Pseudouridine modification of U5 RNA in ribonucleoprotein particles assembled in vitro

1991 ◽  
Vol 11 (12) ◽  
pp. 5998-6006
Author(s):  
J R Patton
Keyword(s):  
Time Course ◽  
Buoyant Density ◽  
Early Time ◽  
Creatine Phosphate ◽  
Stem Loop ◽  
Cell Extracts ◽  
Nuclear Extracts ◽  
Binding Sequence ◽  
Made In

The formation of pseudouridine (psi) in U5 RNA during ribonucleoprotein (RNP) assembly was investigated by using HeLa cell extracts. In vitro transcribed, unmodified U5 RNA assembled into an RNP particle with the same buoyant density and sedimentation velocity as did U5 small nuclear RNP from extracts. The greatest amount of psi modification was detected when a combination of S100 and nuclear extracts was used for assembly. psi formation was inhibited when ATP and creatine phosphate or MgCl2 were not included in the assembly reaction, paralleling the inhibition of RNP particle formation. A time course of assembly and psi formation showed that psi modification lags behind RNP assembly and that at very early time points, Sm-reactive U5 small nuclear RNPs are not modified. Two of three psi modifications normally found in U5 RNA were present in RNA incubated in the extracts. Mutations in the form of deletions and truncations were made in the U5 sequence, and the effect of these mutations on psi formation was investigated. A mutation in the area of stem-loop I which contains the psi moieties or in the Sm binding sequence affected psi formation.

scholarly journals Pseudouridine modification of U5 RNA in ribonucleoprotein particles assembled in vitro.

1991 ◽  
Vol 11 (12) ◽  
pp. 5998-6006 ◽  
Author(s):  
J R Patton
Keyword(s):  
Time Course ◽  
Buoyant Density ◽  
Early Time ◽  
Creatine Phosphate ◽  
Stem Loop ◽  
Cell Extracts ◽  
Nuclear Extracts ◽  
Binding Sequence ◽  
Made In

The formation of pseudouridine (psi) in U5 RNA during ribonucleoprotein (RNP) assembly was investigated by using HeLa cell extracts. In vitro transcribed, unmodified U5 RNA assembled into an RNP particle with the same buoyant density and sedimentation velocity as did U5 small nuclear RNP from extracts. The greatest amount of psi modification was detected when a combination of S100 and nuclear extracts was used for assembly. psi formation was inhibited when ATP and creatine phosphate or MgCl2 were not included in the assembly reaction, paralleling the inhibition of RNP particle formation. A time course of assembly and psi formation showed that psi modification lags behind RNP assembly and that at very early time points, Sm-reactive U5 small nuclear RNPs are not modified. Two of three psi modifications normally found in U5 RNA were present in RNA incubated in the extracts. Mutations in the form of deletions and truncations were made in the U5 sequence, and the effect of these mutations on psi formation was investigated. A mutation in the area of stem-loop I which contains the psi moieties or in the Sm binding sequence affected psi formation.

F-actin serves as a template for cytokeratin organization in cell free extracts

2002 ◽  
Vol 115 (7) ◽  
pp. 1373-1382 ◽  
Author(s):  
Kari L. Weber ◽  
William M. Bement
Keyword(s):  
Time Course ◽  
Early Time ◽  
Dynamic Imaging ◽  
Actin Network ◽  
Intact Cells ◽  
Time Points ◽  
Egg Extracts ◽  
Actin Cables

The microtubule, F-actin, and intermediate filament systems are often studied as isolated systems, yet the three display mutual interdependence in living cells. To overcome limitations inherent in analysis of polymer-polymer interactions in intact cells, associations between these systems were assessed in Xenopus egg extracts. In both fixed and unfixed extract preparations, cytokeratin associated with F-actin cables that spontaneously assembled in the extracts. Time-course experiments revealed that at early time points cytokeratin cables were invariably associated with F-actin cables,while at later time points they could be found without associated F-actin. In extract samples where F-actin assembly was prevented, cytokeratin formed unorganized aggregates rather than cables. Dynamic imaging revealed transport of cytokeratin by moving F-actin as well as examples of cytokeratin release from F-actin. Experimental alteration of F-actin network organization by addition of α-actinin resulted in a corresponding change in the organization of the cytokeratin network. Finally, pharmacological disruption of the F-actin network in intact, activated eggs disrupted the normal pattern of cytokeratin assembly. These results provide direct evidence for an association between F-actin and cytokeratin in vitro and in vivo, and indicate that this interaction is necessary for proper cytokeratin assembly after transition into the first mitotic interphase of Xenopus.

