High Template Activity Research Articles

Analysis of soybean chloroplast DNA replication by two-dimensional gel electrophoresis

Chloroplast DNA replication was studied in the green, autotrophic suspension culture line SB-1 of Glycine max. Three regions (restriction fragments Sac I 14.5, Pvu II 4.1 and Pvu II 14.8) on the plastome were identified that displayed significantly higher template activity in in vitro DNA replication assays than all other cloned restriction fragments of the organelle genome, suggesting that these clones contain sequences that are able to direct initiation of DNA replication in vitro. In order to confirm that the potential in vitro origin sites are functional in vivo as well, replication intermediates were analyzed by two-dimensional gel electrophoresis using cloned restriction fragments as probes. The two Pvu II fragments that supported deoxynucleotide incorporation in vitro apparently do not contain a functional in vivo replication origin since replication intermediates from these areas of the plastome represent only fork structures. The Sac I 14.5 chloroplast DNA fragment, on the other hand, showed intermediates consistent with a replication bubble originating within its borders, which is indicative of an active in vivo origin. Closer examination of cloned Sac I 14.5 sub-fragments confirmed high template activity in vitro for two, S/B 5 and S/B 3, which also seem to contain origin sites utilized in vivo as determined by two-dimensional gel electrophoresis. The types of replication intermediate patterns obtained for these sub-fragments are consistent with the double D-loop model for chloroplast DNA replication with both origins being located in the large unique region of the plastome [17, 18]. This is the first report of a chloroplast DNA replication origin in higher plants that has been directly tested for in vivo function.

Minus-strand initiation by brome mosaic virus replicase within the 3′ tRNA-like structure of native and modified RNA templates

An RNA-dependent RNA polymerase (replicase) extract from brome mosaic virus-infected barley leaves has been shown to initiate synthesis of (−) sense RNA from (+) sense virion RNA. Initiation occurred de novo, as demonstrated by the incorporation of [γ- 32P]GTP into the product. Sequencing using cordycepin triphosphate to terminate (−) strands during their synthesis by the replicase generated sequence ladders that confirmed that copying was accurate, and that initiation occurred very close to the 3′ end. The precise site of initiation was further defined by testing the replicase template activity after stepwise removal of 3′-terminal nucleotides. Whereas removal of the terminal A did not decrease template activity, removal of the next nucleotide (C-2) did. Thus, initiation almost certainly occurs opposite the penultimate 3′-nucleotide (C-2) in vitro. The structure of the double-stranded replicative form of RNA isolated from brome mosaic virus-infected leaves was consistent with such a mechanism occurring in vivo, in that it lacked the 3′-terminal A found on virion RNAs. The specific site of (−) strand initiation and normal template activity were retained for RNAs with as many as 15 to 30 A residues added to the 3′ end. However, only limited oligonucleotide 3′ extensions can be present on active templates. In order to assess the 5′ extent of sequences required for an active template, a 134-nucleotide-long fragment of brome mosaic virus RNA, corresponding to the tRNA-like structure, was generated. This RNA had high template activity, but a shorter 3′ (85-nucleotide) fragment was inactive. RNAs with various heterologous sequences 5′ to position 134 also showed high template activity. Thus, the 3′-terminal tRNA-like structure common to all four brome mosaic virus virion RNAs contains all of the signals required for initiation of replication, and sequences 5′ to it do not play a role in template selection.

X chromosomal organisation and dosage compensation. In situ transcription of chromatin template activity of X chromosome hyperploids of Drosophila melanogaster.

The chromatin template activity of the polytene X chromosomal DNA was assayed by in situ transcription on the fixed polytene chromosomes using E. coli RNA polymerase holoenzyme and 3H-UTP as the monitoring substrate in various 1X2A, 2X2A and 3X2A larvae and 1X2A (+ X fragments) segmental aneuploid larvae of Drosophila melanogaster. The segmental aneuploids contained duplications for the segments 15EF-20F, 11A-20F, 8C-20F and 3E-20F of the X chromosome. Results revealed that a double dose of active loci located in the X chromosome regions 15EF-20F, 11A-20F, and 8C-20F in aneuploids synthesized nearly 40%-70% more RNA than the normal single dose of this region in the wild-type males. The activity per gene dose for the two segments in the aneuploids was also significantly higher than in their male counterpart except for the duplication dp (3E-20F), where the duplicated piece extended from the centromeric heterochromatin to include 85% of the euchromatic portion of the X chromosome. In the case of dp (3E-20F), the X chromosome was transcribed at the lower, "female" level. It may also be noted that some regions of the X chromosome when present in extra copy, especially in dp (8C-20F) influenced the template activity of the X-linked genes inside or outside the duplicated segment. Metafemales (3X2A) have 50% higher template activity of the X chromosomes than their diploid sisters. In this study, metafemales behaved as females with duplication.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in template activity of protein and globin mRNA during Chironomus development

