Synthesis Of Cytoplasmic RNA Research Articles

Multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase.

Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5'-to-3' direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5'-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5' cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5'-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.

Expression of mutated glucocerebrosidase alleles in human cells.

Gaucher disease is a heterogeneous disease characterized by impaired activity of the lysosomal enzyme glucocerebrosidase. This heterogeneity is attributed to a large number of mutations in the corresponding gene. In order to test the biochemical properties of some mutations prevalent among Israeli populations, the normal human glucocerebrosidase cDNA and cDNAs carrying mutations N370S, L444P, D409H, recTL, recNcil, P415R and 84GG were coupled to the T7 RNA polymerase promoter in a vaccinia virus-derived expression vector (pTM-1). Recombinant viruses were produced and used to infect human tissue culture cells. RNA and protein stability, recognition by anti-glucocerebrosidase monoclonal antibodies and intracellular enzymatic activity were measured. The results demonstrated that the D409H allele directed synthesis of cytoplasmic RNA with decreased stability compared with its normal counterpart or other mutated forms. The D409H and L444P mutated proteins had lower stability than that of their normal counterpart, while the recNcil-mutated protein was more stable. Only glucocerebrosidase forms harboring leucine at position 444 were recognized by the anti-glucocerebrosidase monoclonal antibodies used (8E4 and 2C7). Measurements of enzymatic activity of the recombinant proteins in cells loaded with a fluorescent glucosylceramide demonstrated that the N370S mutated enzyme had activity similar to that of the normal enzyme. The other mutated enzymes exhibited varying degrees of activities, generally corresponding to the phenotypes with which they are associated. The results presented demonstrate the use of the vaccinia virus-derived expression system and of loading living cells with fluorescent substrate as efficient tools for studying mutants in Gaucher disease and in other lysosomal diseases.

Transcriptional inhibition by carcinogenic chromate: relationship to DNA damage.

Hexavalent chromium compounds are carcinogenic to humans, are potent inducers of tumors in experimental animals, and can neoplastically transform cells in culture. In this study, the effects of sodium chromate on the expression of the 78-kDa glucose-regulated protein (GRP78) gene and on general transcription were investigated with respect to the DNA damage induced by this agent. DNA single-strand breaks, DNA-protein cross-links, and chromium-DNA adducts were present in CHO cells immediately after 2 h of treatment with sodium chromate. Subsequently, these types of damage were repaired at different rates. Single-strand breaks were essentially repaired after 8 h. By 24 h posttreatment, no cross-links remained in cells exposed to 30 or 150 microM chromate, although cells treated with the 300-microM concentration still contained cross-links at that time. DNA-chromium adducts remained unrepaired for at least 32 h. The moderate constitutive level of GRP78 mRNA was not affected by chromate. Chromate did, however, suppress induction of this gene by tunicamycin in a concentration-and time-dependent manner. Thirty micromolar sodium chromate (96% survival), which caused the least DNA damage, had no effect on GRP78 induction, general RNA synthesis, or mRNA synthesis. Induction of GRP78 was suppressed immediately and 12 h after treatment with 150 microM chromate (54% survival), although there was a partial recovery of induction at 24 h after treatment, which correlated with the repair of DNA-protein cross-links. In contrast, both total cytoplasmic RNA and mRNA synthesis were suppressed by approximately 60-75% for at least 32 h by 150 microM chromate. At the 300-microM concentration (8% survival), where DNA-protein cross-links persisted beyond 24 h, GRP78 induction was totally suppressed for at least 24 h, while total RNA and mRNA synthesis were suppressed by 80-90% for at least 32 h. Overall, the effects of chromate on GRP78 induction correlated most closely with the presence of DNA-protein cross-links, but suppression of total RNA and mRNA synthesis correlated with the presence of DNA-chromium adducts. These results indicate that chromate exerts differential effects on the induction of the GRP78 gene and on general transcription.

