-chain RNA processing, while retention of the last intron to produce gamma B mRNAs is the tissue-specific and regulated pattern of gamma-chain RNA processing. The pattern of rat gamma-chain RNA processing is similar to human, implying that the mechanism is conserved. These data support a mechanism of tissue-specific splice site selection predominating over poly(A) site selection in gamma-chain pre-mRNA processing. The expression of both fibrinogen gamma-chain transcripts in liver, rather than mutually exclusive expression in liver and other tissues, provides a new model for studying tissue-specific alternative 3' end formation regulatory mechanisms.

Animals↗

PubMed1999

Blood interactions with plasticized poly(vinyl chloride): relevance of plasticizer selection.

An investigation has been made of blood interactions with plasticized poly(vinyl chloride) (PVC) biomaterials in tubular form, taking into account the influence on the blood response of the polymer, antithrombotic agent, blood condition and test procedure. In vitro and ex vivo procedures were used to achieve a comparison between PVC plasticized with di- (2-ethylhexyl)phthalate (DEHP) and with tri-(2-ethylhexyl)trimellitate (TEHTM). The blood response was monitored in terms of the measurement of fibrinogen adsorption capacity, thrombin-antithrombin III complex (TAT) and the complement component C3a. Surface characterization of the polymers was performed by X-ray photoelectron spectroscopy (XPS). The data obtained indicate that in comparison with DEHP-PVC, there is a higher reactivity for TEHTM-PVC, which correlates with the plasticizer distribution at the polymer surface.

Journal Article↗