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1.
Alterations in lysine transfer RNA during erythroid differentiation of the Friend cell. 总被引:2,自引:2,他引:2
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The proportion of lysine tRNA represented by the isoacceptor species lysine tRNA4 has previously been shown to be largest in cells with the greatest ability to proliferate. Using reverse phase chromatography (RPC-5), we have analyzed the changes in the relative quantities of lysine tRNA species which occur in different cellular states of the Friend cell, a transformed murine cell infected with Friend erythroleukemia virus complex. This cell undergoes erythroid differentiation when exposed to various chemicals. Lysine tRNA4 comprises 32% of the total lysine tRNA in rapidly dividing, uninduced Friend cells, but only 16% of the total lysine tRNA in uninducase. Friend cells undergoing erythroid differentiation divide more slowly than uninduced cells, and finally cease proliferation, but lysine tRNA4 becomes the major lysine tRNA species (greater than 50%). This does not appear to reflect erythroid properties of the cell, since the lysine tRNA of the mouse reticulocyte contains very little lysine tRNA4. The non-dividing erythroid Friend cell, therefore, represents an exception to the finding that non-dividing cells usually have little or no lysine tRNA4 present. 相似文献
2.
V L Pulito D L Miller S Sassa T Yamane 《The Journal of biological chemistry》1983,258(24):14756-14758
A variety of chemical agents that are known to induce erythrodifferentiation in the Friend virus-induced murine erythroleukemia (MEL) cell have been suggested to mediate DNA cleavage in cultured cells prior to differentiation. The activation of the nuclear enzyme, ADP-ribosyltransferase, depends upon the presence of single strand breaks in DNA. If dimethyl sulfoxide (Me2SO) causes DNA breakage, it would be expected that the activity of ADP-ribosyltransferase would increase. A study of ADP-ribosyltransferase activity during cell growth indicates that both Me2SO-treated and untreated MEL cells exhibit a similar increase in the enzyme activity but the increase in Me2SO-treated cells is delayed by a few hours. When examined at comparable stages of growth, both treated and untreated cells show almost identical levels of enzyme activity. The present data thus do not support the contention that Me2SO induces DNA breakage in the MEL cells. 相似文献
3.
The changes in rate of protein synthesis and cell division and the distribution of polyribosomes and globin mRNA on the polyribosomes of Friend erythroleukemia (FL) cells exposed to 2% DMSO and maintained at low cell density, were examined at different times after exposure to DMSO. The rate of protein synthesis and the capacity of cells to divide declined in concert to 50% of the level found in untreated cell cultures at 24 hours after exposure. Thereafter these rates recovered to 70% of the rate found in untreated control cultures until 96 hours post-exposure and then irreversibly declined as the cells lost the capacity to divide. The proportion of ribosomes present as polyribosomes in cells exposed to DMSO paralleled the capacity of these cells to synthesize protein. The distribution of polyribosomes analyzed by sedimentation in sucrose gradients demonstrated that a discrete, abundant class of polyribosomes composed of pentamers to heptamers appeared as early as 48 hours after exposure to DMSO. The appearance of an abundant class of polyribosomes was correlated with globin synthesis by demonstrating that a discrete class of polyribosomes arises in cells treated with the inducers hexamethylene bisacetamide and hemin. 相似文献
4.
A method for the clonal analysis of murine erythroleukemia cells has been developed which allows the precise characterization of the number of progeny produced by each cell and the degree of differentiation of each progeny cell. The potential of almost every cell in the culture can be monitored because a plating efficiency close to 100% has been achieved. The effects of treatment with an inducer of differentiation (DMSO) on the proliferative capacity of the treated cells have been studied with this technique. Cells from a mass culture treated with inducer give rise to colonies of differentiated progeny when subsequently cloned in the absence of inducer. Colonies exhibiting this phenotype represent the progeny of cells committed to the differentiation pathway by treatment with inducer. We observe that the commitment decision limits the subsequent proliferative capacity of the cell to four additional cell divisions. A quantitative analysis suggests that the commitment decision for each cell is made in a stochastic manner. Irreversible commitment to the expression of differentiated functions occurs with discrete probability per cell generation for many cell generations. The value for this probability is a function of the concentration of inducer (DMSO). A correlative biochemical study suggests that an irreversible commitment decision by a significant proportion of the population precedes or accompanies increases in cytoplasmic globin mRNA levels, one of the earliest detectable biochemical markers for erythroid differentiation in this system.A specific kinetic model based on these considerations has been developed to predict clonal phenotypes as a function of time and probability of commitment. Quantitative predictions based on this model are in excellent agreement with experimental observations. The effectiveness of a stochastic model in predicting the behavior of this system is discussed in relation to the stochastic behavior of normal hematopoiesis and the biochemical mechanisms which control these differentiation programs. 相似文献
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6.
