Characterization of Sertoli cell RNA synthetic activities in vitro at selected times during sexual maturation

In this study, Sertoli cell RNA synthetic activity in vitro was characterized at selected times during sexual maturation. Sertoli cells, isolated from rat testes undergoing the first wave of spermatogenesis and placed in culture for 4 days, exhibited 2-fold increases in soluble ribonucleotide pools and in total RNA concentrations over the age span of 18-35 days. High performance liquid chromatographic analysis of the ribonucleotide pools in Sertoli cells cultured from 18- and 33- to 34-day-old rats revealed that, in addition to the overall age-related doubling of concentrations, uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools were disproportionately increased 4- and 6-fold, respectively. In general, Sertoli cell contained relatively small amounts of UTP in comparison to several other cell types, but exhibited a high ADP:ATP ratio. A uniform 2-fold increase in the base composition of Sertoli cell RNA per mg DNA was observed over the age span of 18-35 days, with no preferential increase in any one specific nucleotide. There was no change in [3H]uridine incorporation (2 h) into RNA per cell (pmol/mg DNA), but decreased specific activity of the RNA (pmol/mg RNA) in Sertoli cells cultured from 35-day-old rats as compared to those from 18- to 19-day-old rats. Similar differences were noted in the specific activity of label incorporated into specific RNA bases. In contrast, the specific activity of the UTP-CTP soluble pool/mg DNA was only slightly increased. These data indicate that processes related to RNA synthesis in the Sertoli cell undergo a number of changes during the period of sexual maturation.     

Utilization of L-cell nucleoside triphosphates by Chlamydia psittaci for ribonucleic acid synthesis.

Long-term, 32-P-labeled L cells were infected with the obligately intracellular parasite Chlamydia psittaci (strain 6 BC). At 20 h postinfection, [3-H]uridine was added, and the infected cells were sampled at intervals for incorporation of the labels into the uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools of the host L cell and the uridine monophosphate (UMP) and cytidine monophosphate (CMP) in 16S ribosomal ribonucleic acid (RNA) of the parasite. The specific activity of the nucleotides was calculated from the ratio of 3-H to 32-P counts in the nucleotides. The rate of approach to equilibrium labeling of UTP and CTP in L-cell pools and UMP and CMP in 16S RNA from the exogenous uridine label was determined from the increase in the ratios of the specific activities of CTP to UTP and CMP to UMP with time. The rate of approach to equilibrium CMP:UMP labeling of the 16S RNA of C. psittaci was consistent with the rate predicted from the kinetics of labeling of the CTP and UTP pools of the host L cell. In analogous experiments, the rate of approach to equilibrium guanosine monophosphate:adenosine monophosphate labeling of 16S RNA from an exogenous [14-C]adenine label was consistent with the rate predicted from the kinetics of labeling of the purine nucleoside triphosphate pool of the host cell. These results support the concept that members of the genus Chlamydia owe their obligate intracellular mode of reproduction to a requirement for energy intermediates which is fulfilled by the host cell. In addition, evidence was obtained that the total acid-soluble purine nucleoside triphosphate pool of L cells accurately represents the precursors of L-cell 18S ribosomal RNA.     

Stimulation of uridine phosphorylation in primary cultures of Xenopus laevis hepatocytes by estradiol-17 beta

The effects of estrogen on the uridine uptake into cells were examined in primary cultures of liver parenchymal cells from Xenopus laevis. The total uptake of [3H]uridine into the estrogen-treated cells and its incorporation into RNA were about 1.5 times higher than the values for control cells. The uptake of [3H]adenosine and its incorporation into RNA were not affected by estrogen. An experiment in which liver parenchymal cells were double labeled with [3H]uridine and [3H]adenosine showed that estrogen elevated the specific radioactivity of the UTP pool 1.4-fold the value found for the control cells, but that of the ATP pool was not altered by estrogen. Short term labeling revealed that estrogen did not significantly alter the rate of the initial uptake of [3H]uridine into the cells, but it did stimulate [3H]uridine phosphorylation about 1.7-fold. Uridine kinase activity measured in cell-free extracts of hepatocytes treated with estrogen had a value 1.6 times that of the control cells. These data indicate that the stimulation of [3H]uridine uptake and phosphorylation in Xenopus laevis hepatocytes in the presence of estrogen is caused by the enhancement of uridine kinase activity.     