Effect of double-strand breaks on homologous recombination in mammalian cells and extracts

1985 ◽  
Vol 5 (12) ◽  
pp. 3331-3336
Author(s):  
K Y Song ◽  
L Chekuri ◽  
S Rauth ◽  
S Ehrlich ◽  
R Kucherlapati
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Double Strand Breaks ◽  
Base Pairs ◽  
In Vitro System ◽  
Strand Breaks ◽  
Cell Extracts ◽  
Nuclear Extracts

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.

scholarly journals Hazard and risk assessment strategies for nanoparticle exposures: how far have we come in the past 10 years?

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 376 ◽  
Author(s):  
David B Warheit
Keyword(s):  
Health Effects ◽  
Ultrafine Particles ◽  
Time Course ◽  
The European Union ◽  
Particle Size Range ◽  
Potential Health ◽  
The Past ◽  
Safety Research ◽  
Made In

Nanotechnology is an emerging, cross-disciplinary technology designed to create and synthesize new materials at the nanoscale (generally defined as a particle size range of ≤10-9 meters) to generate innovative or altered material properties. The particle properties can be modified to promote different and more flexible applications, resulting in consumer benefits, particularly in medical, cosmetic, and industrial applications. As this applied science matures and flourishes, concerns have arisen regarding potential health effects of exposures to untested materials, as many newly developed products have not been adequately evaluated. Indeed, it is necessary to ensure that societal and commercial advantages are not outweighed by potential human health or environmental disadvantages. Therefore, a variety of international planning activities or research efforts have been proposed or implemented, particularly in the European Union and United States, with the expectation that significant advances will be made in understanding potential hazards related to exposures in the occupational and/or consumer environments. One of the first conclusions reached regarding hazardous effects of nanoparticles stemmed from the findings of early pulmonary toxicology studies, suggesting that lung exposures to ultrafine particles were more toxic than those to larger, fine-sized particles of similar chemistry. This review documents some of the conceptual planning efforts, implementation strategies/activities, and research accomplishments over the past 10 years or so. It also highlights (in this author’s opinion) some shortcomings in the research efforts and accomplishments over the same duration. In general, much progress has been made in developing and implementing environmental, health, and safety research-based protocols for addressing nanosafety issues. However, challenges remain in adequately investigating health effects given 1) many different nanomaterial types, 2) various potential routes of exposure, 3) nanomaterial characterization issues, 4) limitations in research methodologies, such as time-course and dose-response issues, and 5) inadequate in vitro methodologies for in vivo standardized, guideline toxicity testing.

The ovotestis of Aplysia californica: anatomy and egg release

1980 ◽  
Vol 58 (12) ◽  
pp. 2220-2229 ◽  
Author(s):  
F. Edward Dudek ◽  
Hampik S. Injeyan ◽  
Bonnie Soutar ◽  
Greg Weir ◽  
Stephen S. Tobe
Keyword(s):  
Time Course ◽  
Aplysia Californica ◽  
Cell Extract ◽  
Continuous Exposure ◽  
Gradual Reduction ◽  
Release Process ◽  
Cell Extracts ◽  
High K ◽  
Scanning Electron

Egg release from the ovotestis of Aplysia californica has been studied using ovotestis fragments and bag cell extracts. Light and scanning electron microscopy showed clusters of follicles surrounded by muscle cells. Mature oocytes observed in egg masses and those released from ovotestis fragments were 90 μm in diameter. The number of mature releasable oocytes was relatively constant throughout the ovotestis, although a gradual reduction occurred with increasing distance from the small hermaphroditic duct.Bag cell induced egg release was detectable in vitro within 30 min and was complete by 180 min. The time course of egg release was similar under conditions of either continuous exposure or a 30-min pulse of bag cell extract. Artificial seawater (ASW) solutions with high K+ (110 mM) did not stimulate egg release unless bag cell extract was present. ASW with no Ca2+ and 3 mM EGTA or ASW containing Co2+ (10 mM) inhibited both bag cell induced and spontaneous (ASW alone) egg release.Therefore, brief exposure to bag cell peptide can trigger the egg release process, which is long lasting (~ 3 h) and Ca2+ dependent. The observation that high K+ did not stimulate egg release challenges the muscle contraction hypothesis of egg release.