Specific changes in Q-ribonucleoside containing transfer RNA species during Friend leukemia cell erythroid differentiation. 总被引:1,自引:0,他引:1
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Changes in specific tRNA isoacceptors during Friend leukemia cell (F.L.C.) erythroid differentiation have been found to be concomitant with differences in the extent of the Q-base modification in certain species of tRNA. Transfer RNA was isolated from F.L.C. cultures after 0, 36, 48, 72, and 96 hr of DMSO induced differentiation. Changes in 17 isoacceptors of tRNAasn, tRNAasp, tRNAhis and tRNAtyr were compared by RPC-5 chromatography. Isoacceptors of these tRNA changed in relative amounts, following consistent trends throughout cell differentiation. The amount and distribution of Q-base containing tRNA isoacceptors was assayed by measuring the quanine-tRNA transferase catalyzed incorporation of [3H]-labeled guanine into tRNA species undermodified in Q-base followed by RPC-5 chormatography of the tRNA. The amount of Q-base containing tRNA species decreased in the first 48 hr after the induction, then increased again, indicating the level of Q-modification is correlated to the process of differentiation. Isoacceptors that lacked the Q-base were eluted late from RPC-5. 相似文献
7.
Incubation of bone marrow cells from anaemic rabbits in the presence of actinomycin D led to a decrease in total protein synthesis and an increase in the relative synthesis of globin. This increase in the proportion of globin was observed with in vivo labelling of cellular proteins and in vitro translation of isolated RNA, which indicates that the messenger RNA for globin is much more stable than the other bone marrow cell messages. This was further shown by pulse-labelling the RNA and characterization of the different species by separation on a cDNA-oligo(dT)-cellulose column. Within 12 h after pulse-labelling the relative levels of globin mRNA had risen 10-fold, while a rapid decrease in the level of the poly(A)-rich RNA fraction was observed. Investigations into the mechanisms of this differential stability indicate that the more metabolically active cells from the early stages of erythropoietic development are more susceptible to inhibitors of RNA synthesis such as actinomycin D and alpha-amanitin. A preliminary study using a lysosomal inhibitor, chloroquine, indicates that there appear to be at least two degradative mechanisms, involving a lysosomal and a non-lysosomal pathway, with selective specificity for different messages. 相似文献
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9.
Alterations in tRNA isoaccepting species during erythroid differentiation of the Friend leukemia cell. 总被引:2,自引:1,他引:2
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The chromatographic profiles of isoaccepting tRNAs were analyzed at five time points during the 96 hr, dimethylsulfoxide induced, erythroid-like differentiation of Friend leukemia cells. Sixty-four isoaccepting species of tRNA for 16 amino acids were resolved by RPC-5 chromatography. The relative amounts of tRNAphe, tRNAile, and tRNAval species were maintained by the cells during differentiation; whereas the relative amounts of some of the isoacceptor tRNAs for the other 13 amino acids changed significantly. Fluctuations in amounts of isoacceptors occurred between 36 and 72 hr after addition of dimethysulfoxide, corresponding to globin mRNA appearance and hemoglobin synthesis, respectively. In most cases, thepredominant tRNA isoacceptors of uninduced cells were retained throughout differentiation. Notable exceptions were tRNA species for threonine, proline, and methionine. Some of the isoacceptors occurring in relatively smaller amounts were not expressed at all times. These changes possibly reflect the cell's functional adaptation of tRNA in differentiation for hemoglobin synthesis. 相似文献
10.
P. R. Harrison D. Conkie T. Rutherford N. Affara M. Allan P. Hissey J. Paul 《Experimental cell research》1978,115(2)
The genetic basis of control of erythroid differentiation in Friend cells has been investigated by studying the dominance relationships of non-inducible variants when fused to the inducible parent from which they are derived. Three non-inducible variants seem to be dominant in causing extinction of induction of hemoglobin in such hybrids, whereas two other non-inducible variants are cis-dominant or recessive. 相似文献
11.
