共查询到20条相似文献,搜索用时 31 毫秒
1.
Procedures are described that permit the detection and isolation of a specific messenger RNA as well as its precursor from total cell extracts. DNA complementary to the mRNA was elongated by the addition of dCMP residues and annealed with labeled cell RNA. The elongated DNA with RNA hybridized to it was isolated by chromatography on a poly(I)-Sephadex column. The method was used to isolate 32P-labeled globin mRNA from labeled Friend cells, a mouse erythroleukaemic cell line, induced with dimethylsulfoxide to synthesize hemoglobin. 32P-labeled globin mRNA isolated by this procedure was estimated to be 80% pure by hybridization analysis and sedimented as a single peak at 10 S. Partial sequences were determined for 16 oligonucleotides derived from the purified 32P-labeled globin mRNA by RNAase T1 digestion. The partial sequences for nine oligonucleotides corresponded to those predicted from the amino acid sequences of α and β globin; the other oligonucleotides were presumably derived from non-translated regions.In order to detect a possible precursor to globin mRNA, RNA from induced Friend cells pulse-labeled with [32P]phosphate for 20 minutes was centrifuged through a sucrose gradient and the resulting fractions were analyzed for globinspecific sequences. Two peaks of globin-specific RNA were detected, a larger one at 10 S, the position of mature globin mRNA, and a smaller one at 15 S. 相似文献
2.
In Xenopus laevis oocytes, 5S RNA is stored in the cytoplasm until vitellogenesis, at which time it is imported into the nucleus and targeted to nucleoli for ribosome assembly. This article shows that throughout oogenesis there is a pool of nuclear 5S RNA which is not nucleolar-associated. This distribution reflects that of oocyte-type 5S RNA, which is the major 5S RNA species in oocytes; only small amounts of somatic-type, which differs by six nucleotides, are synthesized. Indeed, 32P-labeled oocyte-type 5S RNA showed a degree of nucleolar localization similar to endogenous 5S RNA (33%) after microinjection. In contrast, 32P-labeled somatic-type 5S RNA showed significantly enhanced localization, whereby 70% of nuclear RNA was associated with nucleoli. A chimeric RNA molecule containing only one somatic-specific nucleotide substitution also showed enhanced localization, in addition to other somatic-specific phenotypes, including enhanced nuclear import and ribosome incorporation. The distribution of 35S-labeled ribosomal protein L5 was similar to that of oocyte-type 5S RNA, even when preassembled with somatic-type 5S RNA. The distribution of a series of 5S RNA mutants was also analyzed. These mutants showed various degrees of localization, suggesting that the efficiency of nucleolar targeting can be influenced by many discrete regions of the 5S RNA molecule. J. Cell. Biochem. 69:490–505, 1998. © 1998 Wiley-Liss, Inc. 相似文献
3.
Short fragments of DNA (5 S) isolated by denaturation from polyoma replicative intermediates pulse-labeled in vitro were shown to have RNA covalently attached by three criteria: (1) such fragments were slightly denser than bulk viral DNA. (2) They could be labeled directly with α-32P-labeled ribotriphosphates. (3) Alkaline hydrolysis of fragments labeled with α-32P-labeled deoxynucleoside triphosphates showed 32P transfer to 3′ ribonucleoside monophosphates. Except for a preference of transfer from dC, the link showed little sequence specificity. The data are compatible with the notion that all short fragments in replicating viral DNA are initiated by an RNA primer. This RNA is maximally 30 bases long and is rather short-lived. 相似文献
4.
Topography of 16 S RNA in 30 S subunits and 70 S ribosomes accessibility to cobra venom ribonuclease
A ribonuclease extracted from the venom of the cobra Naja oxiana, which shows an unusual specificity for double-stranded RNA regions, was used to obtain new insight on the topography of Escherichia coli ribosomal 16 S RNA in the 30 S subunit and in the 70 S couple. 32P-labeled 30 S subunits or reconstituted 70 S tight couples containing 32P-labeled 16 S RNA have been digested under progressively stronger conditions. The cleavage sites have been precisely localized and the chronology of the hydrolysis process studied.The enzyme cleaves the 16 S RNA within 30 S subunits at 21 different sites, which are not uniformly distributed along the molecule. These results provide valuable information on the 16 S RNA topography and evidence for secondary structure features.The binding of the 50 S subunit markedly reduces the rate of the 16 S RNA hydrolysis and provides protection for several cleavage sites. Four of them are clustered in the 3′-terminal 200 nucleotides of the molecule, one in the middle (at position 772) and one in the 5′ domain (at position 336). Our results provide further evidence that the 3′-terminal and central regions of the RNA chain are close to each other in the ribosome structure and lie at the interface of the two subunits. They also suggest that the 5′ domain is probably not involved exclusively in structure and assembly. 相似文献
5.
