Low Concentrations Of Actinomycin Research Articles

Effects of cell sap, ATP, and RNA synthesis on the transfer of ribosomal proteins into nuclei and nucleoli in a rat liver cell-free system.

To investigate the mechanism for the transfer of ribosomal proteins from cytoplasm into nuclei and nucleoli, the uptake of 3H-labelled ribosomal proteins by the isolated rat liver nuclei was investigated in the system containing ATP, GTP, CTP, UTP, and an energy-regenerating system. In the presences of cell sap, the transfer became temperature-dependent. The concentration of ribosomal proteins was also very important for their specific transfer and 10-15 micrograms of ribosomal proteins/ml were suitable in the presence of 10(7) nuclei. Removal of one of the four nucleoside triphosphates from the complete system containing cell sap, especially that of CTP or UTP, resulted in a marked decrease of the uptake. A low concentration of actinomycin D inhibited significantly the transfer of ribosomal proteins, while alpha-amanitin to a less extent. The results indicate that the uptake of ribosomal proteins by liver nuclei is largely dependent on RNA synthesis especially rRNA synthesis. The transfer was enhanced to some extent by ATP alone. Other nucleoside triphosphates were less effective. Non-hydrolyzable ATP analogues, adenosine 5'-[beta, gamma-methylene]triphosphate and adenosine 5'-[alpha, beta-methylene]-triphosphate were only slightly stimulative. Although ATP enhanced the transfer into the extranucleolar fraction to some extent, the maximal transfer not only into nucleoli but also into the extranucleolar fraction was dependent on the rRNA synthesis. Sedimentation analyses of the nucleolar fraction of rat liver nuclei incubated with [3H]GTP or 3H-labelled ribosomal proteins, showed that small but distinct amounts of the both labelled compounds were incorporated into 60S preribosomal particles although most of them were found in ribonucleoprotein particles below 30S which were previously shown to be degraded products containing larger rRNA precursors [Tsurugi, K., Morita, T., and Ogata, K. (1972) Eur. J. Biochem. 25, 117-128].

Sodium butyrate-induced adhesion in mastocytoma cells.

Sodium butyrate induced adhesion of cultured mastocytoma p-815 cells to the surface of a standard tissue culture grade petri dish. The ratio of the number of adherent cells to that of total cells (adherent plus floating cells) was dependent on the serum concentration and on the dose of sodium butyrate. During approximately the first 6 hr after the addition of sodium butyrate, no cells adhered. The optimum conditions for adhesion were provided by 2 mM sodium butyrate and 15% fetal calf serum, 44 hr after addition of this compound. Morphologically, adherent cells consisted of spindle-shaped and round cells: the latter clustered to the former. Low concentrations of actinomycin D (0.005 microgram/mL) and of cycloheximide (0.5 microgram/mL) inhibited cell adhesion. Adherent cells were easily detached by 0.25% trypsin-0.02% EDTA but not by EDTA alone. Adherent mastocytoma cells which were cultured in the presence of 2 mM sodium butyrate, re-adhered to the surface of the dish. The ratio of adhesion in the second dish, however, was very low (35% after 2 hr incubation). Radioactive iodinated surface proteins of butyrate-treated adherent cells showed two new bands (70,000 and 92,000 D) which were not detected in control cells, but there was no difference in the extent of labeling of high molecular weight protein (250,000 D) between butyrate-treated and control cells.

Polyoma-induced stimulation of nucleoplasmic transcription is paralleled by development of resistance against actinomycin D.

Polyoma virus induced in quiescent, Go-arrested mouse kidney cells a lytic infection. Synthesis of the polyoma T-antigens began 7-8 h after infection and was followed by a mitotic reaction of the host cell comprising stimulated synthesis and accumulation of cellular (mainly ribosomal) RNA and protein and duplication of the host cell chromatin (S-phase). In the present work we focused attention on nucleoplasmic transcription, i.e. synthesis of hnRNA, 5S RNA and tRNA. To inhibit selectively nucleolar transcription we used low concentrations of actinomycin D (act. D). Synthesis of 45S precursor- ribosomal RNA in mock- and polyoma-infected mouse kidney cells was completely blocked by 0.05 micrograms/ml act.D within 2 h. In mock-infected cells also nucleoplasmic transcription was rather sensitive against 0.05 micrograms/ml act.D. Polyoma- induced stimulation of nucleoplasmic transcription began around 12 h and was paralleled by the development of resistance against act.D. Resistance of nucleoplasmic transcription in virus-infected cells was thus similar to that observed by others in uninfected, proliferating mammalian cells. The possible biological implications of these results are discussed.

