scholarly journals Studies on sex-organ development. Changes in the oestrogenic response of the chick Müllerian duct as measured by chromatin template and ribonucleic acid initiation capacity

1979 ◽  
Vol 182 (2) ◽  
pp. 257-269 ◽  
Author(s):  
G K Andrews ◽  
C S Teng
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Average Rate ◽  
Developmental Trend ◽  
Mullerian Duct ◽  
Müllerian Duct ◽  
E Coli ◽  
Chromatin Template

Assays of transcription in vitro, with Escherichia coli RNA polymerase or wheat-germ RNA polymerase II, were used to characterize chromatin templates isolated from the left Müllerian duct of the chick embryo during normal development, and during development in the presence of diethylstilboestrol. Control Müllerian-duct template capacity with E. coli RNA polymerase decreased from 6.42% on day 10 to 4.34% by day 15 of development. Similar results were found with wheat-germ RNA polymerase II. In the presence of rifampicin and heparin, the prokaryotic enzyme transcribed a number-average RNA chain of 670 nucleotide residues, at an average rate of 110 nucleotide residues/min, from Müllerian-duct chromatin of all developmental stages. From day 10 to day 15 there was a 44% decrease in the number of initiation sites for E. coli RNA polymerase on Müllerian-duct chromatin. A 47% decline was observed when these chromatins were transcribed with excess RNA polymerase II in the presence of rifamycin Af/013. Signs of increasing responsiveness to oestrogen developed between days 10 and 16. Embryos exposed to maximally responsive doses of diethylstilboestrol for 2 days showed increases in Müllerian-duct chromatin template capacity, RNA-chain initiation sites, wet weight, protein and RNA. The changes seen in the oviduct of the 1-week-old chick injected for 2 days with diethylstilboestrol were defined as 100% responses. By comparison, the Müllerian duct, after exposure to diethylstilboestrol from day 10 to day 12, from day 13 to day 15 or from day 16 to day 18, showed a 15%, 39% and 72% template response respectively, and a 42%, 56% and 85% initiation-site change respectively. A similar developmental trend was observed in all parameters. It is concluded that oestrogenic responsiveness in the developing Müllerian duct increases from day 10 to nearly maximal values by day 16 of development, and that this transition is paralleled by a progressive restriction of genomic activity.

scholarly journals Studies on sex-organ development. Changes in chromatin structure during spermatogenesis in maturing rooster testis as demonstrated by the initiation pattern of ribonucleic acid synthesis in vitro

1978 ◽  
Vol 170 (2) ◽  
pp. 203-210 ◽  
Author(s):  
C Mezquita ◽  
C S Teng
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Acid Synthesis ◽  
Chromatin Interaction ◽  
E Coli ◽  
Double Stranded Dna ◽  
The Stability ◽  
Kinetics Of

To probe the structural change in the genome of the differentiating germ cell of the maturing rooster testis, the chromatin from nuclei at various stages of differentiation were transcribed with prokaryotic RNA polymerase from Escherichia coli or with eukaryotic RNA polymerase II from wheat germ. The transcription was performed under conditions of blockage of RNA chain reinitiation in vitro with rifampicin or rifampicin AF/013. With the E. coli enzyme, the changes in (1) the titration curve for the enzyme-chromatin interaction, (2) the number of initiation sites, (3) the rate of elongation of RNA chains, and (4) the kinetics of the formation of stable initiation complexes revealed the unmasking of DNA in elongated spermatids and the masking of DNA in spermatozoa. In both cases the stability of the DNA duplex in the initiation region for RNA synthesis greatly increased. In contrast with the E. coli enzyme, the wheat-germ RNA polymerase II was relatively inefficient at transcribing chromatin of elongated spermatids. Such behaviour can be predicted if unmasked double-stranded DNA is present in elongated spermatids.

Trinucleotide AUA and UAU formation catalyzed by wheat germ RNA polymerase II

1989 ◽  
Vol 54 (3) ◽  
pp. 811-818 ◽  
Author(s):  
Aleš Cvekl ◽  
Květa Horská ◽  
Karel Šebesta ◽  
Ivan Rosenberg ◽  
Antonín Holý
Keyword(s):  
Ionic Strength ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Catalytic Mechanism ◽  
Bond Formation ◽  
E Coli ◽  
Substrate Properties ◽  
Rna Polymerase Holoenzyme ◽  
Analogous Mechanism

The elongation of dinucleotides ApU and UpA to trinucleotides ApUpA and UpApU by wheat germ RNA polymerase II was studied at a medium ionic strength (60 mM-KCl). The catalytic mechanism of the first internucleotide bond formation consists in the binding of the primer dinucleotide followed by the binding of NTP ("ordered bibi" reaction), i.e. by an analogous mechanism as found for RNA polymerase holoenzyme from E. coli. In further experiments phosphonate analogues of dinucleotides ApU and UpA were used as the priming dinucleotides. It was shown that analogues U(c)pA and Up(c)A are very poor primers for the synthesis of corresponding trinucleotides; the elongation of analogues A(c)pU and Ap(c)U was not observed at all. The comparison of kinetic constants Kia, KmA, KmB and Vmax as well as the substrate properties of phosphonate analogues indicates the increased specifity of the wheat germ RNA polymerase initiation binding site in comparison with the E. coli holoenzyme.