Jacques Champier Bernard Rousset Michel Berger 《Molecular and cellular biochemistry》1983,56(2):123-129
Summary Bovine thyroid RNA labeled by incubation of slices in the presence of 32P-orthophosphate were fractionated by a two-step procedure. Total RNA were extracted by gel filtration on AcA 22 in the presence of pronase and separated by Sepharose 2B chromatography. A small fraction of heavily-labeled RNA (giant RNA) was obtained in the void volume (peak I); the major fraction of RNA (smaller than 45 S) was retarded on the column (peak II) and had a low specific radioactivity. Labeled and total RNA of peak I and labeled RNA species of peak II had a DNA-like nucleotide composition and were polyadenylated. In contrast, the nucleotide composition of total RNA of peak II was similar to that of ribosomal RNA and had a very low poly (adenylic acid) content. Pulse-chase experiments showed a precursor-product relationship between the two RNA fractions. These data indicate that labeled RNA of peak I and peak II likely correspond to newly-synthetized pre-mRNA and mRNA, respectively. Thyrotropin induced a decrease in the amount of 32P-labeled pre-mRNA and a proportional increase of 32P-labeled mRNA suggesting a stimulatory effect of the hormone on the degradation of pre-mRNA.Abbreviations SDS
sodium dodecyl sulfate
- TIPNS
triisopropylnaphthalene disulfonic acid, sodium salt
- TSH
thyrotropin-stimulating hormone 相似文献
6.
It was reported previously that two spherical flacherie viruses of silkworm, FVS I and FVS II, had been isolated from flacherie silkworm larvae and the nucleic acid of FVS II was RNA as suggested by the experiments of incorporation of [3H]-uracil. In this paper, it has been confirmed by biochemical methods that the nucleic acid of FVS I and FVS II is RNA. FVS I and FVS II were labeled with 32P in flacherie silkworms, and the viruses were analyzed by sucrose density gradient centrifugation. When the 32P-labeled compound in the viruses was treated with 0.5 n KOH, the acid-insoluble 32P-labeled compound changed to acid-soluble compounds. It was determined by paper chromatography and ion-exchange column chromatography that the alkali-decomposed compounds included four ribonucleotides. Therefore, the viral nucleic acid of FVS I and FVS II was determined to be RNA. The correlations between FVS I and FVS II particles were discussed, and it was suggested that FVS I and FVS II might be closely related or were the same viral species. 相似文献
7.
Recently developed methods which allow one to read RNA sequences directly from polyacrylamide gels do not always provide unequivocal results. A combination of primary and secondary in vitro 5′-labeling, as presented here, is methodically and in its results equivalent to fingerprinting and sequencing techniques developed for in vivo labeled RNA. 5 S RNA was used to demonstrate the applicability and reliability of this combination of postlabeling procedures: 5 μg RNA was partially digested, and the resulting overlapping fragments were 5′-32P-labeled with T4 phage-induced polynucleotide kinase in vitro. After two-dimensional polyacrylamide gel electrophoresis and carrier-free electrophoretic elution, the labeled long fragments, obtained in the 10-ng range, were completely degraded with RNase T1 and RNase A, respectively. These digests were again 32P-phosphorylated with T4 kinase and lead to fingerprints which allowed the deduction of the nucleotide sequences of the corresponding long fragments. 相似文献
8.
9.