Gamma radiation inhibits the appearance of induced ornithine decarboxylase activity in Chinese hamster cells.

Ornithine decarboxylase activity of Chinese hamster cells (ODC, EC 4.1.1.17) can be induced in plateau phase by change of medium. Exposure of the cells to gamma radiation before induction reduces the amount of ODC activity induced. The dose-response curve is exponential with a D0 of 106 krad. Exposure of BUdR-substituted cells is more effective in reducing ODC induction at high doses, with a D0 of 38 krad. Cells can recover from the reduction incurred by 74 krad if enzyme induction is delayed for 2 hours after exposure. Treatment of the cells with psoralen-plus-light completely inhibits RNA synthesis without affecting protein synthesis (Heimer, Ben-Hur and Riklis 1977, 1978). Using this procedure it is shown that the effect of gamma radiation on inducible ODC activity is due not only to DNA damage by also involves a post-transcriptional effect. This conclusion is supported by employing a heat shock to inhibit protein synthesis prior to gamma-irradiation of log-phase cells. In such cells the increased activity of ODC upon transfer to 37 degrees C is due primarily to enzyme synthesis using pre-existing RNA species during the first few hours. A low concentration of actinomycin D, which inhibits rRNA synthesis, applied during the recovery period, prevents the recovery of the cells' capacity for maximal ODC induction. This may indicate that, in order to recover, the cells have to repair damage to the ribosomes as well as to DNA.

Regulation of endogenous type C virus expression during normal myeloid cell differentiation Evidence for a role in promoting myeloid cell proliferation and differentiation

A specific regulatory pattern for the expression of endogenous type C virus was found during differentiation of normal myeloblasts from different strains of mice. Endogenous virus was expressed in the myeloblasts. Upon induction of differentiation by the normal macrophage- and granulocyte-inducing protein MGI, there was a rapid enhancement of virus production followed by an inhibition of virus secretion and an intracellular accumulation of viral proteins. There was finally a complete shutoff of viral protein synthesis at the terminal stage of differentiation, so that the mature cells no longer expressed viral proteins. The comparison of normal and fully differentiatable MGI +D + leukemic myeloblasts has shown that the regulatory pattern for virus production during differentiation in the normal myeloblasts was similar to that during differentiation in the leukemic myeloblasts. Ecotropic and xenotropic viruses were detected in the normal myeloblasts, whereas only ecotropic virus was detected in the leukemic myeloblasts. Only the production of ecotropic virus was enhanced upon induction of differentiation of the normal myeloblasts by MGI, whereas there was a shutoff of both xeno- and ecotropic viruses at the terminal stage of differentiation. Dexamethasone, lipopolysaccharide, dimethylsulfoxide, and low concentrations of actinomycin D increased the MGI-induced enhancement of ecotropic virus in both the normal and the leukemic myeloblasts, but did not affect the production of the xenotropic virus. Superinfection of the normal myeloblasts with high titers of their own endogenous ecotropic virus, as in the case of infection with virus from MGI +D + leukemic cells, increased the proliferation of myeloblasts, and in the presence of MGI this then resulted in an increased number of mature cells. It is concluded that the observed pattern for endogenous ecotropic type C virus expression is part of the developmental program of mouse myeloid cell differentiation and that endogenous type C viruses may serve as normal physiological agents for the promotion of myeloblast cell proliferation, which in the presence of the normal inducer of differentiation then results in an increased number of differentiated cells.

Protein synthesis during activation of lymphocytes by mitogens.

