Synthesis Of DNA-like RNA Research Articles

Low-salt and high-salt RNA polymerase activity during preimplantation development in the mouse.

one of the two main forms of the enzyme. A situation of this kind has been found during the development of Rana pipiens embryos (Kohl, Norman & Brooks, 1973); Polymerase I (the nucleolar form) and Polymerase II (nucleoplasmic) behave differently. The levels of Form II do not change during development despite changing patterns of DNA-like RNA synthesis, while Form I increases in the nuclei after gastrulation when ribosomal RNA synthesis begins, suggesting that the expression of the ribosomal cistrons may be controlled in this case by the availability of the corresponding polymerase. We have attempted, therefore, to discriminate between the levels of Polymerases I and II at various developmental stages of the mouse embryo by measuring at two different ionic strengths the enzymatic activity present in embryo homogenates. The activities of RNA polymerases are markedly influenced by the ionic strength. In the presence of excess native DNA, Polymerase I shows optimal activity at 25 to 50 mM-ammonium sulphate, Polymerase II at 100 to 120 min (Gissinger, Kedinger & Chambon, 1974). The technical procedures used were those previously reported (Siracusa, 1973), with the following main differences. Native DNA was used as template in the assay; tritiated uridine triphosphate ([3H]UTP) concentration was raised to 50 μ (NEN, sp. act. 19-8 Ci/mmol), incubation time was prolonged to 90 min. The final incubation mixture (40 μ ) contained 50 mM-tris-HCl, pH 7-9; 2-5 mM-MgCl2 ; 2 mM-MnCl2 ; 0-2 mM-ATP; 0-2 mM-GTP; 0-2 mM-CTP; 50 ^m-[3H]UTP; 0-2 mg native DNA/ml (calf thymus); 12-5% glycerol; 0-05 mM-EDTA; 0-5 mM-dithiothreitol; 2-5 mmNaF; 0-5 mg BSA/ml; 25 or 125 mM-(NH4)2S04. A total of 4140 embryos was used.

Stimulation of ribosomal and DNA-like RNA synthesis by α-methyltryptophan

Rapidly labeled nuclear RNA from rat liver was fractionated by methyl albumin kieselguhr chromatography into the ribosomal and DNA-like RNA components. Injection of 100 mg/kg of α-methyltryptophan into rats stimulated the labeling by [ 3H] orotic acid of both species of nuclear RNA; there was a greater effect on the ribosomal than on the RNA-like fraction. This stimulatory effect of α-methyltryptophan could not be accounted for by an increase in the uptake of the labeled nucleoside by the liver, nor by the increase in the specific activity of the nucleoside and nucleotide pool of the liver.

MRNA synthesis in erythropoiesis: specific effect of erythropoietin on the synthesis of DNA-like RNA.

The erythropoietic mechanism is a gradual process of cell differentiation and proliferation leading from primitive cells (stem cells) to highly differentiated cells (erythrocytes). This phenomenon, which occurs in bone marrow, seems to be essentially the same for different mammals (1).KeywordsSodium Dodecyl SulfateSilk FibroinmRNA SynthesisLabel Amino AcidCulture Bone Marrow CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Early effects of oestradiol-17 on the chromatin and activity of the deoxyribonucleic acid-dependent ribonucleic acid polymerases (I and II) of the rat uterus.

Oestradiol-17beta (1.0mug) was injected intravenously into ovariectomized rats. The earliest detectable hormonal response in isolated uterine nuclei was an increase (10-15min) in RNA polymerase II activity (DNA-like RNA synthesis), which reached a peak at 30min and then decreased to control values (by 1-2h) before displaying a second increase over control activity from 2 to 12h. The next response to oestradiol-17beta was an increase (30-60min) in polymerase I activity (rRNA synthesis) and template capacity of the chromatin. The concentrations of acidic chromatin proteins did not begin to increase until 1h after injection of oestradiol-17beta and histone concentrations showed no significant changes during the 8h period after administration. The early (15min) increase in RNA synthesis in ;high-salt conditions' can be completely eliminated by alpha-amanitin, an inhibitor of the RNA polymerase II. The exact nature of this early increase in endogenous polymerase II activity remains to be determined, e.g. whether it is caused by the increased availability of transcribable DNA of the chromatin or via direct hormonal activation of the enzyme per se.

