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1.
Cloacin DF12 cleavage of Escherichia coli f[3H]MettRNA-AUG-ribosome complexes affects this substrate for in vitro peptide chain termination. Codon-directed release factors' (RF) 1 and 2 release of f[3H]methionine is inhibited by cloacin. Since cloacin inhibits RF1 and -2 binding to ribosomes but not RF-directed f[3H]methionine release from f[3H]met-tRNA-AUG-ribosome complexes when reactions contain 20% ethanol, we conclude that cloacin DF 13 inhibits formation of the termination codon recognition complex. Thus, cleavage of the 3'-OH 49-nucleotide sequence of the 16 S rRNA perturbs the codon-directed binding of RF to ribosomes.  相似文献   

2.
The structures of [3H]pargyline-labeled, flavin-containing polypeptides of monoamine oxidase (MAO) from hybrid NCB20 cells, and their parental cells, A/J mouse brain cells and Chinese hamster brain cells, were analyzed and compared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and limited proteolysis and one-dimensional peptide mapping in SDS gels. After preincubation of mitochondrial preparations with deprenyl or clorgyline, the flavin-containing polypeptide of type A or type B MAO was selectively labeled with [3H]pargyline. SDS-PAGE of [3H]pargyline-labeled mitochondrial samples revealed that the polypeptide with apparent Mr of 62,000 was associated with type A activity in the three types of cells, and that the polypeptide with apparent Mr of 61,000 or 58,000 was associated with type B activity in Chinese hamster brain cells and NCB20 cells or A/J mouse brain cells, respectively. Chymotrypsin digestion of the [3H]pargyline-labeled polypeptides and the peptide mapping in SDS gels from A/J mouse and Chinese hamster brain cells produced identical map patterns between the two type A MAOs, almost the same map patterns (with the exception of one additional peptide fragment) between the two type B MAOs, and different map patterns between type A and type B MAOs. The results of identical treatments of the [3H]pargyline-labeled polypeptides of MAOs in NCB20 cells showed that type A and type B MAO in NCB20 cells were similar to type A MAO of A/J mouse and Chinese hamster brain cells and to type B MAO of Chinese hamster brain cells.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

3.
The degradation rates of kidney rRNA labeled before UNI or sham are unchanged 5 days after the operations (t one-and-a half, 88 h). Therefore, there is no contribution from pre-existing rRNA to the increased amount of rRNA in the stimulated kidney. After labeling with L-(methyl-3H)methionine, the kinetics of incorporation into rRNA precursors, 10-60 min and at the postoperative times of 4, 16, 36, and 96 h. The specific activity of cytoplasmic rRNA after 1-h labeling with L-(methyl-3H)methionine increased occured at 4 or 96 h. Since (a) the rate of degradation of rRNA, (b) the kinetics of incorporation and processing of rRNA precursors, and (c) the rate of RNA synthesis appear unchanged after UNI, the accretion of rRNA must involve decreased degradation of newly synthesized rRNA.  相似文献   

4.
Liver rough endoplasmic reticulum (RER) membranes were isolated from rats given [3H]orotic acid for 48 h (ribosomal RNA [rRNA] label) or for 3 h along with 5-fluoroorotate; this latter procedure permits the labeling of cytoplasmic messenger RNAs (mRNAs) in the absence of rRNA labeling. More than 50% of the labeled mRNA remained attached to membranes of the RER after complete removal of ribosomes with a buffer of high ionic strength in the presence of puromycin. Under similar conditions, membranes retained 40% of their polyadenylate as determined by a [3H]-polyuridylate hybridization assay. Treatment of mRNA-labeled endoplasmic reticulum membranes with pancreatic RNase indicates that the polyadenylate and possibly nonpolyadenylate-pyrimidine portions of the messenger are involved in the binding of mRNA to the membranes. The implication of these results in furthering our understanding of the mechanisms of the translational regulation of genetic expression is discussed.  相似文献   