When lymphocytes purified from peripheral blood are incubated with mitogenic lectins such as phytohaemagglutinin they initiate DNA synthesis, but only after a lag of about 24 h. There are, however, substantial changes in many aspects of lymphocyte metabolism which begin much more rapidly after lectin addition, and these include large increases in the rates of transcription and translation (Ling & Kay, 1975). The increase in the rate of protein synthesis is first detectable about 2 h after lectin addition, and rises to twice the initial rate after 4 h, and to about 10 times the initial rate per cell after 20h (Jagus & Kay, 1979). There is also an increase in the number of ribosomes per cell by 20h, but if this is selectively suppressed by the addition of low concentrations of actinomycin, the increase in protein synthesis is relatively unimpaired (Kay et al., 1969). The increase in protein synthesis is accompanied by a substantial accumulation of polyribosomes. Most of the ribosomes present in unstimulated lymphocytes are monoribosomes not engaged in protein synthesis, and the majority of those that are active are also monoribosomes, but after activation the proportion of ribosomes actively translating mRNA increases steadily to 7540% by 12-20h (Kay el al., 1971; Ahern & Kay, 1975; Cooper & Braverman, 1977). In contrast, there is no significant change in the elongation rate over this period (Kay et al., 1975), so the increase in protein synthesis must be due entirely to an increase in the rate of the initiation step. Determination of the ability of cell-free protein-synthesizing systems from unstimulated and 20 h-activated lymphocytes to form initiation complexes with [ 35Slmethionyl-tRNA~a has shown that the defective initiation of the unstimulated cells is conserved in cell-free systems derived from them (Ahern et al., 1974). Detailed analysis of this system has shown that, although

Effect of protein synthesis inhibitors and low concentrations of actinomycin D on ribosomal RNA synthesis

FEBS LettersVolume 107, Issue 2 p. 281-284 Full-length articleFree Access Effect of protein synthesis inhibitors and low concentrations of actinomycin D on ribosomal RNA synthesis Silvia Iapalucci-Espinoza, Silvia Iapalucci-Espinoza Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956 - 1113 Buenos Aires, ArgentinaSearch for more papers by this authorMaría Teresa Franze-Fernández, María Teresa Franze-Fernández Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956 - 1113 Buenos Aires, ArgentinaSearch for more papers by this author Silvia Iapalucci-Espinoza, Silvia Iapalucci-Espinoza Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956 - 1113 Buenos Aires, ArgentinaSearch for more papers by this authorMaría Teresa Franze-Fernández, María Teresa Franze-Fernández Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956 - 1113 Buenos Aires, ArgentinaSearch for more papers by this author First published: November 15, 1979 https://doi.org/10.1016/0014-5793(79)80390-7Citations: 32AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume107, Issue2November 15, 1979Pages 281-284 ReferencesRelatedInformation

Possible Point Mutation Sites in LSc, 2ab Poliovirus RNA and a Protein Covalently Linked to the 5'-terminus

Poliovirus type I, vaccine strain (LSc, 2ab), which is a temperature- and actinomycin D-sensitive mutant derived from type I Mahoney strain, was grown in HeLa cells in the presence of 32P and a low concentration of actinomycin D. Seven and a half h p.i., genome 32P-RNA was recovered from the purified virion. Analysis of RNase TI digests of the RNA by two-dimensional gel electrophoresis revealed that three possible point mutation sites exist in the large and unique oligonucleotides in the fingerprint. Neither a capping structure nor a nucleotide such as pppNp, ppNp or pNp, was detected by ion exchange column chromatography at pH 5.0 after digestion of virion RNA with RNase T2. Instead, a 32P-labelled compound, which could be digested with Pronase or proteinase K, was eluted at the void volume of the column. Proteinase K digests of the 32P-labelled compound contained pUp or pU as a single labelled c"mpound, when genome RNA was digested with RNase T2 or nuclease P1, respectively, before digestion with the proteinase. Our data locate possible point mutation sites on the genome of a mutant strain (LSc, 2ab) of type I poliovirus and show that a protein (VPg) is covalently bound to the 5'-terminus of RNA. The protein (VPg) of LSc, 2ab strain migrates faster than capsid protein VP4 (mol. wt. 7000 to 8000) in a polyacrylamide gel and is thus similar to the VPg of the wild-type virus.

The screening test of various chemotherapeutic drugs in primary malignant melanoma cells and human malignant melanoma cell line (TM-1).

The sensitivities to various chemotherapeutic drugs of the primary malignant melanoma cell of the esophagus and the malignant melanoma cells established from human skin were studied by determining the incoporation of 3H-thymidine into tumor cells using the tissue culture method. The incorporation of 3H-thymidine was depressed by a low concentration of Actinomycin D in malignant melanoma cells of the esophagus and by a low concentration of Adriamycin or Actinomycin D in the established malignant melanoma cells. When Actinomycin D was clinically used according to the experimental results, the subcutaneously metastasized tumor was remarkably reduced.

