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1.
RNA metabolism in nuclei: selective transport of kappa exons from myeloma nuclei and adenoviral transcripts from infected HeLa nuclei. 总被引:1,自引:1,他引:0
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Two independent systems and several analytical procedures have been used to establish that isolated mammalian nuclei selectively transport mature RNA polymerase I and II products. Murine myeloma nuclei retain physiologic restriction in our transport assay as assessed by the transport of the immunoglobulin kappa light chain mRNA and 18S and 28S rRNAs. Nearly 50% of the total kappa exons are transported as structurally intact mature mRNA molecules while less than 8% of either pulse-labeled or steady state kappa intron sequences are detected in the transported fraction. Ribosomal external transcribed spacer sequences also are absent in transported RNA. Release of cytoplasmic RNA from the outer nuclear membrane during the transport assay accounts for less than 10% of transported RNA. Nuclei isolated from adenovirus-infected HeLa cells at 20 hours post infection retain cellular actin mRNA and transport viral poly A+RNA. Ribosomal RNA is transported from infected nuclei although at a reduced rate compared to transport from mock-infected nuclei. Inhibition of transport of host mRNA is paralleled by the absence of pulse-labeled actin mRNA in the cytoplasm of infected cells. The implications of our transport data in relationship to intranuclear RNA trafficking are discussed. 相似文献
2.
Human myeloma cell line RPMI 8226 synthesizes and secretes a lambda immunoglobulin light chain. Total cellular RNA, obtained from cells grown in culture, was used for the isolation of poly(A)-containing mRNA by oligo(dT)-cellulose chromatography. The poly(A)-containing mRNA was translated in an Ehrlich ascites extract. Immunoprecipitation of the cell-free products showed that a polypeptide chain antigenically related to human lambda chain was synthesized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the cell-free product was larger than the secreted light chain. On tryptic digestion the cell-free product and the secreted light chain exhibited essentially identical peptide patterns except for an additional peptide derived from the cell-free product. We conclude that the lambda mRNA from this human myeloma cell line codes for a precursor to the secreted lambda chain as described for immunoglobulin mRNAs from murine plasmacytomas. 相似文献
3.
This paper describes experiments in which the half-lives of a number of cytoplasmic RNA species have been estimated in a mouse myeloma (MOPC 21) without resort to metabolic inhibitors. Partial purification of the messenger RNA coding for immunoglobulin light chains enabled an estimate of the stability of this species to be made. The procedure chosen was that of a conventional pulse-chase following uniform labelling of cells with [3H]uridine. Centrifugation of the uniformly labelled cells and resuspension in 0·1 mm-uridine resulted in a 75% drop in the specific activity of the UTP pool within 2 hours, followed by a logarithmic decay with a half-life of about 3·5 hours. Exposure of P3K cells to uridine causes them to swell appreciably and centrifugation at the end of the pulse period is followed by a lag phase of 3 hours before the cells re-enter logarithmic growth. Since all chase conditions had certain disadvantages, a comparison of experiments using different chase conditions was undertaken. The stability of the various RNA species did not vary greatly under the different chase conditions. The half-life of the light-chain mRNA is estimated to be 12 to 14 hours, although a value in the range of 5 to 20 hours cannot be excluded. An RNA fraction including the heavy-chain mRNA behaves similarly. Half-lives determined for other RNA species were: 18 S ribosomal RNA (40 to 60 h); 12 S mitochondrial ribosomal RNA (28 to 32 h). Poly(A)-containing RNA from free polyribosomes decays rapidly in the first 5 hours with a half-life of 20 to 30 hours, subsequently. 相似文献
4.