Maternal dietary deficiency in rats and the in vivo synthesis of cytoplasmic, nucleolar and chromosomal RNA in the liver of their 21-day-old progeny

The metabolism of cytoplasmic, nucleolar and chromosomal RNA was studied at various time intervals ranging from 6 to 120 minutes in the liver of the 21-day-old progeny of well-fed and dietary restricted rats. The dietary-restricted group's progeny demonstrated significant increases in the kinetics of labelling of cytoplasmic RNA (p<0.001) at 120 minutes, of nucleolar RNA (p<0.001) at 30, 60 and 120 minutes, and of chromosomal RNA (p<0.001) at 60 and 120 minutes. At 60 minutes, there was a pronounced 10-fold increase in incorporation in the 18S region (mRNA) of the gradient of nucleolar RNA of the dietary-restricted group's progeny, which became 3-fold higher at 120 minutes. Radioactivity was lower in chromosomal RNA at 6 and 30 minutes but was considerably higher at 60 and 120 minutes in the dietary-restricted group's offspring compared to the well-fed pups. These data clearly demonstrate that maternal dietary restriction in rats leads to cellular events which increase the synthesis of RNA in the liver of their 21-day-old progeny.

Acquired thermotolerance following heat shock protein synthesis prevents impairment of mitochondrial ATPase activity at elevated temperatures in Saccharomyces cerevisiae

The complex molecular response of cells to sudden temperature changes is a well-characterized phenomenon. Although it is clear that the induction of heat shock proteins provides protection from heat in all of the organisms so far tested, very little is known about the role that this set of proteins plays in cellular homeostasis. Recently, putative roles for hsp60 and hsp70-like proteins have been proposed in Saccharomyces cerevisiae. hsp70-like proteins have been shown to be necessary for translocation of precursor polypeptides into mitochondria and endoplasmic reticulum, while hsp60 is required for the assembly of precursor polypeptides into oligomeric complexes following incorporation into the mitochondrial matrix. In this paper, we report that a brief temperature shock (44 °C) impairs coupling of oxidative phosphorylation in S. cerevisiae as measured indirectly by the CI-CCP/oligomycin assay. Furthermore, at high temperature oligomycin stimulates rather than inhibits oxygen uptake under nonthermotolerant conditions. Pretreatment of cells for a short period of time at 37 °C, prior to exposure to higher temperatures rescues the capacity to maintain coupling between oxidative phosphorylation and electron transport. Inhibition of cytoplasmic RNA or protein synthesis during heat shock prevents the protection of this mitochondrial activity. We propose that one of the roles of the induction of heat shock proteins (or related activities) is to protect mitochondrial ATPase activity under conditions of further increase in temperature.

Characterization of the thymus leukemia (TL) product encoded by the BALB/c T3c gene by DNA-mediated gene transfer. Comparison to the T13c product and BALB/c leukemia TL.

Using DNA-mediated gene transfer, we have studied the TL protein products encoded by both the T3c and T13c BALB/c genes. Biochemically, the proteins differed in their m.w. and pI points; serologically, although both molecules were recognized by TL alloantiserum, only the T13c protein was recognized by monoclonal TL antibodies. Interestingly, both proteins were serologically and immunochemically recognized by leukemia-specific TL.4 antiserum. The quantity of cell surface T13 was significantly greater than T3 possibly due to the less efficient splicing of T3 transcripts in the L cell nucleus; both genes directed the synthesis of cytoplasmic RNA containing an unspliced intron 3 as assessed by S1 analysis. In toto, the results suggest that T3c is similar or perhaps identical with the novel TL product previously identified on the surface of certain x-ray-induced BALB/c leukemias.

Gametic differentiation in Chlamydomonas reinhardtii: light dependence and gene expression patterns

Gametes of Chlamydomonas reinhardtii synthesize numerous proteins not observed in vegetative cells and vice versa. Gametogenesis induced changes in gene expression were confirmed by SDS-PAGE of in vitro translation products using total RNA from gametes and vegetative cells. Vegetative cells and gametes thus represent two cell types with distinct patterns of gene expression. The generation of mature gametes from liquid cultures of asynchronously growing vegetative cells was dependent on light. This light requirement could not be substituted for by an organic source of energy and carbon, indicating that light serves as a signal in gametogenesis. The light signal was shown to become effective only after preincubation in nitrogen-free medium. This delayed competence for light indicates that the two external signals — nitrogen-starvation and light —may function in sequence. Execution of the light dependent step in gamete formation required cytoplasmic protein synthesis and RNA synthesis.