Co-regulation of type C RNA virus production and cell differentiation in myeloid leukemic cells

Mouse myeloid leukemic cells which differ in their competence to be induced to differentiate by the normal macrophage- and granulocyte-inducing protein MGI have been used to study the relationship between type C RNA virus production and myeloid cell differentiation. Clones which can be induced by MGI to form Fc and C3 rosettes, to synthesize and secrete lysozyme and to differentiate to mature macrophages and granulocytes (MGI +D +) were induced by MGI to produce higher amounts of type C virus. Clones (MGI +D −) that were less inducible by MGI for Fc and C3 rosettes and lysozyme and were not induced to form mature cells were also less inducible for higher virus production. In both types of clones, the increased virus production induced by MGI preceded the induction of rosettes and lysozyme. Clones that were not induced by MGI for rosettes or lysozyme (MGI −D −) showed little or no enhancement of virus production. MGI did not affect virus production in erythroleukemic cells, and erythropoietin did not affect virus production in the myeloid leukemic cells. Dexamethasone, lipopolysaccharide, dimethylsulfoxide and low concentrations of actinomycin D can induce some differentiation-associated properties in some of the clones. With these compounds, there was also a direct relationship between the enhancement of virus production and induction of differentiation-associated properties. Virus released from the three types of clones before or after treatment with MGI or dexamethasone was identified as N-tropic. The enhancement of virus production, as measured by reverse transcriptase activity, was accompanied by an increase in the amount of the viral protein p30, and interferon, which did not inhibit the induction of differentiation in the myeloid leukemic cells, also did not prevent the increase in the amount of p30. After the early enhancement of virus production associated with the induction of differentiation, a shut-off of virus production occurred in the mature cells induced by MGI in MGI +D + clones, whereas clones that did not differentiate to mature cells continued to produce virus. The results indicate that enhancement of virus production appears to be an early step in the induction of differentiation. Once induction has occurred, the lack of virus production in the mature cells suggest that a subsequent shut-off of virus production may be required for the completion of differentiation to mature cells. This relationship between cell differentiation and virus production suggests that type C virus has a regulatory role in myeloid cell differentiation.

Inhibition of nucleoplasmic transcription and the translation of rapidly labeled nuclear proteins by low concentrations of actinomycin D in vivo. Proposed role of messenger RNA in ribosomal RNA transcription.

Inhibition of nucleoplasmic transcription and the translation of rapidly labeled nuclear proteins by low concentrations of actinomycin D in vivo. Proposed role of messenger RNA in ribosomal RNA transcription.

Selective inhibition of collagen synthesis in sea urchin embryos by a low concentration of actinomycin D

The rate of collagen synthesis is significantly inhibited in Strongylocentrotus purpuratus embryos continuously exposed to a low act. D concentration (0.75 μg/ml). Collagen synthesis was determined at the mid-gastrula, diamond-shaped larva and pluteus larval stages by two independent assays. One assay determined the amount of hydroxyproline accumulation, and the other measured the rate of [2, 3- 3H]proline incorporation into collagenase-sensitive peptides. The rate of collagen synthesis was reduced in the three developmental stages, with a 75% reduction in the pluteus stage (96 h). Total protein synthesis as determined by [ 3H]proline incorporation was not inhibited. The results suggest that low act. D concentrations selectively inhibit collagen synthesis. A possible mechanism is discussed.

A new class of small nuclear RNA molecules synthesized by a type I RNA polymerase in hela cells

A new class of previously undetected small RNA molecules with a range of discrete sizes between 6S and 10S has been identified in HeLa cell nuclei. They differ in size and location from the previously described small nuclear RNA species (snRNA). These RNA molecules were initially found by selective RNA labeling in vitro in isolated nuclei. The in vitro products migrate in gel electrophoresis in the region from 6–10S with predominant components between 8S and 10S. They are labeled in the presence of very high concentrations of α-amanitin (150–400 μg/ml), suggesting they are synthesized by a type I polymerase. Unlike the major polymerase I product, ribosomal precursor RNA, however, these molecules are found in the nucleoplasm and their labeling is not affected by pretreatment of cells with low concentrations of actinomycin D (0.04 μg/ml). Their formation by a presumptive polymerase I type of enzyme is the basis of their tentative designation as small nuclear polymerase I (snPI) RNAs. The snPI RNA molecules appear to be associated with chromatin and the nuclear matrix. They can be selectively eluted from nuclei leaving most of hnRNA behind. This association is used as the basis of fractionation procedures which separate these molecules from hnRNA and permit the demonstration of the synthesis of at least the most predominant of these RNA molecules in vivo. w

Free fibosomes and growth stimulation in human peripheral lymphocytes: activation of free ribisomes as an essential event in growth induction.