F Ramirez R Gambino G M Maniatis R A Rifkind P A Marks A Bank 《The Journal of biological chemistry》1975,250(15):6054-6058
Previous studies have shown that mouse fetal erythroid precursor cells isolated by an immunological technique synthesize little or no globin and contain little, if any, globin mRNA, as assayed in a cell-free system (translatable mRNA). After culture for 10 hours in the presence of erythropoietin, there is a marked increase in globin synthesis and in translatable globin mRNA. The present studies were designed to measure directly the content of globin mRNA sequences during erythroid cell differentiation, by molecular hybridization with 3H-labeled DNA complementary to globin mRNA. The results indicate that few, if any, globin mRNA sequences are present in the total RNA of erythroid precursor cells. There is little or no pool of untranslated globin mRNA in these cells. After 10 hours of culture with erythropoietin, there is an increase in globin mRNA content, as ;easured by a change in the Cot1/2 values obtained by cDNA: mRNA hybridization with (Co) representing the concentration of RNA. Between 0 and 22 hours of culture, there is a 250-fold rise, and between 22 and 44 hours, a further 2-fold increase in globin mRNA content. During the 44 hours in culture, the number of cells in culture increases 2- to 3-fold. The number of globin mRNA molecules rises in erythroid precursor cells to an average value of 1800 molecules/cell during 22 hours of culture. In cultures without added erythropoietin, the absolute number of cells decreases, however, cells presumably induced to differentiate by exposure to erythropoietin in vivo continue to differentiate in vitro, accumulating globin mRNA and initiating globin synthesis. 相似文献
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13.
Alterations in polyamine metabolism during embryonal carcinoma cell differentiation in vitro 总被引:2,自引:0,他引:2
Murine embryonal carcinoma (EC) cells can be stimulated to differentiate by several chemical inducers. Since the response of EC cells to induction is likely to occur shortly after exposure to the inducer, we report here the changes that occur in polyamine levels in a number of EC cell lines shortly after exposure to two chemical stimuli, alpha-difluoromethylornithine (DFMO) and retinoic acid (RA). Our results suggest that polyamine levels are important in determining the state of EC cell differentiation, but that reduction in these levels alone is not sufficient to induce differentiation in all the EC cell lines tested. Also, it is apparent that RA does influence levels of polyamines. However, this influence does not seem to be mediated through direct interaction with ODCase. 相似文献
14.
Friend erythroleukemia cells display transient and permanent changes in the composition of their plasma membrane-bound glycoproteins during dimethyl sulfoxide-induced differentiation. The transient changes, as revealed by metabolic labeling with [14C]glucosamine, are most conspicuous around the time during which most cells become committed to terminal differentiation. Permanent changes are revealed by reductive tritiation after oxidation with NaIO4 or galactose oxidase. In differentiated cells one glycoprotein fraction (Mr 150 000) could not be labeled by any of these methods, although it does contain neuraminic acid. We found no evidence in support of the hypothesis that the anomalous behavior of this fraction is caused by an increased degree of O-acetylated neuraminic acid in the plasma membrane of differentiated cells. 相似文献
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16.
Z. Darżynkiewicz F. Traganos T. Sharpless C. Friend M.R. Melamed 《Experimental cell research》1976,99(2):301-309
Friend leukemia (FL) cells grown in the presence of dimethylsulfoxide (DMSO) undergo erythroid differentiation. Acridine orange (AO) binding to DNA and thermal denaturation of DNA in situ were studied in differentiated and non-differentiated FL cells using flow-through cytofluorometry. The differentiated cells bind less AO than do the non-differentiated ones. The difference in AO binding is higher in the spectrum of emission characteristic for AO intercalation (at 530 nm) than for AO stacking (>600 nm) and depends on AO concentration. The difference is abolished after extraction of acid-soluble macromolecules from cells. During stepwise extraction by lowering pH, there is a progressive increase of AO binding to DNA. Most of the increase in AO binding of the non-differentiated cells occurs at pH 2.5-2.0; of the differentiated cells at pH 1.75-1.50. There are differences in sensitivity of DNA in situ to heat-denaturation between differentiated and non-differentiated cells, as evidenced by the variation in height and in position of the melting bands on derivative melting profiles. The changes described suggest that a profound modulation of chromatin structure, perhaps involving altered DNA-histone interactions, occurs during the DMSO-induced erythroid differentiation of FL cells. 相似文献
17.