Compartmentation of uridine 5?-triphosphate occurs during synthesis of cytoplasmic and mitochondrial ribosomal RNAs of cultured tomato cells but not during synthesis of cytoplasmic ribosomal and transfer RNAs

Compartmentation of uridine 5'-triphosphate (UTP) was studied during the nucleolar synthesis of cytoplasmic ribosomal RNA (cyt-rRNA) and the synthesis of cytoplasmic transfer RNA (cyt-tRNA) in the nuclear matrix as well as the synthesis of mitochondrial ribosomal RNA (mt-rRNA) in tomato (Lycopersicon esculentum Mill. cv. Lukullus) cell-suspension culture using the approach of Wiegers et al. (Eur. J. Biochem. 64, 535-540, 1976). Before measurements were made, it was ensured that: (i) there was steady-state labeling of all RNAs studied as well as UTP; (ii) there was stability of cyt-tRNA and cyt-rRNA; (iii) there was no label randomization through degradation of [(3)H]uridine; (iv) there were significant differences in the specific radioactivity of UTP, the final immediate precursor of RNA, after supplying the cells with two different exogenous [(3)H]uridine concentrations.By comparing the steady-state specific radioactivity of UTP with that of cyt-tRNA and cyt-18S rRNA during constant [(3)H]uridine supply, we found that the three molecules had equal specific radioactivities which, however, differed significantly from that of the mt-rRNA. With a 20-fold higher uridine concentration, i.e. a 20-fold lower specific radioactivity of exogenous [(3)H]uridine, the specific radioactivity of cyt-rRNA, cyt-tRNA and UTP decreased proportionally whereas that of mt-RNA increased. These results argue against different UTP pools during synthesis of cyt-rRNA and cyt-tRNA, but indicate compartmentation of UTP during rRNA synthesis in the nucleus and the mitochondria of tomato cells.

Synthesis of globin RNA in enucleated differentiating murine erythroleukemia cells.

In an earlier report (Volloch, V., 1986, Proc. Natl. Acad. Sci. USA., 83:1208-1212) we had presented evidence for the occurrence of the cytoplasmic synthesis of globin mRNA and of RNA complementary to globin mRNA which differed from DNA-dependent transcription by its insensitivity to actinomycin D. In this paper, we describe the use of enucleated differentiating mouse erythroleukemia cells to demonstrate directly the occurrence of cytoplasmic synthesis of both positive- and negative-strand globin RNA. For this purpose, we developed an enucleation procedure which yielded pure cytoplasts from differentiated mouse erythroleukemia cells in the absence of cytochalasin B and selectively permeabilized the cytoplasts to small molecules by treatment with dextran sulfate and saponin. The permeabilized cytoplasts incorporated [3H]dUTP into positive- and negative-strand globin RNA and experiments with mercurated nucleotide substrate suggested that this process involved de novo RNA synthesis rather than limited terminal nucleotide addition. Globin RNA synthesis required Mg++, was inhibited by Mn++, and was unaffected by the addition of Zn++. Studies of its response to inhibitors of DNA-dependent RNA synthesis showed that it differed from that process in its insensitivity to actinomycin D and alpha-amanitin, but that like many other macromolecular biosynthetic reactions it was inhibited by rifamycin AF/ABDP and aurintricarboxylic acid. These observations provide additional evidence for the occurrence of cytoplasmic RNA-dependent RNA synthesis in differentiated cells and show permeabilized enucleated cells to be a useful experimental system for further studies of the characteristics of that process.

Role of exogenous hemin in the synthesis of hemoproteins and nonheme proteins during glucose repression in Saccharomyces cerevisiae

Exogenous addition of hemin to glucose-repressed cells of Saccharomyces cerevisiae stimulates the incorporation of amino acid into cytoplasmic proteins twofold. There was no significant change in the synthesis of total cytoplasmic RNA whereas a 40% increase in the synthesis of poly(A)-containing RNA was observed upon hemin treatment. Cell-free translation of cytoplasmic mRNAs and immunoprecipitation analysis of the translated products with antibodies against subunit V of cytochrome oxidase and the α and β subunits of F 1-ATPase reveals that there is an eightfold enrichment of the mRNA for subunit V of cytochrome oxidase upon hemin treatment. The effect of hemin on the α and β subunits of F 1-ATPase is only marginal, suggesting a differential role for heme in the synthesis of hemoproteins and nonheme proteins during glucose repression.

Activation of putative transposition intermediate formation in tumor cells.