5.
6.
Short-lived, small RNAs in the cytoplasm of HeLa cells   总被引:16,自引:0,他引:16  
G L Eliceiri 《Cell》1974,3(1):11-14
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7.
[35S]--70S ribosomes (150 Ci/mmol) were isolated from E. coli MRE-600 cells grown on glucose-mineral media in the presence of [35S] ammonium sulfate. The labeled 30S and 50S subunits were obtained from [35S] ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+). The activity of [35S]--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70%. The activity of [35S]--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes. The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis. The [35S] ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis. The [35S] label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.  相似文献   

8.
THE RIBOSOMAL RNA OF HAMSTER-MOUSE HYBRID CELLS   总被引:7,自引:2,他引:5       下载免费PDF全文
The ribosomal RNA (rRNA) of a series of hamster-mouse somatic cell hybrids was studied. Mouse 28S rRNA was separated from its hamster counterpart by a two-step procedure involving sucrose gradient centrifugation of ribosomes and polyacrylamide gel electrophoresis of rRNA. Both hamster and mouse types of rRNA were synthesized in the 11 hybrids tested, including hybrids containing only about one-half the haploid number of either mouse or hamster chromosomes. It appears that, for both hamster and mouse rRNA, when the chromosomes of one species constituted the majority of the chromosomes of a hybrid, a disproportionately higher percentage of rRNA of that species was present in the hybrid. Some hybrid clones, having a majority of mouse chromosomes, had a mouse rRNA cell concentration approximately four to five times higher than the concentration expected from linear extrapolation of the value found for the mouse parental cell line.  相似文献   

9.
Two Types of Ribosome in Mouse–Hamster Hybrid Cells   总被引:87,自引:0,他引:87  
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10.
The composition and structure of the complex oligosaccharides of thyrotropin (TSH) and free alpha-subunits are not well established, but are believed to be important determinants of the biological properties of these glycoproteins. We employed a simple double-label technique to learn the relative fucose content of mouse thyrotropin and free alpha-subunits. Thyrotropic tumor minces were incubated simultaneously with [35S]methionine and [3H]fucose. Thyrotropin and free alpha-subunits were labeled with both isotopes, and the ratio of 3H/35S was higher in free alpha-subunits than in thyrotropin; free alpha-subunits were approximately fivefold richer in fucose than was thyrotropin. The 3H/35S ratio was not substantially altered in TSH or free alpha-subunits secreted after a brief incubation with 10(-7) M thyrotropin-releasing hormone. Species which incorporated [3H]fucose were resistant to endoglycosidase H. Thus, mouse free alpha-subunits secreted by thyrotropic tumor are relatively rich in fucose. Double-isotope labeling using an amino acid and a sugar appears to be a useful technique for studies of the glycoprotein hormones.  相似文献   

11.
The incorporation of intracisternally injected L-[methyl-3H]methionine [( 3H]Met) or S-adenosyl-L-[methyl-3H]methionine (Ado[3H]Met) into rat brain AdoMet and phospholipid pools was examined. When [3H]Met was administered, both AdoMet and phospholipid pools were labeled. However, exogenously injected Ado[3H]Met did not serve as a substrate for phospholipid-N-methyltransferases. It was concluded that only Ado[3H]Met formed in situ was utilized to methylate phospholipids and that this process was initiated on the cytoplasmic side of the membrane. The apparent biological half-life in brainstem of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine formed from [3H]Met was 1.4 and 1.7 days, respectively. The half-life of phosphatidylcholine could not be determined due to interference from peripheral sources.  相似文献   