Transcription in nuclei from immunized rabbit lymph node cells

Lymph node cell nuclei from either ovalbumin immunized or non-immunized rabbits were compared for their ability to synthesize RNA. Nuclei from immunized animals incorporated labelled nucleotides 1.15 to 3.05 times more than the nuclei from control animals. In addition, the characteristic A + U G + C ratio increases significantly after immunization. Using α-amanitin, it is shown that both these quantitative and qualitative changes are related mainly to the activity of the class B RNA polymerases present through the nucleoplasm and implicated in the synthesis of DNA-like RNA.

RNA synthesis in Tetrahymena: Temperature-pressure studies

RNA has been extracted from whole cells of heat-synchronized Tetrahymena and fractionated by methylated albumin Kieselguhr chromatography (MAK). It was found that ribosomal precursor RNA (Q 1) and ribosomal RNA are synthesized during the period between the end of the heat treatment and cytokinesis. Moreover, there is an increase in the synthesis of Q 1 RNA during the later half of cell cycle. The synthesis of DNA-like RNA is maintained during the cycle, however the amount of DNA-like RNA associated with cytoplasmic ribosomes increases at later stages during the cycle. The effects of two physical agents, high temperature and high hydrostatic pressure, on RNA synthesis were also investigated. It was established that these agents markedly inhibit the synthesis of both ribosomal RNA and DNA-like RNA. It is proposed that transfer of messenger RNA from the nucleus to the cytoplasm increases as the cells approach cytokinesis and this is related to the synthesis of ‘division protein’. Moreover, the temperature and pressure treatments result in the inhibition of the supply of messenger RNA.

RNA Synthesis as the Basis for EPTC and 2,4-D Antagonism

Three-day-old soybean(Glycine max(L.) Merr., ‘Hawkeye 63′) hypocotyl segments (0.5 to 1.5 cm below the cotyledons) were utilized to evaluate the effects of the antagonistic interaction ofS-ethyl dipropylthiocarbamate (EPTC) and (2,4-dichlorophenoxy)acetic acid (2,4-D) on growth and nucleic acid synthesis. EPTC inhibited growth and RNA synthesis in soybean tissue. The addition of 2,4-D with EPTC was antagonistic to EPTC inhibition of growth and caused an increase in total RNA synthesis. Analysis of soybean tissue nucleic acids by methylated-albumin-kieselguhr(MAK) column chromatography showed that EPTC inhibited ribosomal-RNA (r-RNA), DNA like RNA (D-RNA), and tenaciously-bound-RNA (TB-RNA) synthesis. The combination of 2,4-D with EPTC caused an increase in D-RNA and TB-RNA synthesis compared to the EPTC treatment alone. The 2,4-D-enhanced synthesis of D-RNA and TB-RNA in the presence of EPTC appears to be the basis of the antagonism between EPTC and 2,4-D. Preliminary analysis of r-RNA indicated that EPTC preferentially inhibited the synthesis of 18S r-RNA more than 25S r-RNA and that 2,4-D had no effect on this selective inhibition.

Stimulation of ribosomal and DNA-like RNA synthesis by tryptophan

Fractionation of rapidly labeled nuclear RNA on methyl albumin kieselguhr columns has shown that administration of tryptophan to adrenalectomized rats stimulates the labeling of Q1 (ribosomal) RNA and Q2 RNA (DNA-like) by about 50 % and stimulates TD RNA (major DNA-like RNA fraction) by 120 %.