Comparison of overall rates of RNA synthesis in implanting and delayed implanting mouse blastocysts in vitro

Implanting and delayed implanting mouse embryos were incubatedin vitro with [³H]uridine for 2–24 hr. The size and specific activity of the [³H]UTP pools were determined by means of a double isotope technique using copolymer synthesis with the [³H]UTP in the embryos, exogenous [¹⁴C]ATP, andE. coli RNA polymerase. Using the rate of incorporation of [³H]uridine into acid-insoluble material and the specific activity of the [³H]UTP pools, it was possible to calculate the overall rate of incorporation of uridine into RNA by the embryos. In implanting embryos it was constant for 24 hr. In contrast, the initial rate of uridine incorporation by the delayed implanting embryos was only 31% of that in implanting embryos (i.e., per cell); this increased steadily during the incubation period, reaching 81% of the rate in implanting embryos after 24 hr. This activation of RNA synthesis by delayed implanting embryosin vitro occurred in the absence of any uterine stimulatory factors. Further, it was shown that although 10% mouse serum would support trophoblastic outgrowthin vitro, it did not influence uptake, distribution of label into nucleotides, or rate of uridine incorporation into RNA in either implanting or delayed implanting embryos. Therefore, it is suggested that if depression and activation of metabolic activity in blastocysts are part of the mechanims of delayed implantation, and if trophoblast outgrowthin vitro is analogous to the process of implantationin vivo, then these two aspects of embryo activation are under different controls.     

Stimulation of facilitated [3H]uridine transport by thyroid hormone in GH1 cells. Evidence for regulation by the thyroid hormone nuclear receptor

We have previously shown that 3,5,3'-triiodo-L-thyronine (L-T3) stimulates cell growth and a 4- to 8-fold increase in growth hormone mRNA in GH1 cells. These effects appear to be mediated by a thyroid hormone nuclear receptor with an equilibrium dissociation constant for L-T3 of 0.2 nM and an abundance of about 10,000 receptors per cell nucleus. In this report, we show that L-T3 exerts a pleiotypic effect on GH1 cells to rapidly (within 2 h) stimulate [3H]uridine uptake to a maximal value of 2.5- to 3-fold after 24 h. This results from an increase in the number of functional uridine "transport sites" as shown by studies documenting an increase in the apparent Vmax with no change in the Km, 17 microM. Although the labeling of the cellular uridine pool and pools of all phosphorylated uridine derivatives was increased by L-T3, there was no change in the relative amounts of the individual pools in cells incubated with or without hormone. The intracellular concentration of [3H]uridine was estimated to be similar to that of the medium, suggesting that facilitated transport mediates [3H]uridine uptake. That this increase in [3H]uridine transport was nuclear receptor-mediated is supported by the excellent correspondence of the L-T3 dose-response curve for [3H]uridine uptake and that for L-T3 binding to receptor. Finally, inhibition of protein synthesis by cycloheximide and RNA synthesis by actinomycin D demonstrated that the L-T3 effect required continuing protein and RNA synthesis. These results are consistent with an effect of the L-T3-nuclear receptor complex to increase uridine uptake in GH1 cells by altering the expression of gene(s) essential for the transport process.     