During differentiation of murine erythroleukemia cells, the levels of certain mRNA were observed to change. To characterize the various patterns of changes that occur during differentiation, cDNA libraries made from RNA isolated from uninduced and differentiating cells were screened with labeled cDNA or RNA labeled in vivo for different periods of time. cDNA clones that corresponded to individual mRNAs whose level remained constant, increased, or decreased during differentiation were identified. These clones were used to analyze Northern blots containing RNA from uninduced and differentiated cells. A number of characteristic changes in individual mRNAs in differentiating murine erythroleukemia cells could be identified, such as no change, increase in concentration, increase in concentration and slight change in size, decrease in concentration, decrease in concentration and change in size, appearance of new band(s) of entirely different size, and change in relative concentrations of two related mRNAs. Measurements of rates of mRNA synthesis and degradation suggest that both parameters change during differentiation and that these changes are instrumental in establishing cellular concentration of specific mRNAs. It seems that the changes in mRNA stability observed in differentiating murine erythroleukemia cells may be associated with changes in the primary structure of the transcribed portion of mRNA. The observation that specific mRNA synthesized before and after induction may have very different stabilities at the same point in differentiation supports this hypothesis. 相似文献
18.
The relationship between differentiation of murine erythroleukemia cells (MEL) induced by DMSO and the cell division cycle has been analyzed. We demonstrate that incubation in the presence of DMSO increases the length of the G1 phase of the cell cycle. A method of synchronization of MEL cells by unit gravity sedimentation has been developed and characterized. Using this method, a series of synchronized cell populations covering the entire cell division cycle can be generated simultaneously. Cells synchronized by this technique were challenged with DMSO and analyzed for kinetics of commitment to the differentiation program. Our results indicate that populations of cells in G1 or G2 at the time of addition of inducer give rise to a greater proportion of committed cells than an unfractionated population, while cells in S phase result in a lower percentage of committed cells than the unfractionated population when cultured in DMSO. 相似文献
19.
The base at the first anticodon ("wobble") position of certain eukaryotic tRNA species is either guanine or the hypermodified base queuine. These tRNA species are synthesized with guanine in the wobble position (tRNAG); this guanine can then be replaced with queuine by the action of the enzyme tRNA-guanine ribosyltransferase. In the present report, we show that tRNAG levels increased in response to the induction of erythroid differentiation of murine erythroleukemia (MEL) cells. We also found that tRNA-guanine ribosyltransferase was significantly inhibited by tetrahydrobiopterin. MEL cells showed a transient threefold increase in tetrahydrobiopterin levels 6 to 12 h after exposure of the cells to inducers such as DMSO or tetramethylurea. The increase in tetrahydrobiopterin preceded the increase in tRNAG which in turn preceded the appearance of phenotypic changes characteristic of differentiation. By contrast, a mutant MEL cell line unable to differentiate in response to inducers showed no change in the level of tetrahydrobiopterin or of tRNAG upon exposure to DMSO. N-acetylserotonin, a well-characterized inhibitor of tetrahydrobiopterin synthesis, prevented the DMSO-mediated increase in tetrahydrobiopterin in normal MEL cells. N-acetylserotonin also inhibited the increase in tRNAG levels and the appearance of phenotypic differentiation in these cells. 相似文献
20.
Murine erythroleukemia (MEL) cells are widely used to study erythroid differentiation thanks to their ability to terminally differentiate in vitro in response to chemical induction. At the molecular level, not much is known of their terminal differentiation apart from activation of adult-type globin gene expression. We examined changes in gene expression during the terminal differentiation of these cells using microarray-based technology. We identified 180 genes whose expression changed significantly during differentiation. The microarray data were analyzed by hierarchical and k-means clustering and confirmed by semi-quantitative RT-PCR. We identified several genes including H1f0, Bnip3, Mgl2, ST7L, and Cbll1 that could be useful markers for erythropoiesis. These genetic markers should be a valuable resource both as potential regulators in functional studies of erythroid differentiation, and as straightforward cell type markers. 相似文献