The transcription levels of two families of mouse repetitive elements namely intracisternal A particle (IAP) genes, and B2 sequences were analyzed in different tumor cells and normal tissues. These sequences belong to two major classes of mobile elements present in the mouse genome. The Northern blots containing poly(A) + RNAs from tumor cells and normal tissues were hybridized to the cloned IAP gene and B2 sequence. The content of IAP gene transcripts in tumor cells is much higher than in normal cells. A 10- to 100-fold difference was found. The predominant IAP-gene specific RNAs in all investigated tumor cells were 9.5, 6.8 and 5.3 kb long. Additional RNA species were found in some of the tumors. The active synthesis of small cytoplasmic B2 RNA transcribed by RNA polymerase III was also detected in most tumor cells tested. Usually it was higher than in normal cells. Free closed circular DNAs hybridizing to IAP gene probes were cloned from Ehrlich ascites carcinoma cells. We speculate that the data obtained indicate the enhanced transposition of mobile elements in tumor cells which may be an important factor of tumor progression.

Characterization of 3H-uridine incorporation and messenger RNA synthesis in human monocytes activated to secrete alpha interferon or monocyte-derived fibroblast growth factor.

Human monocytes are multifaceted cells with a wide range of immunoregulatory functions and distinct secretory products. This manuscript reports on initial attempts to identify specific early macromolecular synthetic events associated with various types of human monocyte activation by observing the patterns of RNA synthesis displayed by human monocytes that are exposed to well characterized activating stimuli. It was found that muramyl dipeptide (MDP), an activator of monocyte-derived fibroblast growth factor (MD-FGF) release from monocytes, also stimulates a reproducible increase in human monocyte total 3H-uridine incorporation and cytoplasmic messenger RNA (mRNA) synthesis at 4 h following activation. In contrast, polyriboinosinic acid:polyribocytidilic acid (poly I:C), an excellent stimulator of monocyte alpha interferon (IFN alpha) release, did not cause a change in either 3H-uridine incorporation or cytoplasmic mRNA production at any of the time points tested. Poly I:C was also found to be a poor stimulator of MD-FGF release. Conversely, MDP did not stimulate any detectable IFN release from human monocytes. The discrepancy between the patterns of macromolecular synthesis observed in human monocytes activated to secrete MD-FGF as compared with IFN indicates that divergent postactivation control mechanisms may be operative at the RNA level in the monocyte following activation of these two distinct functions.

Evidence for a role of calmodulin in the regulation of prolactin gene expression.

Epidermal growth factor (EGF) stimulates prolactin (PRL) gene expression in GH3 cells in a Ca2+-dependent manner (White, B. A., and Bancroft, F. C. (1983) J. Biol. Chem. 258, 4618-4622). The present report shows that the phenothiazine, calmidazolium (compound R 24571), blocks the ability of EGF plus Ca2+ to increase levels of PRL mRNA. Calmidazolium inhibition of this response is dose dependent in the range of 0.05-1.00 microM. Total inhibition of the response was consistently obtained at a level of calmidazolium (0.5 microM) that had no effect on total cytoplasmic RNA synthesis, total cytoplasmic protein synthesis, cell viability, or extent of EGF plus Ca2+-induced cell aggregation. The drug inhibited the increase in PRL mRNA when given immediately before or 48 h after treatment with EGF plus Ca2+. Another calmodulin inhibitor, W13, similarly blocked the ability of EGF plus Ca2+ to stimulate PRL mRNA, whereas the less active analog, W12, had little effect. These results implicate Ca2+-binding proteins such as calmodulin in the mechanism of action of EGF in GH3 cells, and, therefore, provide further evidence for a role of intracellular Ca2+ in the regulation of the expression of a specific eukaryotic gene, the PRL gene.