Problems associated with the labelling of RNA with radioactive precursors in vivo (author's transl)]

The radioactivity of RNA, DNA and proteins in the liver, muscles and cerebrum of 30-day-old rats after labelling with [3H]uridine, [14C]uridine, [3H]cytidine or [3H]orotic acid was measured. It was found that after administration of [3H]uridine, the proteins were 5 - 10 times more radioactive than the RNA. After administration of [14C]uridine, the proteins were 1 - 2 times more heavily labelled than the RNA. Hydrolysis of the proteins followed by chromatography of the amino acids revealed that the protein labelling was mostly due to [3H]glutamate. In the liver, [3H]orotic acid produced very specific labelling of the RNA. The radioactivity of the proteins is very slight. However, the specific labelling of the RNA in the muscles and cerebrum is not so pronounced with this precursor. [3H]Cytidine is an ideal precursor for RNA. The labelling of protein in all three organs examined is very slight, and furthermore, the specific activity of the RNA is 10 - 20 times higher than after labelling with uridine. We were also able to show that after labelling with radioactive uridine, the method of isolation of RNA by alkaline hydrolysis gives incorrect results, because [3H]amino acids interfere with the measurement of the specific activity of the RNA. The heavy labelling of proteins by [3H]-uridine must also be taken into account in histoautoradiography, because our experiments showed that in liver, the proteins in the cell nucleus are 3 times as radioactive as the nucleic acids. The particulate components of the cytoplasm are even 20 times more radioactive than the nucleic acids.     

Effects of adenosine 3':5'-monophosphate and related agents on ribonucleic acid synthesis and morphological differentiation in mouse neuroblastoma cells in culture.

A variety of compounds were assessed for their ability to induce morphological differentiation and to affect the synthesis of RNA in uncloned mouse neuroblastoma cells in culture. The stimulation of morphological differentiation in uncloned cells after exposure for 48 hours to concentrations of 3 times 10-7 to 3 times 10-4 M papavarine or 10-9 to 10-3 M dibutyryl adenosine 3':5'-monophosphate (dibutyryl-cAMP) was associated, in part, with a concentration-dependent decrease in incorporation of [5-3H]uridine into ribosomal RNA (rRNA) and heterogeneous RNA (HnRNA). The latter effect on cellular RNA produced by papavarine occurred within 1 hour after its addition to the medium and was associated with impaired uptake of radioactive precursor into uridine nucleotides and reduction in the intracellular concentration of uridine 5'-triphosphate (UTP). Dibutytyl-cAMP produced a decreased in the specific radioactivity of UTP without affecting the concentration of UTP in the tumor cells. The effects of papavarine and dibutyryl-cAMP could be distinguished further by the 50% reduction of acetylcholinesterase activity produced by papavarine, but not by dibutyryl-cAMP. Papavarine did not, however, reduce the cellular level of the soluble enzyme, adenine phosphoribosyltransferase. Sodium butyrate, while producing morphological effects similar to those of papavarine and dibutyryl-cAMP at equimolar concentrations, caused no significant changes in the incorporation of [5-3H]uridine into rRNA and HnRNA; however, acetylcholinesterase activity was stimulated 6- to 7-fold above control levels. In contrast to the other differentiating agents examined, addition of 10-9 to 3 times 10-4 M concentrations of cAMP to the tissue culture medium enhanced morphological differentiation of nueroblastoma cells, and caused a 10- to 20-fold stimulation of the incorporation of [5-3H]uridine into rRNA and HnRNA at concentrations of 10-4 M and higher. This effect observed only at high concentrations of cyclic nucleotide was accompanied by an elevation in the specific acitivty of UTP, These studies suggest that the morphological response of neuroblastoma cells is not necessarily associated with concomitant alterations in the synthesis of RNA with agents other than cAMP. Observed changes in incorporation of [5-3H]uridine into RNA appear in most instances to be due to alterations in the uptake of uridine, and in the pool size and specific radioactivity of UTP.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of (3H] uridine incorporation into RNA and [3H] leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10-21 M). Insulin stimulated the rate of [3H] thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100-1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H] thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of 3H- uridine, [3H] thymidine and [3H] leucine into their respective precursor pools is not responsible for the apparent stimulation of RNA, DNA and protein synthesis.     