Isolation of messenger RNA for an immunoglobulin kappa chain and enumeration of the genes for the constatn region of kappa chain in the mouse 总被引:10,自引:0,他引:10
The messenger RNA for an immunoglobulin light (kappa) chain was isolated from the mouse myeloma MOPC41 and shown to be almost twofold longer than necessary to code for its protein product. DNA complementary to the mRNA was synthesized with the RNA-directed DNA polymerase of Rous sarcoma virus. Although the individual chains of the cDNA2 contained an average of only 270 nucleotides, the cDNA was heterogeneous in molecular weight, allowing the isolation of a fraction of the cDNA 620 nucleotides long. This large cDNA would be long enough to code for nearly all (95%) of the constant region if all the untranslated region of the mRNA were between the 3′ terminal poly(A) and the constant region. On the other hand, if all the untranslated region of the mRNA were at the 5′ terminus, this cDNA would code for 93% of the entire kappa chain.The specificity of nucleotide sequences in the cDNA was documented by molecular hybridization with both template RNA and RNA from various myelomas. The amount of hybridization obtained with myeloma RNA was approximately proportional to the amount of kappa chain protein produced by the various myeloma cells. In addition, there was no hybridization with RNA isolated from either BALB/c mouse liver or Escherichia coli.The genes for the constant region of the kappa chain were enumerated in the mouse genome by annealing cDNA to DNA from mouse liver and MOPC41 myeloma. The haploid genome of both tissues contained three to four genes for the constant region of kappa chain even when tested under conditions that would detect distantly related nucleotide sequences. The fact that there are only a few genes coding for the constant region of kappa chains implies that specialized genetic mechanisms are required for the generation of antibody diversity. 相似文献
5.
Poly(A)+RNA is synthesized during the first hours of pollen germination and is rapidly incorporated into polysomal structures. After
a 2-h pulse with uracil-14C, 42% of the transcribed fraction of polysomal RNA is polyadenylated. Following 4 h of germination the amount of the newly-made
poly(A)+RNA decreases steadily at the rate of about 14% per h, whereas that of rapidly-labelled poly(A)−RNA continues to grow. Beginning 1 h of cultivation the ratio of poly(A)−/poly(A)+RNA increases exponentially. Similarly as in non-polyadenylated mRNA the main portion of the synthesized polysomal poly(A)+RNA sediments at a rate of 4 to 14 S and its mean size decreases slightly with the time of labelling. RNA isolated from nuclei
and cell wall containing pollen tube fraction differed from the polysomal one in higher apeoific radioactivity and the polyadenylated
RNA exhibited higher size distribution. The comparison of the results with earlier observations suggests the involvement of
poly(A)in mRNA translation in pollen tubes. 相似文献
6.
L Miller 《Developmental biology》1978,64(1):118-129
The relative amounts of newly synthesized poly(A)+ and poly(A)? mRNA have been determined in developing embryos of the frog Xenopus laevis. Polysomal RNA was isolated and fractionated into poly(A)+ and poly(A)? RNA fractions with oligo(dT)-cellulose. In normal embryos the newly synthesized polysomal poly(A)+ RNA has a heterodisperse size distribution as expected of mRNA. The labeled poly(A)? RNA of polysomes is composed mainly of rRNA and 4S RNA. The amount of poly(A)? mRNA in this fraction cannot be quantitated because it represents a very small proportion of the labeled poly(A)? RNA. By using the anucleolate mutants of Xenopus which do not synthesize rRNA, it is possible to estimate the percentage of mRNA which contains poly(A) and lacks poly(A). All labeled polysomal RNA larger than 4S RNA which does not bind to oligo(dT)-cellulose in the anucleolate mutants is considered presumptive poly(A)? mRNA. The results indicate that about 80% of the mRNA lacks a poly(A) segment long enough to bind to oligo(dT). The poly(A)+ and poly(A)? mRNA populations have a similar size distribution with a modal molecular weight of about 7 × 105. The poly(A) segment of poly(A)+ mRNA is about 125 nucleotides long. Analysis of the poly(A)? mRNA fraction has shown that it lacks poly(A)125. 相似文献
7.