Effects of ionic strength on the RNA polymerase activities of isolated nuclei and nucleoli of rat liver

With appropriate divalent cation concentrations the RNA polymerase activity of intact nuclei isolated from rat liver exhibits a biphasic stimulatory response to increasing ionic strength. Base ratios and susceptibility to inhibition by low concentrations of actinomycin D indicate that the initial peak of stimulation at 40 m m (NH 2) 2SO 4 represents enhancement of pre-rRNA synthesis whereas the second phase of stimulation at 10-fold higher ionic strength reflects increased synthesis of more DNA-like RNA species. Evidence has been obtained that the initial peak reflects an effect on nucleolar RNA polymerase activity. Isolated nucleoli exhibit only a monophasic stimulation of pre-rRNA synthesis in response to increasing ionic strength; the peak occurs at 60 m m (NH 4) 2SO 4 and appears to correspond to the initial peak observed at 40 m m (NH 4) 2SO 4 with intact nuclei. These findings suggest not only that intact nuclei from rat liver incubated in vitro synthesize a mixture of pre-rRNA and DNA-like RNA, but also that both pre-rRNA and DNA-like RNA synthesis can be stimulated separately by different levels of ionic strength.

Selective inhibition of ribosomal RNA synthesis in HeLa cells by nogalamycin, a dA:dT binding antibiotic

Nogalamycin is known to bind to DNA and inhibit its use as a template for RNA synthesis. Since the antibiotic has been reported to bind to the dA:dT pair of the DNA double helix, it was explored as a possible selective inhibitor of the synthesis of DNA-like RNA. A concentration of 1.39·10 −7 M was found to produce close to 50% inhibition of cytidine incorporation into the nucleic acids of HeLa cells; 30 times this concentration had no significant immediate effect on protein synthesis. The concentration of 1.39·10 −7 M nogalamycin produced some inhibition of the synthesis of all nucleic acids in increasing order of magnitude DNA < DNA-likeRNA < tRNA, 5-S RNA < rRNA. The selectivity for rRNA was pronounced but a little less than that of actinomycin D. No evidence of a marked increase in the rate of degradation of any nucleic acid was found, nor did it prevent the maturation of the small amount of ribosomal precursor RNA formed in its presence. Various hypotheses concerning the unique sensitivity of rRNA synthesis to these drugs were evaluated.

Cyclic variations in protein, DNA and ribosomal RNA synthesis in 6C 3HED lymphoma cell cultures due to essential amino acid depletion

Suspension cultures of 6C 3HED lymphoma cells growing at concentrations between 3·10 6 and 5·10 6 per ml develop gross changes in nucleic acid and protein synthesis within 24 h of dilution of the culture with an equal volume of fresh medium. The effect was shown to be due to depletion of one or more of the essential amino acids by the cells as they double in number. Unlike bacteria in “step-down” culture, DNA synthesis is most affected; its rate of synthesis in each cell in S phase declines and there is some retardation of initiation of S. Of only slightly less magnitude is the selective decline in the transcription of rRNA. The maturation of precursor rRNA also is retarded. A much smaller fall in the rate of synthesis of DNA-like RNA, tRNA and 5-S RNA may be due to alterations in the handling by the cells of the exogenous nucleosides used for tracer analysis (pool effects). Following replenishment of the essential amino acids in the medium all these effects are rapidly reversed. There is a rapid increase in protein synthesis followed by an increase in the rate of DNA synthesis, which is greater in magnitude than the concurrent increase in rRNA transcription. There is an increase in the rate of initiation of DNA synthesis. Also there is an acceleration in the processing of rRNA precursors to the mature rRNA components. Despite little evidence of change in the rate of synthesis of DNA-like RNA as a whole, some subtle alterations within this fraction were revealed by methylated bovine serum albumin kieselguhr chromatography. The correlation between the changes in rate of transcription and the maturation of rRNA which accompany changes in the rate of protein synthesis induced by amino acid deficiency or cycloheximide in these cells, and in other mammalian cells suggests that the process of maturation of rRNA may provide a mechanism to modulate the rate of supply of ribosomes to the cell which is dependent on the rate of protein synthesis.