Metabolism of uridine and determination of liver ribonucleic acid synthesis in developing and adult mice

The metabolism of [5-3H]uridine and the incorporation of the precursor into liver RNA was studied in developing (13-day-old) and adult (45-day-old) mice. Different time-courses of labelling and increased amounts of labelled catabolic products of uridine were found in liver and blood of developing mice compared with adult animals. This is suggested to be a consequence of enlarged metabolite pools resulting from a lower total amount of uracil-degrading enzymes in the developing mice. The labelling of the uracil nucleotides was decreased in the developing liver. However, in spite of a lower specific radioactivity of UTP, the RNA-specific radioactivity of developing liver was increased compared with adult liver. Also the labelling of liver RNA with [6-14C]orotic acid was found to be increased in developing mice, thus indicating a higher rate of RNA synthesis in these animals. A more pronounced difference in liver RNA labelling between the developing and the adult mice obtained with the use of [14C]orotic acid than with [3H]uridine may suggest that the de novo pathway, relative to the salvage pathways, is more important in developing than in adult liver.     

Mechanism of Apparent Transcription Inhibition by Methyl Mercury in Cerebellar Neurons

Abstract: We have investigated the mechanism of inhibition of RNA synthesis by methyl mercury (MeHg) in isolated neonatal rat cerebellar cells. Each of the three component steps involved in the incorporation of exogenous [³H]uridine into cellular RNA was examined separately in whole-cell and/or subcellular preparations. Nuclear RNA polymerase activity was measured in preparations containing both free nuclei and whole cells. Incorporation of [³H]UTP into nuclear RNA was found to be unimpaired at concentrations of MeHg that inhibited whole-cell incorporation of [³H]uridine by > 75%. Cellular uptake of [³H]uridine was assayed in cerebellar cells treated with KCN to deplete ATP levels and block subsequent phosphorylation reactions of transported uridine. Uptake activity under these conditions was unaffected by MeHg. Measurement of intracellular phosphorylation of [³H]uridine indicated that inhibition of this activity closely paralleled that of RNA synthesis. Quantitation of individual uridine nucleotides by polyethyleneimine-cellulose TLC revealed reduced levels of UTP and UDP whereas levels of UMP were elevated, suggesting that impairment of phosphorylation was not the result of cellular ATP depletion but, more likely, a direct effect on phosphouridine kinase enzymes. This mechanism of MeHg-induced inhibition of RNA synthesis was confirmed by assays of uridine phosphorylation using cell-free extracts in which exogenous ATP was supplied.     

Separate pyrimidine-nucleotide pools for messenger-RNA and ribosomal-RNA synthesis in HeLa S3 cells.

Kinetic analyses of mRNA and 28-S RNA labeling [3H]uridine revealed distinctly different steady-state specific radioactivities finally reached for uridine in mRNA and 28-S RNA when exogenous [3H]uridine was kept constant for several cell doubling times. While the steady-state label of (total) UTP and of uridine in mRNA responded to the same extent to a suppression of pyrimidine synthesis de novo by high uridine concentrations in the culture medium, uridine in 28-S RNA was scarcely influenced. Similar findings were obtained with respect to labeling of cytidine in the various RNA species due to an equilibration of UTP with CTP [5-3H]Uridine is also incorporated into deoxycytidine of DNA, presumably via dCTP. The specific radioactivity of this nucleosidase attained the same steady-state value as UTP, uridine in mRNA and cytidine in mRNA. The data indicate the existence of two pyrimidine nucleotide pools. One is a large, general UTP pool comprising the bulk of the cellular UTP and serving nucleoplasmic nucleic acid formation (uridine and cytidine in mRNA, deoxycytidine in DNA). Its replenishment by de novo synthesis can be suppressed completely by exogenous uridine above 100 muM concentrations. A second, very small UTP (and CTP) pool with a high turnover provides most of the precursors for nucleolar RNA formation (rRNA). This pool is not subject to feedback inhibition by extracellular uridine to an appreciable extent. Determinations of (total) UTP turnover also show that the bulk of cellular RNA (rRNA) cannot be derived from the large UTP pool.     