A wheat-germ cell-free protein synthesizing system was established that efficiently translates fourth instar mRNA from Chironomus thummi. The translation products were analyzed by double immunoprecipitation with specific antibodies against Chironomus hemoglobins. The predominant translation products were shown to be globins, comprising about 50% of total protein synthesized. Two globins, globins 2 and 3, which are specific for the fourth instar in vivo, constitute most of the globin produced. The wheatgerm system translates also efficiently total cytoplasmic RNA, purified from animals at successive developmental stages. The kinetics of protein synthesis during development indicate that Chironomus mRNA template activity is low during larval molting and at metamorphosis. RNA from intermolt larvae generally demonstrates high template activity. In the fourth instar two distinct peaks of activity are resolved, one associated with newly molted fourth instars and a second one associated with the prepupal stage. These RNAs direct the synthesis mostly of non-globin proteins. Immunoprecipitation of the translation products shows that globin synthesis is high in intermolt larvae decreasing to very low, or background levels in larvae molting or metamorphosing to pupae. Globins 2 and 3 are synthesized exclusively by newly synthesized globin transcripts in the fourth instar stage. The results show stage-specific translational activity of globin mRNAs during Chironomus development and suggest hormonal control of globin production.

Oligonucleosomes as a model system for chromatin transcription: Transcription with Escherichia coli DNA-dependent RNA polymerase

The transcription of eukaryotic genes that are organized into nucleosomal structures is likely to be influenced by the presence of the chromosomal proteins. We investigate in this paper the transcriptional behavior of series of oligonucleosomes, produced by Micrococcus nuclease digestion of nuclei, with RNA polymerase from Escherichia coli. Electrophoresis of the transcribed RNA shows that the nuclease-generated ends of such oligonucleosomes serve as artificial initiation sites, and that the templates are transcribed in full lengths. Thus the problem of inefficient initiation, which has been encountered with circular minichromosomes, can be circumvented. The influence of the nucleosomal histones on transcription was assessed by comparison with the corresponding deproteinized DNA fragments as templates. Unlike most previous reports on chromatin transcription we find for oligonucleosomes a high template activity ranging from 30 to 100% relative to their corresponding DNA fragments. The ratio of incorporation on oligonucleosomes relative to the deproteinized DNA fragments did not show a dependence on the template length, thus indicating the absence of specific effects due to long templates or to higherorder structures. From the temperature dependence of transcription it can be concluded that at 37 °C the presence of nucleosomes on the template does not affect the elongation rate. Furthermore the elongation process seems to be rate-limiting at this temperature. We propose that the lower template activity of nucleosomes compared with naked DNA can be attributed to a decreased availability of template ends as initiation sites. Stabilizing influences of the nucleosomes on the transcription process were observed: E. coli polymerase after preinitiation shows, at salt concentrations higher than 350 m m, a higher incorporation on oligonucleosomes than on DNA. Below 25 °C the nucleosomes seem to lower the activation energy for the initiation on the nuclease-generated template ends.

Isolation of chromatin fragments carrying nascent RNA chains

Evidence is presented that chromatin fragments, enriched with nascent RNA chains, can be selectively prepared by affinity chromatography. This has been achieved by transcription of pig liver chromatin using mercurated UTP in addition to the other nucleotides. The transcribed chromatin is enzymatically fragmented and treated with thiopropyl-Sepharose. A fraction is obtained which displays a high template activity, contains the newly synthesized RNA and is enriched in non-histone proteins.