Dexamethasone-sensitive and -insensitive responses during in vitro differentiation of friend erythroleukemia cells

We have investigated the mechanism(s) by which dexamethasone inhibit DMSO-induced Friend erythroleukemia cell differentiation in vitro. In particular, we examined the effects of dexamethasone on (a) the early events of differentiation such as cell volume alterations and ‘memory response’ and (b) the onset of biochemical events associated with terminal erythroid cell differentiation. By analysing kinetics of commitment of Freind cells to terminal erythroid differentiation on a clonal basis, we have observed that dexamethasone inhibited the completion of the latent period (time elapsed prior to commitment) and impaired “memory” (ability of inducer-treated cells to continue differentiation after a discontinuous exposure to inducer). Treatment of Friend cells with dexamethasone did not prevent the occurence of DMSO-induced alterations in cell volume. However, dexamethasone treatment prevented a series of biochemical events with terminal Friend cell differentiation. These include the decrease in the rate of both cytoplasmic and nuclear RNA synthesis as well as the induction of cytidine deaminase activity and hemoglobin synthesis. These data indicate the dexamethasone-sensitive process(es) operate during the early stages of Friend cell differentiation and that they are responsible for the inhibition of terminal erythroid maturation. These dexamethasone-sensitive processes, however, appear to be different from those regulating cell volume alterations during the earyl steps of DMSO-induced Friend cell differentiation.

The Hypersensitive Reaction in Tobacco Leaf Tissue infiltrated with Pseudomonas pisi 5. Inhibition of RNA Synthesis in Mesophyll Cells

AbstractThe synthesis of mesophyll cell RNA in tobacco leaf tissue infiltrated with the incompatible bacterium Pseudomonas pisi was investigated by light and electron microscope autoradiography of H3‐uridine uptake.Interpretation of the light microscope quantitative data was complicated by the oscillation in levels of cytoplasmic RNA synthesis that resulted from the physical process of fluid infiltration (seen in the control tissue). Direct comparison with the control showed that the presence of bacteria resulted in a rapid decrease in the synthesis of host cell RNA. This effect was clear within the first 1½h (induction period) of the hypersensitive reaction, where it was particularly marked in the cytoplasm of palisade cells. Electron microscope autoradiography showed that the presence of bacteria did not cause complete cessation of RNA synthesis in mitochondria and chloroplasts.The early inhibition of RNA synthesis preceeded fine structural (degenerative) change, and should be regarded as one of the primary events associated with the induction of host cell necrosis.

The inhibition of vaccinia virus replication by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB): an effect at the assembly stage.

The step sensitive to DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) in vaccinia virus replication has been investigated. Ninety microM-DRB extensively inhibited the yield of vaccinia virus after infection of Ehrlich ascites tumour cells. DRB did not inhibit cytoplasmic vaccinia DNA replication. Cytoplasmic viral RNA synthesis (both early and late) was also apparently unaffected and the virus RNAs thus synthesized were normal sized. The expression of early, intermediate and late proteins was not detectably impaired by DRB in vaccinia virus-infected cells. DRB inhibited vaccinia virus replication at the assembly stage since most of the virus DNA remained in a DNase-sensitive form in the infected cells and the virus was therefore not normally coated with virus proteins.

Coordinate regulation of two cytoplasmic RNA species transcribed from early region 2 of the adenovirus 2 genome.

Early region 2 (E2) of the adenovirus 2 genome specifies a 72,000-dalton DNA-binding protein that is required for viral DNA replication. Electron microscopy studies have detected two major forms of 20S E2 mRNA, one species with a 5' leader from map position 75 and a second form having a leader from position 72 (Chow et al., J. Mol. Biol. 134:265-303, 1979). Only the species with a leader from position 75 was detected at early times; however, both forms were found at late times. We have analyzed the temporal regulation of E2 expression by documenting mRNA accumulation in the cytoplasm. Kinetic studies of pulse-labeled RNAs demonstrated a peak of E2 cytoplasmic RNa synthesis at 10 to 12 h, coinciding with the time of maximal synthesis of the 72,000-dalton DNA binding protein and viral DNA. To estimate the relative abundances of the two major E2 RNA species at various times during infection, total E2 cytoplasmic and polysomal 20S RNAs were isolated by hybridization-selection with specific DNA probes. The leader sequences in the selected RNAs were then quantitated by further RNA-DNA hybridization. We found that the elevated accumulation rate for E2 cytoplasmic RNA at late times reflected an increase in formation of both major species. Moreover, for all time points examined 66% of the mRNA species had a 5' end from map position 75, and 33% had a 5' terminus from position 72. Continuous labeling experiments provided evidence that both RNA forms have comparable half-lives. The results suggest that the two major species encoded by E2 are regulated in a coordinate fashion late in infection.