Inhibition of cell division in amoebae: the incorporation of tritiated precursors into Amoeba proteus after the injection of non-homologous cytoplasm.

The injection of non-homologous cytoplasm into any strain of large free-living amoebae leads to a 60% inhibition of division amongst recipient cells. When the post-microsomal supernatant fraction of Amoeba discoides was injected into A. proteus, this inhibition of division was as high as 95%. The incorporation of tritiated precursors, either [3H]uridine or 3H-amino acids, into these inhibited amoebae was studied at various times after the injection of the inhibitory material using autoradiography. When cells were grown in [3H]uridine, autoradiographs indicated that RNA synthesis had ceased 2 days after the injection of non-homologous material. However, if [3H]uridine was injected into the inhibited cells, some synthesis of RNA could be detected up to 4 days after the injection of inhibitor. These results suggested that uptake of [3H]uridine was impaired and that one site of action of the inhibitory molecules was RNA synthesis for membrane components. Experiments with a variety of 3H-amino acids suggested that protein synthesis continued for at least 9 days after the injection of non-homologous cytoplasm, and that in these cells some informational RNA molecules were long-lived. There seemed to be accumulation of material containing [3H]lysine in the nuclei of control cells taken at random from cultures, and this was seen in the nuclei of inhibited cells 1 day after injection. However, 2 days after the injection of inhibitor, no accumulation of [3H]lysine-containing material was found in the nuclei.     

Ribonucleic acid labelling and nucleotide pools during compensatory renal hypertrophy

During the first 48h of compensatory renal hypertrophy induced by unilateral nephrectomy, RNA content per cell increased by 20-40%. During this period, rates of RNA synthesis derived from the rates of labelling of UTP and RNA after a single injection of [5-(3)H]uridine showed no change in the rate of RNA synthesis (3.1nmol of UTP incorporated into RNA/min per mg of RNA). ATP and ADP pools were not changed. The rate of RNA synthesis was considerably in excess of the increment of total RNA appearing in the kidneys. With [5-(3)H]uridine as label, only continuous infusion for 24h could produce an increase (60%) in the specific radioactivity of renal rRNA in mice with contralateral nephrectomies. With a single injection of [methyl-(3)H]methionine used to identify methyl groups inserted into newly synthesized rRNA, the specific radioactivity of this rRNA was unchanged 5h after contralateral nephrectomy, increased by 60% at 9-48h, and returned to normal values at 120h. Most RNA synthesized in both nephrectomized and sham-nephrectomized mice has a short half-life. Since total cellular RNA content increases in compensatory hypertrophy despite unchanged rates of rRNA synthesis, the accretion of RNA might involve conservation of ribosomal precursor RNA or a change in rate of degradation of mature rRNA.     

Exogenous nucleosides stimulate RNA synthesis in resting cells of a culture of scarlet rose

RNA synthesis in response to exogenous nucleoside precursors was studied in a suspension culture of rose cells. Exponentially growing and resting cells were prelabeled with [3H] uridine, an excess of unlabeled uridine added, and subsequent isotopic incorporation into nuclear and ribosomal fractions measured. The data were compared to control values in cells continuously labeled in the absence of unlabeled uridine. Addition of uridine to the growing culture reduced the further uptake, and incorporation of [3H] uridine into RNA. In contrast, in resting cells, the addition of uridine (or, purine nucleosides) enhanced the apparent utilization of [3H] uridine in RNA synthesis by 2- to 4-fold.     

Effect of puromycin on synthesis, processing, and nucleocytoplasmic translocation of rRNA in Tetrahymena pyriformis.

1. Treatment of Tetrahymena pyriformis with various concentrations of puromycin results in a more pronounced inhibition of [3H]uridine accumulation in stable RNA than of protein synthesis. 2. At a concentration of 500 micrograms/ml, which is almost completely inhibitory to [3H]uridine incorporation in vivo, puromycin has no influence on the incorporation of [3H]UTP into RNA in isolated macronuclei. Pretreatment of the cells with the antibiotic, however, reduces the activity of RNA polymerases in isolated nuclei to less than 30%. 3. In puromycin-treated cells a small amount of pre-rRNA is synthesized but not processed into cytoplasmic rRNAs. 4. Puromycin reduces the nucleocytoplasmic translocation of pre-existing RNA to about 25% of the control rate within 5 min, resulting in an accumulation of relatively stable rRNA precursor molecules in the macronucleus.     