Interaction between polyamines and nucleic acids or phospholipids 总被引:10,自引:0,他引:10
K Igarashi I Sakamoto N Goto K Kashiwagi R Honma S Hirose 《Archives of biochemistry and biophysics》1982,219(2):438-443
The binding of polyamines to DNA, RNA, and phospholipids has been studied by gel filtration and sucrose density gradient centrifugation. Spermine was found to bind more to a GC-rich DNA. Among RNAs containing double-stranded region [poly(AU), poly(GC), and ribosomal RNA], the binding of spermine was nearly equal. Among the single-stranded RNAs, the binding of spermine was in the order poly(U) > poly(C) > poly(A). An increase in K+ or Mg2+ concentration resulted in a great decrease in spermine binding to DNA and in a slight decrease in spermine binding to RNA. Therefore, in the presence of more than 2 mm Mg2+ and 100 mm K+, the binding of spermine to RNA was greater than that to DNA. No significant difference in spermine binding was observed between 16 S ribosomal RNA and 30 S ribosomal subunits, suggesting that ribosomal proteins did not affect significantly the binding of spermine to ribosomal RNA. The binding of spermine to microsomes was dependent on phospholipids. The binding strength was in the order phosphatidylinositol > phosphatidylethanolamine > phosphatidylcholine. 相似文献
8.
The metabolism of a poly(A) minus mRNA fraction in HeLa cells 总被引:40,自引:0,他引:40
About 30% of HeLa cell mRNA lacks poly(A) when labeled in the presence of different rRNA inhibitors. Our method of RNA fractionation precludes contamination of the poly(A)? mRNA with large amounts of poly(A)+ sequences. The poly(A)? species is associated with polyribosomes, has an average sedimentation value equal to or greater than poly(A)+ mRNA, and behaves like the poly(A)+ mRNA in its sensitivity to EDTA and puromycin release from polyribosomes. There is very little, if any, hybridization at Rot values characteristic of abundant RNA sequences between the poly(A)? RNA fractions from total cytoplasm or from polyribosomes and 3H-cDNA made to poly(A)+ RNA. This indicates that poly(A)? mRNA does not arise from poly(A)+ mRNA by nonadenylation, deadenylation, or degradation of random abundant mRNA sequences. The rate of accumulation of poly(A)? mRNA larger than 9S in the cytoplasm parallels the accumulation of poly(A)? mRNA. The poly(A)? mRNA is maintained as approximately 30% of the total labeled mRNA in a short (90 min) and in a long (20 hr) time period. These data indicate that poly(A)? mRNA is not short-lived nuclear or cytoplasmic heterogeneous RNA contamination, and that the half-life of the poly(A)? mRNA may parallel that of the poly(A)+ mRNA. Cordycepin appears to almost completely (95%) inhibit poly(A)+ mRNA while only partially (60%) inhibiting the poly(A)? mRNA. The origin of the cordycepin-insensitive mRNA has not been ascertained. 相似文献
9.
The pattern of poly(A)-associated [poly(A)+] RNA synthesis was studied in rabbit cerebral cortex in the period following a single electroconvulsive shock (ECS). Labeled uridine was injected into the brain 2 and 4 hr after ECS and the animals sacrificed 1 hr later. Total and poly(A)+ RNA were then prepared from cortical nuclei and microsomes and analyzed. The amounts of newly synthesized total and poly(A)+ RNA in nuclei and microsomes appeared to be close to the control. However, the pattern of newly synthesized poly(A)+ nuclear RNA appeared to be still displaced toward the high molecular weights as it was in the early post-ECS period. The result indicates a long-lasting disturbance of brain poly(A)+-RNA metabolism by ECS. 相似文献
10.
Polyadenylic acid [poly (A)] is detected, characterized and quantitated in dry radish embryo axis RNA using a 3H poly (U) probe. The amount of poly (A) gradually decreases after the onset of soaking, and, after a few hours, recovers to the initial level. This variation is shown to result from the addition of two opposed phenomena: the decay of stored poly (A) and the accumulation of newly synthesized poly (A). Stored poly (A), as well as the in vivo protein synthesis coded for by preformed mRNA, decreases during early germination with a half-life of two hours. As a whole, these results demonstrate that at least a fraction of the stored mRNA is translated as soon as the seed is soaked and that its role is rapidly taken over by newly-made mRNA.Abbreviations Poly (A) (+) RNA
polyadenylated RNA
- Poly (A)
polyadenylic acid
- Poly (U)
polyuridylic acid 相似文献
11.