A comparative analysis of the effect of actinomycin- d, α-amanitin and cordycepin on new RNA synthesis and N-acetyltransferase induction by isoproterenol in cultured rat pineals

1. 1. Actinomycin- d (0.05 μg/ml) inhibited nuclear (n) and cytoplasmic (c) RNA synthesis by 70–80%. There was no effect, however, on isoproterenol mediated induction of N-acetyltransferase (NAT). 2. 2. Cordycepin inhibited (n) and (c) RNA synthesis by 1 and 63%, respectively, while NAT induction was inhibited by 23%. 3. 3. α-Amanitin inhibited (n) and (c) RNA synthesis by 47 and 45%, respectively, while NAT induction was inhibited by only 34%. 4. 4. These same three inhibitors were tested for their comparative effects on the synthesis of poly (A) rich messenger RNA and 3H-leucine incorporation into TCA-precipitable protein. Results showed that inhibition of NAT induction correlated only with inhibition of total protein synthesis.

Inhibition of protein synthesis by vaccinia virus. II. Studies on the role of virus-induced RNA synthesis.

Cytoplasmic RNA synthesis can be detected in vaccinia virus-infected HeLa cells in the presence of 2 micrograms/ml but not 20 micrograms/ml of actinomycin D. When RNA synthesis is observed protein synthesis is inhibited in infected, treated cells. We had previously noted that such a correlation may also be observed in infected, cycloheximide-treated cells. If actinomycin D (20 micrograms/ml) is added to these cells at various times after infection and treatment, the inhibition of protein synthesis seen upon removal of cycloheximide does not continue beyond the point to which it had developed before the actinomycin D was added. These results indicate that the inhibition of protein synthesis can be correlated with the amount of cytoplasmic RNA synthesized in infected cells and that this RNA synthesis and the subsequent inhibition of protein synthesis can be prevented by sufficiently high concentrations of actinomycin D. The cytoplasmic RNA which is synthesized does not appear to consist of double-stranded RNA nor of extensive self complementary regions. The cytoplasmic RNA synthesized in infected, cycloheximide treated cells appears to consist of early virus mRNA which can function as mRNA in vitro in a cell-free system derived from normal cells. An examination of the phosphorylation of ribosomal proteins shows six additional phosphoproteins in infected cells, two of which may be observed in infected cycloheximide-treated cells, suggesting that phosphorylation of ribosomal proteins cannot be directly correlated with the inhibition of overall protein synthesis seen in infected cycloheximide-treated cells.

RNA synthesis in avian erythroid cells

In nucleated erythroid cells, as in other vertebrate cells, less than 2% of the newly made RNA enters the cytoplasm. Posttranscriptional selection is therefore likely to play an important role in differential gene expression. The work reported here defines the relative roles of transcription, nuclear-cytoplasmic flow, and mRNA turnover in determining the distance of polysomal mRNAs in avian erythroid cells. Cells were preincubated with glucosamine to reduce endogenous pools of UTP and were then labeled with [ 3H]uridine. The specific activity of the UTP pools during the incubation was measured after high-pressure liquid chromatography, and the absolute rates of synthesis of nuclear and cytoplasmic RNA were determined. Under the labeling conditions described, the UTP pool reached a specific activity of 600 dpm/pmole within 5 min and remained between 600 and 800 dpm/pmole for 60 min. The total rate RNA synthesis was 0.9 fg/min/cell. The rate of synthesis of cytoplasmic RNA was 1.7% of the total. Comparison of these figures with published data for L cells indicates that these terminally differentiated, postmitotic cells are much more restricted in transcription than L cells, but similar in the efficiency of converting nuclear transcripts to cytoplasmic RNA. The rate of appearance and size distribution of polysomal RNAs were also determined. About 45% of the newly synthesized poly(A) + polysomal RNA has a sedimentation coefficient of approximately 9 S and was identified as globin mRNA by hybridization to recombinant plasmid DNA containing chicken β-globin mRNA sequence. Each species of globin mRNA is calculated to arrive on polysomes at about 1 molecule per minute per cell and each species of nonglobulin mRNA at about 0.02 molecules per minute per cell. These differences in the rates of appearance do not suffice to explain the 200-fold difference in the concentrations of individual globin and nonglobin poly(A) + mRNA species determined in hybridization experiments. The nonglobin poly(A) + mRNAs in these cells must therefore either be less stable than the globin mRNA or have accumulated for a shorter time than the globin mRNA.