The relationship between the turnover of UTP and RNA synthesis in cultured cells treated with aflatoxin B1.

Methods have been adapted to measure the specific activity of UTP in cells in monolayer culture. In HeLa cells labelled with [3H]uridine and treated with aflatoxin B1 there was reduced radioactivity in crude acid extracts, but the toxin did not affect the radioactive incorporation into UTP. Using cells in which the UTP was pre-labelled, the subsequent addition of aflatoxin B1 inhibited UTP incorporation into RNA. Accordingly aflatoxin B1 did not inhibit the uptake of uridine or the latter's conversion to UTP but inhibited the incorporation of UTP into RNA.     

An autoradiographic method of detectingA. laidlawii andM. hyorhinis in cell cultures

Summary The autoradiographic investigation of L cells and Chinese hamster cells for the presence of mycoplasmas (A. laidlawii andM. hyorhinis) using uridine/uracil (UdR/U) testing is a rapid and reliable method suitable for the serial checking of even a small number of cells. It depends on a reduced incorporation of [³H]uridine and an increased uptake of [³H]uracil into the RNA of mycoplasma-infected cells, shown in autoradiograms by the density of the grains and their distribution. Results obtained by the autoradiographic technique correspond approximately to specific activity values of RNA-infected cells after the incorporation of [³H]uridine and [³H]uracil.     

Lymphokines inhibit macrophage RNA synthesis

The effects of lymphokine (LK) preparations on the incorporation of [3H]uridine into macrophage RNA were investigated. Supernatants from murine spleen cells activated in vitro by alloantigens or Con A, and shown to contain macrophage-activating factor (MAF), were used as the source of LK. It was observed that such LK preparations contain factor(s) causing a profound inhibition of [3H]uridine incorporation into the RNA of proteose-peptone-elicited peritoneal macrophages. Such RNA-labeling inhibitory factor (RIF) was absent in control supernatants from nonstimulated cultures, and showed activation curves similar to that of MAF. RIF activity was not due to altered permeability of macrophages to [3H]uridine nor to the changes in the specific activity of the pool of RNA precursors, but rather reflected an altered metabolism of RNA. The inhibition of RNA synthesis was dependent upon the presence of nanogram amounts of LPS as a costimulator. Moreover, the response to RIF appeared to be genetically controlled since macrophages from C3H/HeJ mice were not affected by RIF, while C3H/HeN mice were fully responsive. In parallel cultures of macrophages, LK were also tested for their MAF activity, and a strong similarity between the biological conditions in which MAF and RIF activities were expressed could be demonstrated. The assay for RIF provides a new and convenient parameter for measuring macrophage-sensitive LK activity that might be very useful for monitoring purification or for screening of T-cell hybridoma supernatants.     

Utilization of uridine for RNA synthesis in the insect cell line CP-1268 derived from the codling moth, Laspeyresia pomonella.

The utilization of [3H]-5-uridine by CP-1268 cells was studied. Uridine was rapidly transported into these cells by a concentration dependent, saturable process. Exogenous uridine rapidly equilibrated with cellular nucleotide pools and virtually all of the uridine transported into the cells was phosphorylated. Uridine incorporation into RNA was studied by continuous and pulse-labeling techniques in the prescence or absence of actinomycin D and cordycepin. These studies have shown that the pattern of unstable RNA precursor and relatively stable RNA product relationship known to exist in mammalian cells similarly exists in insect cells in vitro. This pattern varied markedly with pulse-labeling time and required the addition of RNA inhibitors to block reincorporation of intracellular labeled metabolites during the chase.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.