Preparation of projection-less particles from influenza virus and their messenger activities in prokaryotic and eukaryotic systems

A fraction of projection-less particles was prepared from influenza A/Dunedin/4/73 and A/Victoria/3/75 (X-47) (H3N2) by detergent treatment and extraction into ether at 0 degrees C. The activity of this material in stimulating protein synthesis in vitro was studied and compared with that of isolated virion RNA using a) an RNA-dependent E. coli system, and b) a wheat germ system. In the bacterial system the purified RNA had the highest template activity, while in the eukaryotic system the disrupted particle preparation was by far the most active. Translation products were formed with immunological and electrophoretic properties similar to those of several influenza virion proteins. The experiments indicate that, when added in the form of disrupted projection-less particles, RNA from influenza A2 virus is utilized as a template by eukaryotic ribosomes.

Isolation and characterization of rDNA-containing chromatin from nucleoli

Fractions corresponding to ‘euchromatin’ and ‘heterochromatin’ were isolated from Novikoff hepatoma nucleoli by a modification of the DNase II method of Gottesfeld et al. [18, 19]. Nucleolar chromatin [36] was digested with DNase II, and ‘euchromatin’ was obtained as a soluble fraction following differential precipitation of ‘heterochromatin’ with Mg 2+. In this ‘euchromatin’ fraction, the rDNA was enriched approx. 8.5-fold over the unfractionated nucleolar chromatin, as determined by DNA-RNA hybridization. On 8–80% glycerol gradients, the ‘euchromatin’ separated into three discrete subfractions as with euchromatin obtained from the bulk chromatin [18, 19]. Two subfractions which sedimented at 14S and 20S, respectively, were consistently found after DNase II digestion (to 15 min) which contained all histone fractions. All euchromatin subfractions were enriched in rDNA and had a high protein/DNA ratio, a high RNA-DNA ratio, and a high template activity with exogenous RNA polymerase I. Fraction B with a sedimentation of 14S was most enriched in the parameters related to rRNA synthesis. The Mg 2+-precipitable fractions reported to be ‘heterochromatin’ [18, 19] not only contained substantial amounts of ribosomal genes, but also had higher template activities than unsheared chromatin. These results imply (1) nucleolar ‘euchromatin’ has a structural heterogeneity similar to that of the euchromatin containing active single copy sequences; (2) at least a portion of rDNA in ‘euchromatin’ is protected by proteins, including histones, which does not preclude active transcription of rDNA; (3) none of the currently used criteria for ‘euchromatin’ alone defines the chromatin containing rDNA; and (4) an appreciable enrichment of rDNA occurs in isolated euchromatin but not all the rDNA was separated into ‘euchromatin’ by the DNase II method.

Structurally altered and transcriptionally activated rat prostate chromatin induced by androgens

The effect of androgens on the structure and transcriptional function of rat prostate chromatin was investigated by the following studies: template activity in transcription, distribution of euchromatin and heterochromatin, titration with polylysine, thermal denaturation and circular dichroism spectra. Following a single intraperitoneal injection of testosterone to rats, the template activity of prostate chromatin, assayed in rat liver RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6) II reaction, increased by 68% 2 h (CT 2) after hormone administration; while the template activities of prostate chromatins from rats receiving testosterone for 4 (CT 4), 6 (CT 6) and 12 h (CT 12) were slightly lower than that of the castrated control (C). Similar patterns were also observed in polylysine binding by, and distribution of euchromatin and heterochromatin in, the various prostate chromatins. The template activity of prostate chromatin in rat liver RNA polymerase II reaction was contributed exclusively by its euchromatin fraction. Furthermore, CT 2 euchromatin was also found to have the highest template activity than CT 4, CT 6, CT 12 and C euchromatins. Derivative melting profiles of the various chromatins all showed increased melting of DNA regions bound by nonhistone proteins with concomitant decreases in histone-bound regions of DNA. Circular dichroism spectra of these chromatins showed conformational changes in proteins, as indicated by a reduction in amplitude at 210–220 nm, for CT 2 chromatin, and increases for CT 4, CT 6 and CT 12 chromatins when compared with the control. These results indicate that androgen action in gene activation is related to structural alterations of prostate chromatin induced by androgens.

