Isolation of ribosomal RNA precursors from Physarum polycephalum

Ribosomal RNA synthesis in Physarum polycephalum was studied by labeling intact microplasmodia with [³H]uridine. Labeled, high-molecular-weight RNA species were found in a 30,000 S structure released by phenol extraction at room temperature. RNA was released from the structure by further phenol extraction at 65–70 °C. If the labeling period was 15 min or longer, the labeled RNA was seen by polyacrylamide gel electrophoresis to be of two major types, a heterodisperse collection of 45-35 S molecules and a 26 S species. If the labeling was carried out for 30 min in the presence of cycloheximide, the major labeled species had an electrophoretic mobility corresponding to 40 S. Studies of the labeling kinetics, methylation, and base composition of these RNA molecules indicate that they are precursors to ribosomal RNA. The molecular weights of the homogeneous 40 and 26 S precursors are 3.0 × 10⁶ and 1.45 × 10⁶ daltons, respectively, in comparison with molecular weights of 1.29 × 10⁶ and 0.68 × 10⁶ daltons for the completed ribosomal RNA's.     

快速提取、纯化叶绿体4.5SrRNA的方法

我们采用植物叶与热缓冲液、苯酚直接混合(约65℃)匀浆,离心抽提和乙醇沉淀后,得到植物叶总RNA。经聚丙烯酰胺凝胶电泳分离、纯化,即可得到叶绿体4.5S rRNA,此法不仅操作简单,而且得率高。 同时,经过对同一植物的不同组织或不同细胞组分,如根、细胞质、叶绿体和叶绿体核糖体小分子RNA的提取与鉴定,以简便的方法证明了4.5S rRNA是叶绿体核糖体成份,也证明了我们所采用的提取、纯化4.5SrRNA方法的可靠性。     

RNA composition of Junin virus.

Junin virus grown in BHK-21 cells was labeled with [3H]uridine and highly purified by differential and isopycnic centrifugation. The RNAs extracted with phenol and analyzed by polyacrylamide gel electrophoresis were shown to be composed of four large species (33, 28, and 18S) and three small ones (4, 5, and 5.5S). This pattern was similar to ones already reported for other arenaviruses. However, there was a striking difference when the virus labeling was performed in the presence of actinomycin D. Labeling of viral rRNA was as much as 60% of the levels obtained in the absence of the drug under conditions in which cellular rRNA's were inhibited by 95% or more.     

The functional analysis of 4.5S RNA in ribosomal translocation.

4.5S RNA is a stable 114-nucleotide RNA of the bacterium Escherichia coli (E. coli). We found that 4.5S RNA have the ability of binding EF-G using gel mobility shift assay. Increasing in the concentration of GDP increase the binding activity of 4.5S RNA to EF-G. Based on these data, we propose that 4.5S RNA release EF-G from ribosome.     

Evidence for mannolipids as intermediates in mannose transfer to thyroid rough microsomal glycoproteins.

Thyroid rough microsomes catalyzed the transfer of mannose from GDP-mannose to endogeneous glycoprotein(s) and to glycolipids comprising a recently described dolichol phosphomannose extractable with usual organic solvents and a material tentatively identified as an oligosaccharide lipid. The labeling of the two lipids was consistent with a role in mannose transfer to glycoprotein(s). When partially purified dolichol phospho[14C] mannose was incubated with rough microsomes, a part of the label appeared in the second lipid, suggesting a role as intermediate, and less rapidly in glycoprotein(s). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis did not allow to ascertain whether or not the glycoproteins receiving label from these sugar lipids comprised thyroglobulin precursors.     

Kinetics of the processing of the precursor to 4.5 S RNA, a naturally occurring substrate for RNase P from Escherichia coli.

A study was made of the cleavage by M1 RNA and RNase P of a non-tRNA precursor that can serve as a substrate for RNase P from Escherichia coli, namely, the precursor to 4.5 S RNA (p4.5S). The overall efficiency of cleavage of p4.5S by RNase P is similar to that of wild-type tRNA precursors. However, unlike the reaction with wild-type tRNA precursors, the reaction catalyzed by the holoenzyme with p4.5S as substrate has a much lower Km value than that catalyzed by M1 RNA with the same substrate, indicating that the protein subunit plays a crucial role in the recognition of p4.5S. A model hairpin substrate, based on the sequence of p4.5S, is cleaved with greater efficiency than the parent molecule. The 3'-terminal CCC sequence of p4.5 S may be as important for cleavage of this substrate as the 3'-terminal CCA sequence is for cleavage of tRNA precursors.     