C T Garrett D S Wilkinson J W Tweedle H C Pitot 《Archives of biochemistry and biophysics》1973,155(2):342-354
5-Fluoroorotic acid treatment lowered the (Guanine + Cytosine)/(Adenine + Uracil) base ratio of 32P-labeled microsomal RNA from a control value of 1.36 to 1.00. Low doses of actinomycin D, which are effective in inhibiting ribosomal RNA synthesis without significantly affecting messenger RNA synthesis, caused a similar decrease in the base ratio. Microsomal RNA labeled by [3H]orotate in the presence of 5-fluoroorotic acid had approximately the specific radioactivity but twice the hybridization efficiency of RNA labeled in its absence. Evidence is presented that this RNA (1) has a different structure from that of ribosomal RNA, (2) hybridizes to DNA with an efficiency consistent with that of other published studies of polysome-associated messenger RNA, and (3) possesses sequences which are present in other samples of liver microsomal RNA but not in kidney microsomal RNA. These properties differ from those known to be exhibited by 18 S and 28 S ribosomal RNA. Electrophoretic analysis of this [3H]orotate-labeled microsomal RNA indicated that the analogue greatly inhibited precursor incorporation into ribosomal RNA but had little or no effect on incorporation into messenger RNA. Ribosomal RNA and polyadenylate-rich nonribosomal RNA were prepared from total polyribosomes by phenol extraction at pH 7.6 and pH 9.0, respectively. 5-Fluoroorotic acid inhibited [3H]orotate or 32Pi incorporation into the pH 7.6 fraction much more effectively than incorporation into the pH 9.0 fraction. A subfraction of the pH 9.0 RNA which was retained by a polythymidylate-cellulose column had a greatly increased adenylate content. 相似文献
10.
Characterization of bacteriophage T4 and D RNA, a low-molecular-weight RNA of unknown function 总被引:1,自引:0,他引:1
The nucleotide sequence of T4 band D RNA, a stable RNA species encoded by bacteriophage T4, has been deduced from analysis of the 32P-labeled RNA and comparison with the DNA sequence of the T4 genome in the region encoding the RNA. The sequence is: pA-U-G-A-G-A-A-A-C-C-G-G-G-U-C-G-C-U-A-C-C-G-G-U-A-A-G-U-C-G-U-C-G-G-A-C-U-G-A-U-G-G-U-U-C-C-C-U-G-A-G-U-A-A-G-G-A-A-U-U-G-C-G-U-U-A-A-U-A-A -U-C-U-U-U-G-C-G-U-U-U-A-U-U-G-A-U-G-C-C-C-U-C-U-U-A-C-A-U-C-A-C-A-G-C-A-G-A-A-A-C-G-G-C-G-C-A-C-C-AOH. Band D RNA is 120 nucleotides long, and contains no modified nucleotides. The sequence can be arranged in a secondary structure consistent with the results of limited digestion with nuclease S1, but shows no striking similarities to tRNAs. While a biological function for band D RNA is unknown, similar molecules are encoded by bacteriophages T2 and T6, indicating that the molecule has been preserved during evolution. This retention may reflect a significant function for the RNA. 相似文献
11.
12.
13.
14.
Replication of bacteriophage MS2. X. Phage-specific ribonucleoprotein particles found in MS2-infected Escherichia coli 总被引:5,自引:0,他引:5
Phage-specific RNA-protein complexes formed during the MS2 infection process were examined. The fate of 32P-labeled parental viral RNA was followed to determine what RNA-protein interactions developed early in infection. In order to identify phage-specific ribonucleoprotein complexes at later times in infection, their protein or RNA components were labeled selectively with radioisotopes after suppression of bacterial macromolecular syntheses with Miracil D (Burroughs Wellcome and Co.). 相似文献
15.
The DNA duplex (designated [A]) corresponding to the nucleotides 1 to 20 of the major yeast alanine transfer RNA (Fig. 1) has been synthesized. The first step involved the T4 ligase-catalyzed joining of d-(5′-32P)-C-C-G-G-A-A-T-C (segment 4, Fig. 1) to the dodecanucleotide, d-(5′-OH)-T-G-G-T-G-G-A-C-G-A-G-T (segment 1, Fig. 1), in the presence of the complementary decanucleotide d-(5′-OH)-C-C-G-G-A-C-T-C-G-T (segment 3, Fig. 1). The resulting icosanucleotide, d-(5′-OH)-T-G-G-T-G-G-A-C-G-A-G-T-C-C-G-G-A-A-T-C, was isolated free from the decanucleotide (segment 3). The synthesis of [A] was then completed by the ligase-catalyzed joining of 5′-32P or 33P-labeled hexanucleotide d-(5′-P)-C-C-A-C-C-A (segment 2) to the 5′-32P or 33P-labeled decanucleotide, d-(5′-P)-C-C-G-G-A-C-T-C-G-T (segment 3), in the presence of the above icosanucleotide. 相似文献
16.