Messenger ribonucleoprotein complexes of cryptobiotic embryos of Artemia salina.

Poly(A)-containing ribonucleoprotein (poly(A)+-RNP) particles in the post-mitochondrial supernatant of cryptobiotic embryos of Artemia salina were characterized by hybridization to [3H]-poly(U). By sucrose isopycnic centrifugation, approximately 2/3 of poly(A)+-RNPs was found to band at 1.27-1.30 (g/cm3) and the rest 1+/3 at 1.20-1.23 (g/cm3) and below 1.20 (g/cm3). The 1.27-1.30 RNPs could be separated into two density classes, 1.27-1.28 and 1.30 (g/cm3) respectively. The latter RNP class was apparently complexed with ribosomal components because they were completely converted to the former RNP class (free RNPs) by 25 mM EDTA treatment. Further, the 1.30 (g/cm3) RNPs were resolved into several RNP species having sedimentation coefficients above 50 S. which were transformed mostly to 20-30 S rnps in the presence of 25 mM EDTA. The free 20-30 S RNPs contained 8-14 S poly(A)+-RNAs, having the highest template activity in a wheat embryo cell-free system, whereas the 1.20-1.23 poly(A)+-RNPs consisted of 10 S and 16 S RNPs, both of which contained 4 S poly(A)-containing sequences without any template activity.

Translation of mRNA for glutamate dehydrogenase and spectrophotometric procedure to follow the enzyme biosynthesis.

Heterogeneous poly (A)-mRNA fraction was isolated from rat liver microsomes using phenol-chloroform extraction, millipore filtration and poly (U)-agarose affinity chromatography. Obtained fractions were characterized with respect to their secondary structure and poly (A) content. Isolated poly (A)-mRNA fraction contained high template activity for glutamate dehydrogenase in cell-free systems with microsomes or polysomes. A spectrophotometric procedure to follow enzyme biosynthesis was also developed.

Studies on the stability of chromatin from hen oviduct

The stability of sheared chromatin from the magnum portion of hen oviduct has been examined during storage at 0 °C using several parameters. Chemical composition and ultraviolet spectra were unchanged after 1 month while changes in melting profiles were seen after 2 weeks. Gel electrophoretic patterns of histones indicated slight proteolysis after 4 weeks with no apparent degradation occurring at earlier times. Acidic protein gel patterns remained unchanged during the period of study. Rapid changes (within 1 to 3 days) were seen in chromatin size distributions as measured in sucrose gradients and in chromatin template activity. The mean chromatin size decreased with storage, an increasing percentage of the total population sedimenting as a 17S component. This change in chromatin size was accompanied by an increase in the amount of free protein floating on the top of gradients. Several experiments were conducted to investigate the nature of the chromatin changes occurring at early storage times. Changes in chromatin size and template activity could be attributed to neither proteolysis nor DNase activity. Diisopropylfluorophosphate, a known nuclear protease inhibitor, did not inhibit the rate of change in chromatin size with storage. Furthermore, no proteolysis was detectable by gel electrophoresis during storage times at which significant changes in chromatin size and template activity had occurred. When the size of chromatin DNA was examined, no changes were seen with storage nor were there differences in the size of DNA from fast and slow sedimenting chromatin fractions. When template activities of fast and slow sedimenting chromatin fractions were compared, the slow sedimenting fractions were found to be the most template active. The template activity of a slow sedimenting, high template activity fraction was examined with storage and was found to increase with time.

Phenobarbital stimulation of housefly chromatin template activity

The effect of phenobarbital on the chemical composition and template activity for Escherichia coli RNA polymerase of housefly chromatin is examined. Chromatin isolated from the resistant Fc housefly strain after administration of phenobarbital has a much higher template activity as compared to control chromatin. The effect is minimal in a susceptible housefly strain. Phenobarbital treatment alters the composition of the RNA polymerase product which results in an increase of the (A + U) (C + G) ratio with chromatin from the Fc strain as template, while the (A + G) ( C + U) and (A + U) ( C + G) ratios are decreased when chromatin of the susceptible strain is used. A substantial portion of the intracellular phenobarbital is physically bound to nuclei and microsomes, but most of it is associated with cytosolic macromolecules. Less than 0.2% is covalently bound to cellular macromolecules. Phenobarbital apparently binds to a small mol. wt cytosolic macromolecule(s) as shown by sucrose density gradient centrifugation and Sephadex G-25 column chromatography.