Inhibition of purified wheat germ DNA dependent RNA polymerase by pyran copolymer

1980 ◽  
Vol 58 (4) ◽  
pp. 295-298 ◽  
Author(s):  
Robert Greene ◽  
Benjamin Munson
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Divalent Cations ◽  
Ion Concentration ◽  
Chain Growth ◽  
Size Dependent ◽  
Dna And Rna ◽  
Dependent Inhibition

The inhibition of DNA and RNA polymerases in vitro by pyran copolymer has been shown to be related to its affinity for divalent cations. The present investigation was designed to explore further the nature of this inhibition using completely purified eukaryotic RNA polymerase II from wheat germ. Inhibition was determined as a function of divalent ion concentration and, as previously seen with less pure enzyme preparation, was greatest at low (Mn2+) and least at higher concentrations. No inhibition was observed at concentrations greater than 4.8 mM MgCl2 in the presence of 10 μg pyran/mL. The inhibition by pyran copolymer was exerted immediately unlike other polyanions, such as heparin and polynucleotides. This indicates that it stops RNA chain growth immediately as do known chelators of divalent cations. The size of pyran copolymer was shown to affect the extent of inhibition when different sized polymers were fractionated from a heterogenous single lot. However, when sized fractions were obtained from different lots we could not show a size-dependent inhibition. Although the mechanism by which pyran copolymer exerts its biological effect is unknown, it may well be related to its association with cations. The inhibition of enzymes requiring these cations appears to be a sensitive method of observing such an association.

Cibacron Blue inhibition of prokaryotic and eukaryotic DNA-dependent RNA polymerases

1990 ◽  
Vol 55 (11) ◽  
pp. 2769-2780
Author(s):  
Aleš Cvekl ◽  
Květa Horská
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Rna Polymerase Iii ◽  
Enzymic Activity ◽  
Rna Polymerases ◽  
Cibacron Blue ◽  
E Coli ◽  
Polymer Formation ◽  
Calf Thymus

A comparison was drawn between the action of Cibacron Blue F3GA on the enzymic activity of DNA-dependent RNA polymerases from different sources, e.g. Escherichia coli, calf thymus and wheat germ (polymerase II). Sensitivity towards this inhibitor was determined for polymer formation and primed abortive synthesis of trinucleotide UpApU. In case of E. coli polymerase and wheat germ polymerase II the dye inhibits both polymer formation and abortive synthesis. Calf thymus polymerase II is inhibited only in the polymerisation step. The primed initiation reaction was found to be resistant towards the dye. In case of E. coli polymerase and wheat germ polymerase II the sensitive step is the formation of internucleotide bond whereas in case of calf thymus polymerase II the translocation of the enzyme is influenced. An analysis of kinetic data indicates more than one binding site for the dye on RNA polymerase II from calf thymus and wheat germ. Cibacron blue does not inhibit specific transcription catalyzed by RNA polymerase III from human HeLa cells and mouse leukemia L1210 cells.

scholarly journals Identification of Rkr1, a Nuclear RING Domain Protein with Functional Connections to Chromatin Modification in Saccharomyces cerevisiae

2007 ◽  
Vol 27 (8) ◽  
pp. 2800-2811 ◽  
Author(s):  
Mary A. Braun ◽  
Patrick J. Costa ◽  
Elia M. Crisucci ◽  
Karen M. Arndt
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Synthetic Lethality ◽  
Chromatin Modification ◽  
Ring Domain ◽  
Functional Connections ◽  
Nuclear Ring ◽  
Chromatin Template ◽  
Paf1 Complex

ABSTRACT Proper transcription by RNA polymerase II is dependent on the modification state of the chromatin template. The Paf1 complex is associated with RNA polymerase II during transcription elongation and is required for several histone modifications that mark active genes. To uncover additional factors that regulate chromatin or transcription, we performed a genetic screen for mutations that cause lethality in the absence of the Paf1 complex component Rtf1. Our results have led to the discovery of a previously unstudied gene, RKR1. Strains lacking RKR1 exhibit phenotypes associated with defects in transcription and chromatin function. These phenotypes include inositol auxotrophy, impaired telomeric silencing, and synthetic lethality with mutations in SPT10, a gene that encodes a putative histone acetyltransferase. In addition, deletion of RKR1 causes severe genetic interactions with mutations that prevent histone H2B lysine 123 ubiquitylation or histone H3 lysine 4 methylation. RKR1 encodes a conserved nuclear protein with a functionally important RING domain at its carboxy terminus. In vitro experiments indicate that Rkr1 possesses ubiquitin-protein ligase activity. Taken together, our results identify a new participant in a protein ubiquitylation pathway within the nucleus that acts to modulate chromatin function and transcription.

A wheat-germ nuclear fraction required for selective initiation in vitro confers processivity to wheat-germ rna polymerase II

Plant Science ◽  
1989 ◽  
Vol 64 (1) ◽  
pp. 31-38 ◽  
Author(s):  
Laure De Mercoyrol ◽  
Claudette Job ◽  
Steven Ackerman ◽  
Dominique Job
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Nuclear Fraction

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

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