12.
Methionine metabolism forms homocysteine via transmethylation. Homocysteine is either 1) condensed to form cystathionine, which is cleaved to form cysteine, or 2) remethylated back to methionine. Measuring this cycle with the use of isotopically labeled methionine tracers is problematic, because the tracer is infused into and measured from blood, whereas methionine metabolism occurs inside cells. Because plasma homocysteine and cystathionine arise from intracellular metabolism of methionine, plasma homocysteine and cystathionine enrichments can be used to define intracellular methionine enrichment during an infusion of labeled methionine. Eight healthy, postabsorptive volunteers were given a primed continuous infusion of [1-13C]methionine and [methyl-2H(3)]methionine for 8 h. Enrichments in plasma methionine, [13C]homocysteine and [13C]cystathionine were measured. In contrast to plasma methionine enrichments, the plasma [13C]homocysteine and [13C]cystathionine enrichments rose to plateau slowly (rate constant: 0.40 +/- 0.03 and 0.49 +/- 0.09 h(-1), respectively). The enrichment ratios of plasma [13C]homocysteine to [13C]methionine and [13C]cystathionine to [13C]methionine were 58 +/- 3 and 54 +/- 3%, respectively, demonstrating a large intracellular/extracellular partitioning of methionine. These values were used to correct methionine kinetics. The corrections increase previously reported rates of methionine kinetics by approximately 40%.  相似文献   

13.
Novikoff cells in culture were labeled with L-[methyl-3H]methionine and [U-14C]uridine in the presence of (a) TubHcy2, (b) AdoHcy, (c) homocysteine, (d) tubercidin, or (e) without any additions. Only in cultures labeled in the presence of TubHcy were undermethylated cap structures observed to represent a significant portion of [3H]methyl radioactivity. Novikoff cells in culture were then simultaneously labeled with L-[methyl-3H]methionine and [32P]orthophosphate in the presence or absence of TubHcy. Total cytoplasmic, polysomal and monosomal poly(A)-containing RNAs were analyzed. Both monosomal and polysomal mRNA fractions from TubHcy-treated cells contain partially methylated cap structures, suggesting that 2'-O-methylation of the nucleoside adjacent to the pyrophosphate linkage in caps is not required for transport, ribosomal binding or translation. Comparison of nuclear and cytoplasmic cap structures from normal and inhibited cultures indicate that an altered mRNA population is generated in the presence of TubHcy.  相似文献   

14.
A microsomal preparation from chondroitin 4-sulfate-synthesizing cultured mouse mastocytoma cells was incubated with UDP-[3H]GalNAc, UDP-GlcA, and 3'-phosphoadenylylphosphosulfate (PAPS) for 30 s at 10 degrees C and with UDP-[14C]GlcA, UDP-GalNAc, and PAPS for 4 h at 37 degrees C for synthesis of 3H- and 14C-labeled chondroitin/chondroitin sulfate. The latter incubation provided more than 100 times as much product as did the short incubation at 10 degrees C. Upon chromatography of the isolated labeled glycosaminoglycans on a Sepharose CL-6B column, most of the [14C]glycosaminoglycan from the 4 h, 37 degrees C incubation was excluded from the column, indicating that this nascent glycosaminoglycan had been polymerized fully. In contrast, most of the [3H]glycosaminoglycan from the 30 s, 10 degrees C incubation was mostly retarded upon cochromatography on this same column, indicating that the nascent glycosaminoglycan was still growing in size. The labeled fractions representing chondroitin/chondroitin sulfate of varying sizes were analyzed for degree of sulfation by degradation with chondroitin ABC lyase followed by paper electrophoresis of the products. Results indicated that the [14C]chondroitin/chondroitin sulfate formed in the 4-h incubation was 60-70% sulfated. Incomplete chains of [3H]chondroitin/chondroitin sulfate formed in the 30-s incubation were also sulfated as much as 20-25%. As the size of the [3H]chondroitin/chondroitin sulfate increased, there was a concomitant increase in sulfation. These results demonstrate that in this microsomal system sulfation takes place while the nascent chondroitin glycosaminoglycan chains are still actively growing in length, although the sulfation lags somewhat behind the polymerization. This not only indicates a common membrane location for both polymerization and sulfation of chondroitin but also demonstrates that the sulfation of chondroitin by these mastocytoma cells may occur during the process of glycosaminoglycan polymerization rather than subsequent to completion of the glycosaminoglycan chains.  相似文献   