During the initial ten hours of growth in lymphocytes stimulated by phytohemagglutinin, the cells are converted from a state in which over 70% of all ribosomes are inactive free ribosomes, to one in which over 80% of ribosomes are in polysomes or in native ribosomal subunits. In this initial period, there was a neglible increase in total ribosomal RNA due to increased RNA synthesis, and abolition of ribosomal RNA synthesis with low concentrations of actinomycin D did not interfere with polysome formation. Therefore, the conversion is accomplished by the activation of existing free ribosomes rather than by accumulation of newly synthesized particles. The large free ribosome pool of resting lymphocytes is thus an essential source of components for accelerated protein synthesis early in lymphocyte activation, before increased synthesis can provide a sufficient number of new ribosomes. Free ribosomes accumulate once more after 24 to 48 hours of growth, when RNA and DNA synthetic activity are maximal. This reaccumulation of inactive ribosomes at the peak of growth activity may represent preparation for a return to the resting state where cells are again susceptible to stimulation. Activation of free ribosomes to form polysomes appears to involve modification of at least two steps: (a) dissociation of free ribosomes with stabilization as native subunits, and (b) adjustment of a rate-limiting step at initiation.

In the absence of ribosomal RNA synthesis, the ribosomal proteins of HeLa Cells are synthesized normally and degraded rapidly

It is possible to solubilize essentially the total protein complement from a lysate of HeLa cells. When such an extract is displayed on a two-dimensional polyacrylamide gel (pH 5·0 × sodium dodecyl sulfate) it is possible to isolate and identify 45 ribosomal proteins, five histones, and numerous other proteins. Using this method we have measured directly the synthesis of ribosomal proteins in the presence of actinomycin D. The results show that the synthesis of ribosomal proteins does not depend on concurrent transcription of ribosomal precursor RNA. Furthermore, if ribosomal precursor RNA synthesis is blocked for 25 hours by 10 ng of actinomycin/ml, ribosomal protein synthesis as well as histone synthesis continues normally. This suggests either that messenger RNA for ribosomal proteins is exceedingly long-lived or that the synthesis of mRNA for ribosomal proteins can be uncoupled from the synthesis of ribosomal precursor RNA. Ribosomal proteins synthesized in the presence of either high or low concentrations of actinomycin are unstable. They disappear, presumably through degradation, with half-lives of 30 to 100 minutes, depending on the individual proteins. Under these conditions, histones and several other cell proteins are stable.

Evidence for an extranucleolar mechanism of actinomycin D action.

SINCE the original observation of Perry1 that a low concentration of actinomycin D (0.04 μg ml−1) selectively inhibits ribosomal RNA (rRNA) synthesis in vivo, this concentration of drug has been used by numerous investigators to selectively inhibit the synthesis of rRNA in cells in culture. Since nucleolar ribosomal DNA contains a high amount of deoxyguanosine and deoxycytosine bases2 and actinomycin D selectively binds to deoxyguanosine3, it has been thought that actinomycin D preferentially binds to nucleolar DNA to inhibit the transcription of 45S ribosomal RNA precursor.

The RNA polymerase activity of the preimplantation mouse embryo

The activation of the mouse embryo genome has been studied during early cleavage, in vivo. Individual embryos, prepared as whole mounts, were assayed for endogenous RNA polymerase activity. RNA synthesis was detected by autoradiography as the incorporation of [3H]UMP into an acid-insoluble product. No RNA polymerase activity could be detected in the pronuclei of one-cell embryos. Radioactive incorporation was first evident in the nuclei of two-cell embryos. This appeared to be confined to the nucleoplasm and could be abolished by alpha-amanitin but not by low concentrations of actinomycin D. Polymerase activity which was not affected by alpha-amanitin was first detected in the four-cell embryo, predominantly at the peripheries of the nucleoli. Nucleolar labelling increased markedly between subsequent cleavages, reaching a peak in early morulae. In one- and two-cell embryos, label incorporation could be found in the nucleus of the persisting polar body.