Different binding of poly(A)-containing and poly(A)-free fractions of nuclear ribonucleic acid to ribosomes from rat liver

Total nuclear RNA extracted from nuclei of rat liver cells by phenol/chloroform in the presence of sodium dodecyl sulphate was separated by combined gel filtration on Sepharose 4 B and affinity chromatography on poly(U) Sepharose into fractions differing in their molecular weights and contents of poly(A) sequences. The poly(A)-containing 45-S RNA became labelled most rapidly if rats were administered [ 3H] orotic acid. This fraction showed a high template activity when added to postmitochondrial supernatants of the Krebs ascites tumour. Fractions of nRNA, free of poly(A) sequences, had no stimulating effect on protein synthesis in this system. The 45-S RNA-containing poly(A) was readily bound to crude polyribosomes from rat liver at 0°C and both ATP and GTP were necessary for this reaction. Sucrose gradient analyses provided evidence that this RNA species is bound predominantly to 80-S ribosomes. No binding was obtained with polyribosomes washed with 0.5 M KCl. The binding ability of washed polyribosomes was restored by the addition of the ribosomal wash fraction or rat liver cytosol. Crude polyribosomes bound significantly lower quantities of nRNA species free of poly(A) when compared with poly(A) · 45-S RNA. The label was scattered through the whole ribosomal sedimentation pattern with no predominant peaks and the binding reaction required neither soluble factors nor nucleotide cofactors. The labelling kinetics and high template activity of poly(A) · 45-S nRNA indicate that this fraction contains precursors of cytoplasmic mRNA. Requirements for soluble factors and nucleotide cofactors in the binding of this RNA species to 80-S ribosomes suggest that this binding, unlike that of other nRNA species, has a specific mechanism resembling that of mRNA binding during peptide initiation.

Characterization of ribonucleic acids from the venom glands of Crotalus durissus terrifucus (Ophidia, Reptilia) after manual extraction of the venom. Studies on template activity and base composition.

RNA synthesis in the venom glands of Crotalus durissus terrificus was stimulated by the manual extraction of the venom (milking). RNA was extracted from venom glands activated by milking and fractionated by centrifugation through sucrose density gradients. Template activity for protein synthesis and base composition of the RNA fractions were studied. RNA fractions that sediment between 18S and 4S had the highest template activity. The base composition analysis indicated that the 28S and 18S rRNA have a C+G content of 65.4 and 58% respectively. The ;melting' temperature (T(m)) of DNA in 0.15m-NaCl-0.015m-trisodium citrate, pH7.0, was 85 degrees C, corresponding to a C+G content of 38%. The base ratio of the RNA fractions that showed a high template activity was intermediate between that of rRNA and homologous DNA. The possible role of these fractions in the synthesis of the two main toxins (crotoxin and crotamine) of the South American rattlesnake's venom is discussed.

ISOLATION OF CAULIFLOWER CHROMATIN AND ITS TEMPLATE ACTIVITY FOR RNA SYNTHESIS

The apical portion of the cauliflower inflorescence has a high DNA content. Soluble chromatin can readly be isolated, showing that the apical portion is a suitable source for the mass preparation of isolated chromatin. Two methods for the determination of the histone content of isolated chromatin were used. RNA synthesizing capacities were also examined. The degree of template activity does not depend on amount of histone estimated in the TCA-precipitate, but does depend on the amount of histone determined in chromatin in the soluble state. Condensed- and extended-chromatins were separated and extended chromatin had four times as high template activity as did condensed chromatin.

Role of subunits of 60 to 70S avian tumor virus ribonucleic acid in its template activity for the viral deoxyribonucleic acid polymerase.