Changes in the patterns of synthesis of ribonucleic acid species in immature rat uterus in response to oestrdiol-17 beta.

1. After treatment of immature rats with diethylstilboestrol, the wet weight and RNA content of uterine tissue increased rapidly, reaching a peak at 40hr. After an initial lag of a few hours, the acid-soluble ribose and protein contents also rose to maxima at 40hr. No increase in DNA content occurred until at least 24hr. after treatment. 2. The RNA from immature rat uterus isolated at various times up to 6hr. after administration of oestradiol-17beta was labelled by injecting [(3)H]uridine and [(3)H]guanosine intraperitoneally 30min. before the animals were killed. It was fractionated on columns of kieselguhr coated with methylated serum albumin and the radioactivity in fractions corresponding to transfer RNA, 7s RNA, ribosomal RNA, Q(1)-RNA, Q(2)-RNA and DNA-like RNA was determined. 3. The radioactivity of the whole RNA increased steadily for 6hr. after hormone treatment. The earliest changes occurred in the Q(1)-RNA (ribosomal RNA precursor), whereas at longer time-intervals the radioactivity of the ribosomal RNA, 7s RNA and transfer RNA increased by four- to five-fold. The radioactivity of the DNA-like RNA increased by about 50%, but only at the longer time-intervals. 4. It is concluded that one of the earliest changes in response to oestradiol treatment is a major increase in synthesis of ribosomal RNA followed later by a similar increase in synthesis of transfer RNA and by a much smaller increase in synthesis of DNA-like RNA. The change in synthesis of ribosomal RNA in immature rat uterus may represent one of the most important responses to oestradiol treatment.

The sequential stimulation of uracil-rich and guanine-rich RNA species during cortisone induction of hepatic enzymes

A mixture of 5- 3H-uridine and 8- 14C-guanine was employed to evaluate hepatic RNA synthesis at various times following a single administration of cortisone. During the first hour of hormone action, which precedes hepatic enzyme induction, uridine-rich RNA was selectively synthesized; subsequently a hormonal stimulation of guanine-rich RNA synthesis occurred. These findings are compatible with early hormonal stimulation of the synthesis of DNA-like RNA (high A+U) followed by synthesis of ribosomal RNA (high G+C). These double-labeled tracer experiments provide some insight into the biochemical processes underlying hormonal enhancement of hepatic enzyme synthesis and the subsequent augumentation of hepatic ribosomal content.

Studies on gene activity in synchronized cultures of mammalian cells

Cultures of KB cells synchronized by excess thymidine treatment were used to study the synthesis of DNA-like RNA (D-RNA) and the enzymes lactic dehydrogenase and fumarase during the mammalian cell cycle. Hybridization competition experiments were employed to determine whether the populations of D-RNA molecules synthesized during two different portions of the mammalian cell cycle were completely different. No difference could be demonstrated suggesting that at least some of the same species of D-RNA were synthesized during both time periods examined. An investigation of the pattern of synthesis of the enzymes lactic dehydrogenase and fumarase in synchronized cells indicated that both enzymes were synthesized continuously. The possibility that some mammalian genes function continuously during interphase is discussed.

Control of nucleic acid synthesis in human diploid cells undergoing contact inhibition