Preparation and sequencing of the cloacin fragment of Streptomyces aureofaciens 16S RNA

A fast method for isolation of a 3'-terminal fragment of Streptomyces aureofaciens 16S RNA was developed. The procedure involves reaction of 70S ribosomes with cloacin DF13 and subsequent fractionation of the reaction mixture by polyacrylamide gel electrophoresis. The cloacin fragment was eluted from the gel and used directly for 3'-end labeling with cytidine-3',5'-[5'-32P]bisphosphate. The labeled RNA fragment was sequenced by the enzymatic method. It consists of 50 nucleotides and has the sequence 5'-GUCGUAACAAGGUAACCGUACCGGA-AGGUGCGGUUGGAUCACCUCCUUUCOH. The differences from the E. coli and Bacillus sequences and their possible influence on the rate and specificity of polypeptide synthesis are discussed.     

Purification and some properties of a specific nuclease which cleaves transfer RNA precursors from the posterior silk gland of Bombyx mori.

A specific endonuclease involved in the processing of tRNA precursors was isolated and partially purified from the posterior silk gland of Bombyx mori, and designated as RNase P.Bmo. This enzyme was shown to catalyze the conversion of 4.5 S precursor RNA to 4.1 S RNA by trimming the 5'-additional segment from the precursor RNA. RNase P.Bmo required divalent cations, Mg2+ or Mn2+. In the presence of these divalent cations, K+ or NH4+ activated the RNase P.Bmo reaction. Optimum pH was observed around 8.0. Ribosomal RNA's and mature tRNA from the silk gland were not cleaved by RNase P.Bmo. A 4.5 S precursor RNA fraction containing formycin, an adenosine analog, was less susceptible to RNase P.Bmo than the normal one. These results indicate that RNase P.Bmo has a high substrate specificity. An additional nuclease(s) was isolated. This activity was assumed to remove the extra 3'-segment of the 4.5 S precursor RNA.     

Newcastle Disease Virus-Specific RNA: Polyacrylamide Gel Analysis of Single-Stranded RNA and Hybrid Duplexes

Newcastle disease virus-specific [(3)H]uridine-labeled 18S RNA was resolved by polyacrylamide gel electrophoresis into several components with molecular weights from 450,000 to 840,000. The analysis of 35 and 24S virus-specific RNA also revealed several components in each sedimentational class. The conversion of 18S RNA into double-stranded form by hybridization with an excess of unlabeled virion RNA improved the resolution in polyacrylamide gels and revealed at least six distinct components. The same six classes of hybrid duplexes were revealed when (32)P-labeled 50S virion RNA was hybridized with an excess of 18S RNA. The applicability of polyacrylamide gel electrophoresis of hybrid duplexes to the analysis of viral genome structure is discussed.     

Respiratory syncytial virus proteins: identification by immunoprecipitation.

The proteins of respiratory syncytial virus have not been clearly identified due to the lability of the virus and difficulties in its purification. We have pulse-labeled respiratory syncytial virus with [35S]methionine and [35S]cysteine and analyzed cell lysates by polyacrylamide gel electrophoresis. Five 35S-labeled viral proteins ranging in molecular weight from 21,000 to 73,000 (VP73, VP44, VP35, VP28, and VP21) were easily discernable above background cellular proteins. Treatment of the infected cells with 0.15 M NaCl before labeling suppressed host cell protein synthesis and allowed clearer visualization of the five viral proteins by polyacrylamide gel electrophoresis. Three glycoproteins (VGP 92, VGP 50, and VGP 17) were also identified after labeling with [3H]glucosamine. Five of these polypeptides (VP51, VP44, VP35, VP28, and VGP92) were shown to be antigenically active because they could be immunoprecipitated with anti-respiratory syncytial virus antibody produced in New Zealand white rabbits, cotton rats, and humans before analysis by polyacrylamide gel electrophoresis.     

Detection of glycoproteins in the Acanthamoeba plasma membrane

In the present study we have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by 125I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB3H4 and galactose oxidase/NaB3H4 labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with Mr of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presence of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with [35S]methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.     

The synthesis and processing of a nuclear RNA precursor to rat pregrowth hormone messenger RNA.