M C Lechner M T Freire B Groner 《Biochemical and biophysical research communications》1979,90(2):531-536
The biosynthesis of a cytochrome P450 peptide sub-unit by the in vitro translation of total hepatic poly (A)+ mRNA in an heterologous cell-free-system is described. The ability of the liver poly (A)+ RNA preparations from normal and phenobarbital induced rats to promote protein synthesis and the identification of in vitro synthesized proteins revealed the presence of a cytochrome P450 peptide sub-unit presenting the same apparent molecular weight of the native peptide. This fact demonstrates that rat liver poly (A)+ mRNA fraction contains an important amount of cytochrome P450 peptide messages. Total poly (A)+ RNA from rats in an early phenobarbital induction stage exhibits a higher cytochrome P450 template activity in good agreement with the enhancement of this hemeprotein concomitantly observed in vivo, in the liver microsomes, it is also concluded that cytochrome P450, peptide sub-unit, induced in rat liver by phenobarbital, is translated in its mature form. 相似文献
12.
M Green T Zehavi-Willner P N Graves J McInnes S Pestka 《Archives of biochemistry and biophysics》1976,172(1):74-89
The mRNA's for both the heavy chain (H315) and the light chain (L315) of the mineral oil-induced plasmacytoma-315 myeloma protein have been isolated and partially purified from both total cellular RNA and RNA derived from membrane-bound polysomes. The yields of both L315 mRNA and, in particular, of H315 mRNA were increased when total cellular RNA was used as starting material. Total poly(A)-containing mRNA and partially purified mRNA obtained by preparative sucrose gradient sedimentation stimulated protein synthesis in cell-free extracts derived from Ehrlich ascites tumor cells or wheat germ. Cell-free products antigenically and structurally related to both the authentic L315 and H315 secreted by intact cells were synthesized in the Ehrlich ascites cell-free system in response to the appropriate mRNA's. Only the L315 mRNA was efficiently translated in the cell-free system derived from wheat germ. 相似文献
13.
The complexity of nuclear RNA, poly(A)hnRNA, poly(A)mRNA, and total poly(A)RNA from mouse brain has been measured by saturation hybridization with nonrepeated DNA. These DNA populations were complementary, respectively, to 21, 13.5, 3.8, and 13.3% of the DNA. From the RNA Cot required to achieve half-sturation, it was estimated that about 2.5–3% of the mass of total nuclear RNA constituted most of the complexity. Similarly, complexity driver molecules constituted 6–7% of the mass of the poly(A)hnRNA. 75–80% of the poly(A)mRNA diversity is contained in an estimated 4–5% of the mass of this mRNA. Poly(A)hnRNA constituted about 20% of the mass of nuclear RNA and was comprised of molecules which sedimented in DMSO-sucrose gradients largely between 16S and 60S. The number average size of poly(A)hnRNA determined by sedimentation, electron microscopy, or poly(A) content was 4200–4800 nucleotides. Poly(A)mRNA constituted about 2% of the total polysomal RNA, and the number average size was 1100–1400 nucleotides. The complexity of whole cell poly(A)RNA, which contains both poly(A)hnRNA and poly(A)mRNA populations, was the same as poly(A)hnRNA. This implies that cytoplasmic polyadenylation does not occur to any apparent qualitative extent and that poly(A)mRNA is a subset of the poly(A)hnRNA population. The complexity of poly(A)hnRNA and poly(A)mRNA in kilobases was 5 × 105 and 1.4 × 105, respectively. DNA which hybridized with poly(A)mRNA renatures in the presence of excess total DNA at the same rate as nonrepetitive tracer DNA. Hence saturation values are due to hybridization with nonrepeated DNA and are therefore a direct measure of the sequence complexity of poly(A)mRNA. These results indicate that the nonrepeated sequence complexity of the poly(A)mRNA population is equal to about one fourth that observed for poly(A)hnRNA. 相似文献
14.