scholarly journals Transcription Termination and 3′-End Processing of the Spliced Leader RNA in Kinetoplastids

1999 ◽  
Vol 19 (2) ◽  
pp. 1595-1604 ◽  
Author(s):  
Nancy R. Sturm ◽  
Michael C. Yu ◽  
David A. Campbell
Keyword(s):  
Binding Site ◽  
Transcription Termination ◽  
Small Nuclear Rna ◽  
Stem Loop ◽  
Spliced Leader ◽  
Nuclear Extracts ◽  
Sequence Elements ◽  
Rna Genes

ABSTRACT Addition of a 39-nucleotide (nt) spliced leader (SL) bytrans splicing is a basic requirement for all trypanosome nuclear mRNAs. The SL RNA in Leishmania tarentolae is a 96-nt precursor transcript synthesized by a polymerase that resembles polymerase II most closely. To analyze SL RNA genesis, we mutated SL RNA intron structures and sequence elements: stem-loops II and III, the Sm-binding site, and the downstream T tract. Using an exon-tagged SL RNA gene, we examined the phenotypes produced by a second-site 10-bp linker scan mutagenic series and directed mutagenesis. Here we report that transcription is terminated by the T tract, which is common to the 3′ end of all kinetoplastid SL RNA genes, and that more than six T’s are required for efficient termination in vivo. We describe mutants whose SL RNAs end in the T tract or appear to lack efficient termination but can generate wild-type 3′ ends. Transcriptionally active nuclear extracts show staggered products in the T tract, directed by eight or more T’s. The in vivo and in vitro data suggest that SL RNA transcription termination is staggered in the T tract and is followed by nucleolytic processing to generate the mature 3′ end. We show that the Sm-binding site and stem-loop III structures are necessary for correct 3′-end formation. Thus, we have defined the transcription termination element for the SL RNA gene. The termination mechanism differs from that of vertebrate small nuclear RNA genes and the SL RNA homologue in Ascaris.

Nitrogenase inactivation by oxygen and enzyme turnover in Anabaena cylindrica

1983 ◽  
Vol 29 (10) ◽  
pp. 1286-1294 ◽  
Author(s):  
Marcia A. Murry ◽  
Patrick C. Hallenbeck ◽  
Diane Esteva ◽  
John R. Benemann
Keyword(s):  
Time Course ◽  
Acetylene Reduction Activity ◽  
Anabaena Cylindrica ◽  
Reactive Material ◽  
Chloromethyl Ketone ◽  
Aerobic Conditions ◽  
Reduction Activity ◽  
Cell Extracts

Nitrogenase is known to be irreversibly inactivated by oxygen in vivo and in vitro. In time-course experiments using Anabaena cylindrica, cultures treated with antibiotics and incubated under various O2 tensions, in vivo acetylene reduction activity and immunologically determined Fe–Mo protein (component I) were lost at rates directly related to O2 tension. Activity was lost at a faster rate than cross-reactive material. The half-life of cross-reactive material was 25 h under microaerophilic conditions, 12 h under aerobic conditions, and 6.6 h under an O2 tension of 245% of air saturation. In vitro, cross-reactive material was lost in O2 exposed, but not in anaerobically prepared, crude cell extracts. Loss of cross-reactive material was prevented by freezing and by α-N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of histidine-residue proteases. These results indicate that nitrogenase is continuously inactivated by O2, hydrolyzed, and resynthesized during growth of this heterocystous cyanobacteria under aerobic conditions.

scholarly journals Production of cartilage link protein by human granulosa-lutein cells

2002 ◽  
Vol 175 (2) ◽  
pp. 505-515 ◽  
Author(s):  
GW Sun ◽  
H Kobayashi ◽  
M Suzuki ◽  
N Kanayama ◽  
T Terao
Keyword(s):  
Time Course ◽  
Matrix Protein ◽  
Cultured Cells ◽  
Extracellular Matrix Protein ◽  
Regulatory Function ◽  
Human Ovary ◽  
Vitro Fertilization ◽  
Link Protein ◽  
Cell Extracts