Hans Söderlund Ulf Pettersson Björn Vennström Lennart Philipson Michael B. Mathews 《Cell》1976,7(4):585-593
A virus-coded low molecular weight RNA (5.2S), which migrates slightly faster on polyacrylamide gels than the well characterized adenovirus-specific 5.5S RNA, has been isolated from cells infected with adenovirus type 2. Hybridization-competition experiments and RNA fingerprints indicate that the two virus-associated (VA) RNAs differ in their primary structures. The gene for 5.2S RNA is located to the right of the gene for 5.5S RNA, on the I strand of a DNA segment which extends between positions 30.3 and 32.2 on the map of adenovirus type 2 DNA.Both 5.5S and 5.2S RNA can be detected early after infection and also in the presence of cytosine-arabinoside or cycloheximide. After the onset of viral DNA replication, the synthesis of 5.2S RNA levels off, whereas 5.5S RNA is synthesized in increasing amounts. Both 5.2S and 5.5S RNAs are synthesized in isolated nuclei by an enzyme which resembles RNA polymerase III in its sensitivity to α-amanitin. In isolated nuclei, both RNA species are labeled with β-32P-labeled GTP, which suggests that they are initiated at separate promoter sites. 相似文献
17.
Specific nucleotide sequences in HeLa cell inverted repeated DNA: enrichment for sequences found in double-stranded regions of heterogeneous nuclear RNA 总被引:13,自引:0,他引:13
W R Jelinek 《Journal of molecular biology》1977,115(4):591-601
Inverted repeat DNA was isolated from HeLa cell nuclei and transcribed in vitro with Escherichia coli RNA polymerase in the presence of [alpha-32P]nucleoside triphosphates. The RNA products were digested with T1 ribonuclease and subjected to separation in two dimensions. The pattern of the prominent oligonucleotides was almost indistinguishable from that seen when the double-stranded regions from 32P-labeled HeLa cell heterogeneous nuclear RNA were fingerprinted in a similar manner. The sequences of several of the largest prominent T1 ribonuclease-generated oligonucleotides were determined and were found to agree with those isolated from the double-stranded heterogeneous nuclear RNA that migrated to the same positions in the fingerprints. The most prominent component of the inverted repeat DNA appears to be sequences that are transcribed into double-stranded regions in heterogeneous nuclear RNA molecules. 相似文献
18.
InEscherichia coli, NADP+-specific isocitrate dehydrogenase (EC 1.1.1.42) may undergo a phosphorylation catalyzed by a cAMP-independent protein kinase, with a concomitant decrease in catalytic activity. In this report, we describe the purification and amino acid sequence of a32P-labeled peptide obtained from in vivo32P-labeled isocitrate dehydrogenase. The32P-labeled peptide was isolated from a tryptic digest and found to contain seven amino acids, including a single serine residue. Following automated Edman degradation and reversephase high-pressure liquid chromatography of the phenylthiohydantoin-amino acids, the sequence of this peptide was established to be-Ser(P)-Leu-Asn-Val-Ala-Leu-Arg. 相似文献
19.
Studies of the binding of Escherichia coli RNA polymerase to DNA. V. T7 RNA chain initiation by enzyme-DNA complexes 总被引:9,自引:0,他引:9
Initiation of T7 RNA chains by Escherichia coli RNA polymerase-T7 DNA complexes has been followed using incorporation of λ-32P-labeled ATP and GTP to determine the relation between the enzyme binding sites and RNA chain initiation sites on the T7 genome. If the period of RNA synthesis is limited to less than two minutes, the stoichiometry of RNA chain initiation can be measured in the absence of chain termination and re-initiation. About 70% of the RNA polymerase holoenzyme molecules in current enzyme preparations are able to rapidly initiate a T7 RNA chain. The ratio of ATP- to GTP-initiated T7 RNA chains is not altered by variations in the number of enzyme molecules added per DNA, nor by alterations in the ionic conditions employed for RNA synthesis. This suggests that RNA chain initiation sites are chosen randomly through binding of RNA polymerase to tight (class A) binding sites on T7 DNA. 相似文献
20.
32P-labeled ribonucleic acid (RNA) from purified Sindbis virus was examined for the presence of hidden breaks. Viral RNA was treated with acid at pH 2.9 or with formaldehyde and was analyzed on sucrose gradients or by polyacrylamide gel electrophoresis. The sedimentation pattern and mobility on polyacrylamide gels of the 42S RNA was unaffected by heating and quick cooling and was not altered by denaturing agents such as dimethyl sulfoxide and urea. No evidence that Sindbis RNA is a polyaggregate of fragments was obtained. It is concluded that the genome consists of a continuous length of single-stranded polynucleotide. 相似文献