In order to define the control mechanisms operative in nucleic acid synthesis in diploid cells in vitro, the synthesis of the nucleic acids of embryonic, human fibroblasts was compared during replication and contact inhibition. Nucleic acid synthesis was measured by the rate of incorporation of isotopically labeled precursors and during a short time interval (4 per cent of doubling time) the nucleic acids were separated by column chromatography. Autoradiography was used as an adjunct to assess the heterogeneity of synthesis in the cell population. Several days after the formation of a monolayer, which induces a state of contact inhibition of replication, the following changes in nucleic acid synthesis ensued: 1. 1. DNA synthesis virtually ceased. 2. 2. The synthesis of tRNA and ribosomal RNA were coordinately repressed to a level which was 1 10 that of the replicating cell. 3. 3. The synthesis of DNA-like RNA, including mRNA, was much less reduced, to a level 1 4 that in replicating cells. 4. 4. Use of the quantitative data on rates of cell replication and assuming that ribosomal RNA was synthesized at a basal level in the contact inhibited cell, the half-life of ribosomal RNA in these cells was calculated as 5.75 days. A similar calculation using in addition the distribution of isotope between DNA-like RNA and rRNA at 1 h, yielded a value for the half-life of the average DNA-like RNA molecule, of 7.9 h. These values are similar to those derived from other mammalian systems. 5. 5. During prolonged maintenance of the diploid cell culture differentiation and slow cell replication occurred. This resulted in the formation of a primitive connective tissue. No significant differences were detected in the patterns of nucleic acid synthesis of the cells in early contact inhibition or in the late stage of an organized fibrous tissue membrane. 6. 6. The stimulation of RNA synthesis and subsequent release from contact inhibition of a small proportion of cells induced by fresh medium was examined. The stimulation of precursor incorporation into RNA was most marked with tRNA, and the decreasing order of response was 5s RNA and other low molecular weight RNA fractions, rRNA, and the least was DNA-like RNA. The stimulatory effect was marked in the 1st hour following the changing of the medium. Since the effect was not abolished by inhibition of protein synthesis its mechanism resembled a derepression of the cistrons involved. The selectivity for different RNA types ruled out a precursor pool effect for anything more than a minor role. There was little change in the sedimentation characteristics of the DNA-like RNA in fresh medium stimulated cells. 7. 7. The subsequent cell replication involved only a small proportion of the cells and showed no obvious relationship to the initial stimulation of RNA synthesis. 8. 8. The study has thus defined several control systems operative in tRNA and rRNA synthesis in diploid cells in vitro.

Patterns of nucleic acid synthesis in the early mouse embryo

Nucleic acid synthesis was examined in the early mouse embryo during cleavage, blastulation, and outgrowth of the blastocyst on an artifical substratum. Rates of synthesis were inferred from the rate of incorporation of isotopically labeled precursors into DNA, sRNA, rRNA, and DNA-like RNA, separated by MAK chromatography. The following conclusions emerged: (1) Nucleic acid synthesis in the 2-blastomere egg was undetectable by the methods used. (2) Incorporation into all nucleic acid fractions was detectable at the 8-cell stage. (3) Measured on a per cell basis, there was abrupt acceleration of the precursor incorporation into rRNA in the morula to a level that was maintained up to the blastocyst stage. Further elevation of incorporation accompanied in vitro attachment and outgrowth of the blastocyst. (4) sRNA and DNA-like RNA showed a gradual steady increase in the rate of incorporation per cell up to the late blastocyst stage. Following blastocyst outgrowth there was a disproportionately great increase in the synthesis of DNA-like RNA. (5) The incorporation of thymidine- 14C into DNA virtually ceased in the late blastocyst; but resumed on outgrowth. (6) The incorporation into nucleic acids rose 19 hours after providing all the nutritional factors previously found to be necessary for blastocyst outgrowth. (7) The DNA-like RNA was synthesized at a low rate relative to rRNA when the early cleavage stages were compared with blastocyst outgrowths, or with fibroblasts and other cell types in cell culture. This may imply that m-RNA is long lived in the early stages of cleavage.