Heating the 60 to 70S ribonucleic acid (RNA) of Rous sarcoma virus (RSV) destroys both its subunit structure and its high template activity for RSV deoxyribonucleic acid (DNA) polymerase. In comparative analyses, it was found that the template activity of the RNA has a thermal transition of 70 C, whereas the 60 to 70S structure dissociates into 30 to 40S and several distinct small subunits with a T(m) of 55 C. Analysis by velocity sedimentation and isopycnic centrifugation of the primary DNA product obtained by incubation of 60 to 70S RSV RNA with RSV DNA polymerase indicated that most, but perhaps not all, DNA was linked to small (<10S) RSV RNA primer. Sixty percent of the high template activity of 60 to 70S RSV RNA lost after heat dissociation could be recovered by incubation of the total RNA under annealing conditions. The template activity of purified 30 to 40S subunits isolated from 60 to 70S RSV RNA was not enhanced significantly by annealing. However, in the presence of small (<10S) subunits also isolated from 60 to 70S RNA, the template activity of 30 to 40S RNA subunits was increased to the same level as that of reannealed total 60 to 70S RNA. It was concluded that neither the 30 to 40S subunits nor most of the 4S subunits of 60 to 70S RSV RNA contribute much as primers to the template activity of 60 to 70S RSV RNA. The predominant primer molecule appears to be a minor component of the <10S subunit fraction of 60 to 70S RSV RNA. Its electrophoretic mobility is similar to, and its dissociation temperature from 60 to 70S RSV RNA is higher than that of the bulk of 60 to 70S RSV RNA-associated 4S RNA. The role of primers in DNA synthesis by RSV DNA polymerase is discussed.

Effect of extraction temperature on template activity of polysomal RNA from fetal, neonatal, and adult rat liver

Polyribosomes were prepared from adult rat liver and the RNA was extracted from the polysomes at various temperatures. The ability of these RNA fractions to serve as templates in the polymerization of amino acids, i.e., mRNA, was tested in a heterologous cell-free system from E. coli D 10, a mutant which is devoid of RNase I. Electrophoresis of the adult liver polysomal RNA extracted at 37, 45, 55, 65, and 70 ° on polyacrylamide gels and fractionation on linear sucrose gradients indicated the presence of species of RNA with molecular weights in excess of 28S ribosomal RNA. This represented aggregation of the latter. The highest template activity was associated with the RNA extracted at 55 °. In this regard, the 28S region of the sucrose gradient was the most active although activity was seen throughout most of the gradient. Polyribosomes were also prepared from fetal and neonatal rat liver; the RNA was extracted at 55 °. Both fetal and neonatal rat liver polysomal RNA exhibited at least a 2-fold greater total template activity than adult material. The template activity of neonatal and fetal RNA fractions from sucrose gradients had very similar patterns although fetal RNA was slightly more efficacious. With these fractions, highest total template activity was also seen in the 28S region of the gradients. The maximal specific template activity, i.e., amount labeled amino acid incorporated per μg RNA, however, occurred in the region between 6 and 18S RNA.

Estradiol-mediated template activity of mouse and rat uterine chromatin: An unsolved problem

The capacity of mouse and rat uterine chromatin to serve as template for RNA synthesis in vitro using added Escherichia coli RNA polymerase was investigated. Intraperitoneal administration of estradiol to mice decreases the apparent template activity of uterine chromatin compared with that of control chromatin. In rats, however, estradiol increases the activity, in agreement with previous findings. Using a highly sensitive test, the existence of a ribonuclease in both mouse and rat uterine chromatin could be demonstrated. Any chromatin of relatively high template activity shows relatively low ribonuclease activity and vice versa, suggesting that during in vitro RNA synthesis degradation of RNA takes place. These observations question the validity of the dogma that an in vitro test, with added bacterial RNA polymerase, of the template activity of a given mammalian chromatin is a reliable measure of its gene activity.

Discrepancies between cytophotometric Feulgen values and deoxyribonucleic acid content.

Diploid nuclei of different cell types of the granulocytopoietic and erythropoietic series showed different Feulgen values. The highest values were found in the most immature nuclei, and the lowest values in the most mature cells. Extraction of acid-soluble nuclear proteins brought the different values to the same level. Examples of similar findings in other cell types, as described in the literature, are discussed. The Feulgen reaction and its colored product depend on the functional state of the chromatin in such a way that nuclei with high template activity of deoxyribonucleic acid show high Feulgen values whereas mature, dense nuclei with low template activity have lower Feulgen values, although both kinds of nuclei contain identical amounts of deoxyribonucleic acid.