Link protein (LP), an extracellular matrix protein in cartilage, stabilizes aggregates of hyaluronic acid (HA) and proteoglycans, including aggrecan and inter-alpha-trypsin inhibitor (ITI). We have shown previously that cartilage LP is present in the maturing rat and mouse ovary. In the present study, we have employed immunohistochemistry to examine the anatomical distribution of cartilage LP in the human ovary. The expression of cartilage LP was selectively detected in the cells within the granulosa compartment of the preovulatory dominant follicle. The HA-positive granulosa-lutein cells were found to be a cartilage LP-positive subpopulation. We subsequently studied the in vitro expression of cartilage LP in cultured human granulosa-lutein cells obtained at oocyte retrieval for in vitro fertilization. Analysis of cultured cells by enzyme-linked immunoaffinity assay, Western blotting and immunofluorescence microscopy revealed that gonadotropin stimulates cartilage LP production. Time-course studies indicated that the cartilage LP production was induced as early as with gonadotropin stimulation for 2 h, and the effect was sustained up to 8 h. Western blot analysis further revealed the presence of the macroaggregates composed of HA, ITI and cartilage LP in the gonadotropin-stimulated granulosa-lutein cell extracts. Collectively, the present results raise the possibility that cartilage LP forms extracellular structures that may have a regulatory function in the developing follicle in the human ovary.

Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo

1988 ◽  
Vol 8 (1) ◽  
pp. 361-370
Author(s):  
S Ganguly ◽  
P A Sharp ◽  
U L RajBhandary
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Wild Type ◽  
Cell Extracts ◽  
Amber Suppressor ◽  
Gene Transcripts ◽  
A Site ◽  
Made In

We describe the results of our studies of expression of a Saccharomyces cerevisiae amber suppressor tRNA(Leu) gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro. Parallel studies were carried out with the wild-type (Su-) tRNA(Leu) gene. Extracts from HeLa or CV1 cells transcribed both tRNA(Leu) genes. The transcripts were processed correctly at the 5' and 3' ends and accurately spliced to produce mature tRNA(Leu). Surprisingly, when the same tRNA(Leu) genes were introduced into CV1 cells, only pre-tRNAs(Leu) were produced. The pre-tRNAs(Leu) made in vivo were of the same size and contained the 5'-leader and 3'-trailer sequences as did pre-tRNAs(Leu) made in vitro. Furthermore, the pre-tRNAs(Leu) made in vivo were processed to mature tRNA(Leu) when incubated with HeLa cell extracts. A tRNA(Leu) gene from which the intervening sequence had been removed yielded RNAs that also were not processed at either their 5' or 3' termini. Thus, processing of pre-tRNA(Leu) in CV1 cells is blocked at the level of 5'- and 3'-end maturation. One possible explanation of the discrepancy in the results obtained in vivo and in vitro is that tRNA biosynthesis in mammalian cells involves transport of pre-tRNA from the site of its synthesis to a site or sites where processing takes place, and perhaps the yeast pre-tRNAs(Leu) synthesized in CV1 cells are not transported to the appropriate site.

In vitro transcription of Drosophila rRNA genes shows stimulation by a phorbol ester and serum

1993 ◽  
Vol 13 (2) ◽  
pp. 934-941
Author(s):  
Y Chao ◽  
M Pellegrini
Keyword(s):  
Phorbol Ester ◽  
Core Promoter ◽  
In Vitro Transcription ◽  
Rrna Genes ◽  
Control Element ◽  
Start Site ◽  
Cell Extracts ◽  
Pol I ◽  
Nuclear Extracts

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum both stimulate rapid increases in the transcription of Drosophila rRNA genes in vivo. Here we report that this stimulation is observed in in vitro transcription assays using nuclear extracts from cells treated with TPA or serum. Experiments in which extracts from TPA- or serum-treated cells were mixed with extracts from cells grown in serum-restricted medium showed that there was an increased RNA polymerase I (Pol I) activity present in the cell extracts from treated cells. We used a series of plasmids that had been deleted in the region 5' to the start site of rRNA transcription to determine which sequences were necessary to support the increased transcription seen in extracts from stimulated cells. DNA templates that contain sequences between -150 and +32 (with +1 as the Pol I transcription start site) show dramatic increases in transcription with TPA- and serum-stimulated cell extracts; however, templates that contain 5' sequences to -60 or -43 show at most one-third of the stimulation level of transcription in nuclear extracts from treated cells in comparison with untreated cell extracts. The 5' deletion to -34 abolishes the stimulation effect and drops the basal-level transcription by 20-fold. These results indicate that the regulation of Pol I transcription in Drosophila cells by serum and TPA requires two DNA elements, sequences from -150 to -60 (upstream control element) and sequences from -43 to -34 (a portion of the core promoter.

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