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.

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.

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 Abortive intermediates in transcription by wheat-germ RNA polymerase II. Dynamic aspects of enzyme/template interactions in selection of the enzyme synthetic mode

1990 ◽  
Vol 269 (3) ◽  
pp. 651-658 ◽  
Author(s):  
L de Mercoyrol ◽  
J M Soulié ◽  
C Job ◽  
D Job ◽  
C Dussert ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Length Distribution ◽  
Enzyme Concentration ◽  
Single Step ◽  
Chain Elongation ◽  
Polymeric Product ◽  
Chain Length Distribution ◽  
Kinetics Of

At constant enzyme concentration and with the full set of nucleotide substrates dictated by template sequence, the chain-length distribution of polymeric product varies with template concentration in reactions catalysed by wheat-germ RNA polymerase II. Under the same conditions, but in the presence of a single ribonucleoside triphosphate, the rate of condensation of the triphosphate substrate to a dinucleotide primer also exhibits a complex dependence with the template concentration. This effect is observed using poly[d(A-T)] as a template. For both reactions there are two extreme types of behaviour in each of which transcription appears to involve a single enzyme synthetic mode, characterized by either a high (at low template concentration) or a low (at high template concentration) probability of releasing the transcripts. A strong correlation is found between these two pathways, such that conditions favouring the abortive release of trinucleotide products in the single-step addition reaction are associated with the synthesis of short-length RNA species in productive elongation, and reciprocally. A model previously developed by Papanicolaou, Lecomte & Ninio [(1986) J. Mol. Biol. 189, 435-448] to account for the kinetics of polymerization/excision ratios with Escherichia coli DNA polymerase I, and by Job, Soulié, Job & Shire [(1988) J. Theor. Biol. 134, 273-289] for kinetics of RNA-chain elongation by wheat-germ RNA polymerase II provides an explanation for the observed behaviour with the plant transcriptase. The basic requirement of this model is a slow equilibrium between two states of the polymerization complex with distinct probabilities of releasing the product. In the presence of Mn2+, and under conditions allowing the synthesis of poly[r(A-U)], one of these states is involved in the formation of oligonucleotides shorter than 15 bases, whereas the other catalyses the polymerization of chains longer than 40 bases.

scholarly journals The use of rat liver nucleoplasm for the characterization of heterogeneous nuclear ribonucleic acid synthesis in vitro

1978 ◽  
Vol 176 (3) ◽  
pp. 715-725 ◽  
Author(s):  
T J C Beebee
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rat Liver ◽  
Dna Sequences ◽  
Acid Synthesis ◽  
Rna Molecules ◽  
Liver Nuclei

1. A nucleoplasmic fraction rich in endogenous RNA polymerase II activity was isolated from rat liver nuclei and conditions were determined under which elongation of RNA molecules initiated in vivo continued at maximal rates in vitro. 2. Elongation rates in vitro were calculated to be about 0.25 nucleotide/s and there were about 7 × 10(3) RNA molecules in the process of being elongated by form-II RNA polymerase per original nucleus. 3. Evidence was obtained suggesting that transcription-dependent release of RNA polymerase II molecules from the template occurred during the incubations in vitro. 4. The nascent RNA was tightly associated with protein and banded as ribonucleoprotein in caesium salt gradients. 5. RNA molecules labelled in vitro were up to 13000 nucleotides in length, but consisted of long unlabelled chains transcribed in vivo with only short labelled sequences added in vitro, and without significant polyadenylation. 6. Hybridization of transcripts in the presence of a vast excess of DNA demonstrated that both form-II RNA polymerase and another enzyme, resistant to low alpha-amanitin concentrations, were synthesizing RNA molecules complementary to both reiterated and unique DNA sequences in the genome.

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
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