Messenger RNA turnover in mouse L cells 总被引:36,自引:0,他引:36
The turnover of polyadenylic acid-containing messenger RNA and histone messenger RNA, which lacks poly(A), was studied in exponentially growing mouse L cells by measuring the kinetics of approach to steady-state uridine labeling. Constant specific activity of precursor pools was verified by showing that the data for stable RNA components, like ribosomal RNA and transfer RNA, follow theoretically predictable curves. In agreement with a previous report by Greenberg (1972), the data for poly(A)-containing mRNA (poly(A)(+)mRNA) follow theoretical curves for a class of molecules turning over with first-order (stochastic) kinetics. Cells growing with doubling times of 13·5 hours at 37 °C and 41 hours at 30 °C exhibited mean lifetimes for their poly(A)(+)mRNA of 15 hours and 42 hours, respectively, suggesting a parallelism between growth and turnover rates. The kinetic data for histone mRNA are not indicative of a stochastic process. Rather, they suggest an age-dependent decay or a zero-order (ordered) turnover with a mean lifetime of about six hours. One model, which gave a good fit to the data, considers that the histone messages persist for a fixed duration of the cell cycle, e.g. the DNA synthetic phase, and are then destroyed in a “sensitive period” after this phase. These results are discussed with regard to the possible implications of the poly(A) sequences in messenger RNA aging. 相似文献
15.
A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(A)+ mRNA, poly(A)? histone mRNA, and poly(A)? nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)+ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)? nonhistone mRNAS than do embryos. Some differences in the proteins encoded by poly(A)+ and poly(A)? RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)+ and poly(A)? RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous comigration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)? mRNA, encoding a 70,000–80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)+ nonhistone mRNA, poly(A)? histone mRNA, and poly(A)+ histone mRNA. Amphibian ovary RNA appearently lacks an abundant poly(A)? nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding histone-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay. 相似文献
16.
The stored poly(A) + RNA from zoospores of the aquatic fungus Blastocladiella emersonii represents 2.5% of the total RNA and has a model MW of 425,000 daltons and an average poly(A) isostich of 32 bases. The poly(A) + RNA also represents 2.5% of the total RNA from early growth phase cells and has a modal MW of 360,000 daltons and an average poly(A) isostich of 38 bases. The poly(A) + RNA from spores and 2-hr plants contains a structure resistant to RNases T1, T2, and A, which can be labeled with 32PO4 and which will bind to DBAE-cellulose. These characteristics strongly suggest that both the zoospore poly(A) + RNA and the 2-hr cell poly(A) + RNA are capped at the 5′ end; and, hence, it is unlikely that capping is involved in the control of protein synthesis during germination.Approximately 80% of the poly(A) + RNA of the spore is located in the membrane-enclosed ribosomal nuclear cap, and more than 90% of the poly(A) + RNA within the cap is found in the 80S monoribosome and heavier fractions.Synthesis of new poly(A) + RNA occurs very early during zoospore germination, and the labeled poly(A) + RNA rapidly enters the newly organized polysomes. The labeling data for early germination also suggest that cytoplasmic polyadenylation occurs. 相似文献
17.