RNA metabolism of fertilized Ascaris lumbricoides eggs during uterine development

1. 1. During their passage through the uterus fertilized Ascaris eggs do not cleave, but have an active RNA metabolism. The total RNA/egg also increases substantially. 2. 2. In the newly fertilized egg there is a massive synthesis of rRNA and possibly a smaller synthesis of DNA-like RNA, almost all of which is localized in the nuclear sub-cellular fraction. Evidence is presented to show that the male genome is exclusively responsible for this synthesis. As the egg passes through the uterus the rate of rRNA synthesis decreases, and in eggs taken from the distal uterus only DNA-like RNA seems to be synthesized. 3. 3. The results were based on labelling of RNA with 3H-uridine or phosphoric acid- 32P, base composition analysis of total and newly synthesized RNA, sedimentation analysis, and determination of protein-synthesizing capability. Adenine-guanine ratios were especially useful in the identification of newly synthesized RNA. 4. 4. Aspects of Ascaris development such as the high rate of oocyte production and yolk formation, shell formation and the long delay before cleavage of the fertilized egg, and chromosome diminution during cleavage are discussed in relation to the RNA metabolism.

Nucleic acid and protein synthesis associated with the induction of nitrate reductase activity in radish cotyledons.

1. RNA and protein synthesis was studied during the incubation of excised radish cotyledons in nitrate, conditions that induced nitrate reductase activity in the tissue. 2. Synthesis of total RNA and protein, as measured by the incorporation of radioactive precursor, was significantly stimulated in the presence of nitrate (compared with chloride control), but was decreased in the presence of ammonium nitrate, which induced higher enzyme activity. 3. Synthesis of RNA and protein was required for induction of enzyme activity, as determined by using the inhibitors actinomycin D, puromycin and cycloheximide. 4. On the basis of 5-fluorouracil inhibition, the synthesis of only DNA-like RNA was required for induction, but no differences, either quantitative or qualitative, were observed in DNA-like RNA synthesis in the presence or absence of induction. 5. A 100-fold purification of the nitrate reductase activity showed no increase in nitrate reductase protein, nor any increased incorporation of radioactive precursor into nitrate reductase protein in the induced versus the control system. Such results suggested that the protein synthesis required for induction may be for a protein other than nitrate reductase.

DNA-associated acidic proteins

After previous removal of acid-soluble nuclear proteins and of RNA, 0.5 M hot perchloric acid extracts DNA together with associated acidic proteins from Ehrlich ascites cell nuclei. The specific activity of these DNA-associated proteins, after pulse-labeling of cells kept in minimal medium, is higher than the specific activity of other nuclear proteins. Their specific activity is proportional to the synthesis of RNA with DNA-like base composition and is not correlated with synthesis of ribosomal precursor RNA and DNA. Centrifugation in CsCl showed that labeled amino acids are associated with DNA in the form of protein molecules and not in the form of amino acids. Labeling experiments indicated that DNA-associated proteins are a mixture of proteins which are labeled independently of each other. Despite their close association with DNA, the synthesis of these proteins is inhibited to the same extent as synthesis of all other proteins by actinomycin D, puromycin and ribonuclease. This indicates that DNA-associated proteins are synthesized in the usual way, by translation of RNA on the ribosomal system. The presence of these proteins is not limited to Ehrlich ascites cells; DNA-associated proteins were also found in various organs of rats. Their specific activity is low in liver in vivo during synthesis of ribosomal precursor RNA. In the liver of starved animals where RNA synthesis is switched to synthesis of RNA with a DNA-like base composition the specific activity of DNA-associated proteins is substantially increased. All of these findings support the proposition that DNA-associated proteins are involved in the control of DNA-like RNA synthesis on DNA template.

Synthesis of DNA-like RNA in synchronized cultures of mammalian cells

1. Cultures of KB cells, synchronized by excess thymidine treatment were used to study the synthesis of DNA-like RNA (D-RNA) and total RNA during the various periods of the mammalian cell cycle. The rate of incorporation of [8- 14C]hypoxanthine into RNA was used as a measure of the rate of total RNA synthesis. D-RNA was assayed by the hybridization of 3H-labeled RNA with DNA. 2. The results obtained indicate that D-RNA is a relatively constant fraction of the RNA synthesized at all times in the cell cycle. On the basis of this result, together with the observation that total RNA synthesis continues throughout interphase, it is concluded that D-RNA is synthesized during all 3 periods of interphase, G 1, S and G 2.