15.
Polyadenylated messenger RNA from mouse kidney labeled in vivo exhibited a pattern of methylation distinct from that of rRNA and tRNA. After mice were given L-[methyl-3H]methionine, 4% of the polyribosomal RNA label was bound to oligo (dT)-cellulose; 20-24% of orotate- or adenine-labeled polyribosomal RNA eluted in the poly(A)+ RNA fraction under similar conditions. [3H]Methyl radioactivity was not incorporated into low molecular weight (5-5.8 S) rRNA, indicating the extent of nonmethylpurine ring labeling was negligible. [3H]Methyl-labeled poly(A)+ RNA sedimented heterogeneously in sodium dodecyl sulfate containing gradients similarly to poly(A)+ mRNA labeled with [3H]orotic acid. Based on an average molecular length of 2970 nucleotides, renal mRNA was estimated to contain 8.6 methyl moieties per molecule. Analysis of alkaline-hydrolyzed RNA sampled by DEAE-Sephadex-urea chromatography provided estimates of the relative amounts of base and ribose methylation. Although 83% of the [3H]methyl radioactivity in rRNA was in the 2'-0-methylnucleotide fraction, no methylated dinucleotides were found in mRNA. In poly(A)+ mRNA 60% of the [3H]methyl label was in the mononucleotide fraction; the remainder eluted between the trinucleotide and tetranucleotide markers and had a net negative charge between -4 and -5. The larger structure, not yet charcterized, could result from two or three consecutive 2'-0-ribose methylations and is estimated to contain 2.6 methyl residues. Alternatively, the oligonucleotide could be a 5'-terminal methylated nucleotide species containing 5'-phosphate(s) in addition to the 3'-phosphate moiety resulting from alkaline hydrolysis. Either structure could have a role in the processing or translation of mRNA in mammalian cells.  相似文献   

16.
A simple method for the isolation of rat liver cells is described. The cells are shown, by an isotope dilution method, to maintain a constant rate of protein synthesis for 8 h of incubation. Antibodies to purified rat liver cytochrome oxidase were raised in rabbits and used to investigate the labeling of cytochrome oxidase in isolated rat liver cells and in vivo. The data demonstrate the occurrence of a precursor of the subunits of cytochrome oxidase that are synthesized in the cytoplasm. 1. Dodecylsulfate gel electrophoresis of the immunoprecipitates from isolated rat liver cells that had been labeled with [35S]methionine for 1 h showed a single radioactive peak with a molecular weight of 50000. 2. Judged by the effects of cycloheximide and chloramphenicol the labeled protein is synthesized on cytoplasmic ribosomes. 3. After labeling for 1 h in vivo with [3H]leucine the labeled protein appears to be exclusively associated with the hepatic microsomal fraction. 4. Ouchterlony double-diffusion analysis demonstrated immunological relationship between the precipitates from microsomes and cytochrome oxidase. In addition to the precipitates derived from mitochondria and microsomes immunoprecipitates were also obtained from the cytosol in comparable amounts; these again were immunologically related. The occurrence of large amounts of precursor(s) (or degradation products) of cytochrome oxidase in rat liver fractions is interpreted in terms of a regulatory pool for amino acid homeostasis in the organism.  相似文献   

17.
Labeling of sulfolipids in Nitzschia alba was studied after growth of the cells in media containing L-[35S]cystine, L-[35S], L-[35S]cysteine, L-[35S]-methionine or a mixture of L-[Me-3H]methionine and L-[35S]methionine, [35S]Cysteine or [35S]cystine labeled the deoxyceramide sulfonate and the sulfonium analog, phosphatidylsulfocholine (and its lyso derivative) but not the sterol sulfate nor the sulfoquinovosyl diglyceride; [35S]methionine labeled only the phosphatidylsulfocholine and its lyso derivative. With the [35S]- and [Me-3H]methionine mixture (3H/35S ratio 1.0) the phosphatidylsulfocholine had a 3H/35 S ratio of 1.5 indicating that both sulfonium methyl groups were derived from methionine. Probable biosynthetic pathways for these novel sulfolipids are discussed.  相似文献   