Kinetic study of protein unfolding and refolding using urea gradient electrophoresis 总被引:8,自引:0,他引:8
T E Creighton 《Journal of molecular biology》1980,137(1):61-80
When total cytoplasmic RNA from mouse Friend cells is fractionated using oligo(dT)-cellulose or poly(U)-Sepharose chromatography, approximately 20% of the messenger RNA activity (as measured in the reticulocyte lysate cell-free system) remains in the unbound fraction, even though this contains < 0.5% of the poly(A) (as measured by titration with poly(U)). This RNA, operationally defined as poly(A)?, is found almost entirely in polysome structures in vivo. Its major translation products, as shown by one-dimensional sodium dodecyl sulphate-containing gels, are the histones and actin. Two-dimensional gels (isoelectric focusing: sodium dodecyl sulphate/gel electrophoresis) show that, with the exception of the mRNAs coding for histones, poly(A)? mRNA encodes similar proteins to poly(A)+ mRNA, though in very different abundances. This is directly confirmed by the arrest of the translation of the abundant poly(A)? mRNAs after hybridization with a complementary DNA transcribed from poly(A)+ RNA.RNA sequences which are rare in the poly(A)+ RNA are also found in poly(A)? RNA, as shown by hybridizing a cDNA transcribed from poly(A)+ RNA to total and poly(A)? polysomal RNA. That this does not simply represent a flow-through of poly(A)+ RNA is indicated by (i) the lack of poly(A) by hybridizing to poly(U) in this fraction, (ii) the fact that further passage through poly(U)-Sepharose does not remove the hybridizing sequences, (iii) the very different quantitative distribution of proteins encoded by poly(A)+ and poly(A)? RNAs. We also think that it does not result from removal of poly(A) from polyadenylated RNAs during extraction because RNAs prepared using the minimum of manipulations give similar results. The distribution of both total mRNA and α and β globin mRNAs between poly(A)+ and poly(A)? RNA does not change significantly during the dimethyl sulphoxide-induced differentiation of Friend cells. 相似文献
18.
Summary Previous studies have shown that aldosterone increases transepithelial active Na+ transport after a latent period of about 60 min and incorporation of3H-uridine into polyadenylated RNA (poly(A)(+)RNA) (putatively poly(A)(+)mRNA) as early as 30 min after aldosterone addition. To assess the physiological importance of this pathway, the effects of 3deoxyadenosine and actinomycin D were compared in studies on the urinary bladder of the toadBufo marinus. 3deoxyadenosine (30 g/ml) only partially, though significantly, inhibited the aldosterone-dependent increase in Na+ transport measured as short-circuit current (scc). The incorporation of3H-uridine into poly(A) (+)RNA was inhibited by 70 to 80%. In contrast, Actinomycin D (2 g/ml) totally inhibited the aldosterone-dependent increase in scc, and the incorporation of3H-uridine into poly(A)(+)RNA by 68 to 75%. 3deoxyadenosine or actinomycin D alone had no significant effects on baseline scc, while inhibiting poly(A)(+)RNA to the same extent. The differential effects of deoxyadenosine and actinomycin on aldosterone-dependent Na+ transport may be related to their different sites of action on RNA synthesis: both drugs inhibited, to a similar extent, cytoplasmic poly(A)(+)mRNA; however, 3deoxyadenosine, in contrast to Actinomycin D, failed to inhibit poly(A)(-)RNA, sedimenting between 4S and 18S (putatively poly(A)(-)mRNA). We conclude that the mineralocorticoid action of aldosterone during the first three hours depends on the synthesis of both poly(A)(+)mRNA and poly(A)(-)mRNA. 相似文献
19.
20.
《The International journal of biochemistry》1981,13(1):83-90
- 1.1. A fraction of heavy polysomes has been isolated from rapidly dividing osmotic dependent yeast cells. Poly A + RNA purified from this fraction represent 30% of the total polysomal poly A + RNA and consist of high molecular weight molecules.
- 2.2. Both centrifugation analysis in denaturing conditions and electron microscopic studies showed the existence of mRNA with molecular weight up to 5 × 106 dallons in polysomal fractions.
- 3.3. Competition hybridization experiments suggested a considerable sequence homology between high and low molecular weight poly A + RNA purified from heavy and light polysomal fractions, respectively.
- 4.4. These results suggest that in rapid growing yeast cells some of the abundant mRNA might be synthesized by a mode different from the monocistronic mRNA synthesis.