Studies on bacteriophage T7 DNA synthesis in vitro. II. Reconstitution of the T7 replication system using purified proteins.

DNA synthesis in vitro using intact duplex T7 DNA as template is dependent on a novel group of three phage T7-induced proteins: DNA-priming protein (activity which complements a cell extract lacking the T7 gene 4-protein), T7 DNA polymerase (gene 5-protein plus host factor), and T7 DNA-binding protein. The reaction requires, in addition to the four deoxyribonucleoside triphosphates, all four ribonucleoside triphosphates and is inhibited by low concentrations of actinomycin D. Evidence is presented that the priming protein serves as a novel RNA polymerase to form a priming segment which is subsequently extended by T7 DNA polymerase. T7 RNA polymerase (gene 1-protein) can only partially substitute for the DNA-priming protein. At 30 degrees C, deoxyribonucleotide incorporation proceeds for more than 2 hours and the amount of newly synthesized DNA can exceed the amount of template DNA by 10-fold. The products of synthesis are not covalently attached to the template and sediment as short (12S) DNA chains in alkaline sucrose gradients. Sealing of these fragments into DNA of higher molecular weight requires the presence of E.coli DNA polymerase I and T7 ligase. Examination of the products in the electron microscope reveals many large, forked molecules and a few "eye"-shaped structures resembling the early replicative intermediates normally observed in vivo.

Comparison of the effects of zinc deprivation and actinomycin D on ribonucleic acid synthesis by stimulated lymphocytes.

1. EDTA inhibited incorporation of [3H]uridine into RNA of lymphocytes, but did not decrease uptake into the cold-acid-soluble fraction of the cells. The inhibition by EDTA was largely reversible by simultaneous addition of Zn2+. 2. Low concentrations pf actinomycin D (3 ng/ml) added at the time of stimulation of the cells inhibited [3H]uridine incorporation into RNA, but concentrations of 50-100 ng/ml were required to produce the same degree of inhibition if addition of actinomycin D was delayed until just before the incorporation was measured. This difference in sensitivity did not reg within the cells. 3. When added immediately before phytohaemagglutinin, actinomycin D (3 ng/ml) and EDTA produced similar time-courses of inhibition of uridine incorporation. 4. Uridine incorporation at 32h was inhibited when actinomycin D (3 ng/ml) or EDTA was added just before stimulation of the cells, but was only slightly affected when they were added at 32h. At intermediate times the incorporation of uridine remained sensitive to addition of EDTA for longer than it was sensitive to actinomycin D. 5. Polyacrylamide-gel separation of RNA synthesized in EDTA-treated cultures in the presence or absence of added Zn2+ showed that lower availability of Zn2+ resulted in a decreased rate of transfer of radioactivity from 32S to 28S rRNA and decreased survival of 28S rRNA relative to 18S rRNA. 6. Close similarities have been shown to exist between the effects of EDTA and low concentrations of actinomycin D. Not all the effects of EDTA could be explained by postulating that Zn2+ was a constituent of RNA polymerase I, nor were the effects of actinomycin D readily explained by previously suggested mechanisms of action of this antibiotic.

Induction of Glutamine Synthetase in Cultures of Embryonic Neural Retina: COMMENTS ON THE USE OF ACTINOMYCIN D

Abstract Selected aspects of the induction of glutamine synthetase by hydrocortisone in cultures of embryonic neural retina were re-examined. The results clarify inconsistencies raised in a recent report (Schwartz, R. J. (1973) J. Biol. Chem. 248, 6426–6435). We show that (a) the extensive histological deterioration of retina cultures attributed to actinomycin D in the above report does not occur under our conditions and was presumably due to some suboptimal culture condition(s); (b) the kinetics of glutamine synthetase induction described in the above report are aberrant and therefore cannot be used as a basis for studies of regulatory processes; (c) contrary to the above report, glutamine synthetase in embryonic retina can be repeatedly induced and deinduced by consecutive application and withdrawal of the steroid inducer; (d) inadequate procedure may account for failures to obtain the characteristic effects of high and low concentrations of actinomycin D in this system. It appears that Schwartz's negative results reflected the pathology of deteriorating retina tissue cultures and therefore are not applicable to the analysis of normal regulatory mechanisms in the induction of glutamine synthetase. Our findings invalidate Schwartz's key arguments for criticizing previous studies on the induction of glutamine synthetase in the retina and on control mechanisms in other inducible enzyme systems.