18.
Ribosomes prepared from wheat or spinach leaves, when incubated with [3H]puromycin, cannot be separated from all the resulting peptidyl-[3H]puromycins by centrifugation. The adherence of these labeled peptidyl-puromycins to ribosomes may lead to ambiguous results when antibodies are added to identify the peptidyl-puromycins. The ambiguties can be avoided in two ways: (1) by examining the small proportion of peptidyl-[3H]puromycins actually released into a postribosomal supernatant fraction; and (2) by promoting the release of all peptidyl-[3H]puromyeins by including neutral detergents in the [3H]puromycin incubation mixture before adding ribosomes. Addition of antibody specific for ribulose diphosphate carboxylase (RuDPCase; EC1.1.1.3.9) to peptidyl-[3H]puromycins released from 70S and 80S ribosomes by either of these methods gives reproducible minimum estimates of the proportion of ribosomes engaged in the synthesis of this enzyme. Physical properties of the peptidyl-[3H]puromycins are also discussed.  相似文献   

19.
The insertion of newly synthesized proteins into the outer membrane of Escherichia coli has been examined. The results show that there is no precurser pool of outer membrane proteins in the cytoplasmic membrane because first, the incorporation of a [35S]methionine pulse into outer membrane proteins completely parallels its incorporation into cytoplasmic membrane proteins, and second, under optimal isolation conditions, no outer membrane proteins are found in the cytoplasmic membrane, even when the membranes are analysed after being labeled for only 15 s. The [35S]methionine present in the outer membrane after a pulse of 15 s was found in protein fragments of varying sizes rather than in specific outer membrane proteins. This label could however be chased into specific proteins within 30--120 s, depending on the size of the protein, indicating that although unfinished protein fragments were present in the outer membrane, they were completed by subsequent chain elongation. Thus, outer membrane proteins are inserted into the outer membrane while still attached to ribosomes. Since ribosomes which are linked to the cell envelope by nascent polypeptide chains are stationary, the mRNA which is being translated by these ribosomes moves along the inner cell surface.  相似文献   

20.
Cells with and without hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity were used to examine the transfer of purine metabolites through the medium and via cell contacts. HGPRT- Chinese hamster and human fibroblasts were able to incorporate 3H-labeled purine metabolite(s) from medium in which mouse HGPRT+ B82 cells had been grown for 24 h with [3H]hypoxanthine, but mouse A9 fibroblasts that were deficient in HGPRT, adenine phosphoribosyltransferase (APRT), and methylthioadenosine phosphorylase (MTAP) were unable to incorporate these metabolites. This suggests that in recipient cells incorporation is due to [3H]MTA, which has been shown previously to be the major 3H-labeled purine metabolite to accumulate in B82 medium, being cleaved by MTAP to [3H]adenine, which is phosphoribosylated by APRT to [3H]AMP. Incorporation by recipient cells of metabolites from the medium is referred to as contact-independent metabolite transfer (CIMT). In autoradiograms of B82/A9 cocultures that were labeled with [3H]hypoxanthine, grains were found over A9 that were not in contact with B82, although A9 did not act as recipients of CIMT. This is termed proximity-dependent metabolite transfer (PDMT). Both CIMT and PDMT interfered with the assessment of nucleotide exchange between HGPRT+ and HGPRT- cells through cell contacts, which is referred to as contact-dependent metabolite transfer (CDMT). These problems were unique to HGPRT+ mouse L cells. However, HGPRT- mouse L cells, A9, could be used as potential recipients. A9 were positive recipients of CDMT with only one of five cell lines tested, which suggested that these cells were selective communicators. CDMT could not be studied with [3H]guanine because the nuclei of HGPRT- cells became labeled.  相似文献   

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