A recombinant DNA plasmid, pBR322-GH1, which contains about 80% of the sequences of rat pregrowth hormone (pGH) mRNA, allowed an analysis of nuclear RNA from GH3 cells for possible precursors of cytoplasmic pGH mRNA. A single 20-22S RNA SPECIES ABOUT 2-3 TIMes larger than pGH mRNA was detected in nuclear RNA from GH3 cells labeled for 5 min. with 3H-uridine. After longer label times a 12S RNA indistinguishable in size from cytoplasmic 12S pGH mRNA became the predominant labeled RNA complementary to the plasmid pBR322-GH1. Both of these nuclear RNA species contained poly (A). Kinetic analysis of the labeling of nuclear and cytoplasmic pGH mRNA sequences showed that the 20S and 12S nuclear RNA molecules were labeled before significant labeling of cytoplasmic pGH mRNA was detected, and also indicated that there is complete conservation of nuclear pGH mRNA sequences in the production of cytoplasmic pGH mRNA. These results indicate that cytoplasmic pGH mRNA is generated by nuclear processing of a larger nuclear RNA molecule.     

Brain transfer RNA

Transfer RNAs were isolated from rat and calf brains and their nucleosides were analysed by tritium derivative technique. Qualitative changes in the minor nucleoside components were compared on the fluorograms which showed differences in the intensities of spots. Cerebellar and cortical tRNAs were also compared, but revealed no significant quantitative differences in their methylated constituants despite 60% higher methyltransferase activity observed in cerebellum compared to cerebral cortex. An overall similarity was noticed between the relative proportions of the major and minor nucleosides of tRNAs derived from rat or calf brain, expressed as mol %. Brain tRNA was also analysed by two-dimensional polyacrylamide gel electrophoresis which showed qualitative and quantitative changes during postnatal development.     

Mapping labeled sites in Escherichia coli ribosomal RNA: distribution of methyl groups and identification of a photoaffinity-labeled RNA region putatively at the peptidyltransferase center

We have developed a method for the rapid localization of sites of ribosomal RNA labeling to limited regions (approximately 200 bases). The method is based on the formation and polyacrylamide gel electrophoretic separation of hybrids between restriction fragments of rrnB DNA and isotopically labeled rRNA and the subsequent determination of radioactivity across the gel. Using [3H]adenine-labeled rRNA as a control sample, we optimized experimental conditions with respect to a number of variables, including rRNA:DNA stoichiometric ratio, temperature of the annealing step, and levels of nucleases. An important result is that different rRNA X DNA hybrid fragments are obtained in different yields. The method was then applied to analyses of C3H3-labeled rRNA, giving results in good accord with known and proposed sites of rRNA methylation, and of rRNA that has been photoaffinity-labeled with 5-azido-2-nitrobenzoyl-[3H]Phe-tRNAPhe, a probe directed toward the peptidyltransferase center. The latter study showed a single major site of RNA labeling, falling within bases 2445-2668 of 23S rRNA. The extent of labeling was shown to be dependent on light-induced formation of a reactive intermediate and to be decreased in the absence of poly(uridylic acid) or in the presence of puromycin. The location of this major site of labeling is consistent with recent results obtained with an analogous tRNA photoaffinity label [Barta, A., Steiner, G., Brosius, J., Noller, H. F., & Kuechler, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3607-3611] and with related genetic and biochemical studies of antibiotic interaction with ribosomes suggesting that the peptidyltransferase center falls within region V (bases 2043-2625) of 23S rRNA.     

Concentrations of 4.5S RNA and Ffh protein in Escherichia coli: the stability of Ffh protein is dependent on the concentration of 4.5S RNA.

We measured the concentrations of both 4.5S RNA and Ffh protein under a variety of growth conditions and found that there were 400 molecules of 4.5S RNA per 10,000 ribosomes in wild-type cells and that the concentration of Ffh protein was one-fourth of that. This difference in concentration is 1 order of magnitude less than that previously reported but still significant. Pulse-chase labeling experiments indicated that Ffh protein is unstable in cells carrying ffh on high-copy-number plasmids and that simultaneous overproduction of 4.5S RNA stabilizes Ffh protein. Our analyses show that free Ffh protein is degraded with a half-life of approximately 20 min. We also tested whether three previously isolated suppressors of 4.5S RNA deficiency could reduce the requirement for Ffh protein. Since the two sffE suppressors do not suppress the Ffh requirement, we suggest that 4.5S RNA either acts in a sequential reaction with Ffh or has two functions.     

A new series of RNAs associated with the genome of spleen focus forming virus (SFFV) and poly(A)-containing RNA from SFFV-infected cells.

A series of low molecular weight RNAs (4.5 to 5.5S) as well as other 4 to 7S RNAs were dissociated from genomic RNA of spleen focus forming virus (SFFV) by heating. On two dimensional polyacrylamide gel electrophoresis, this series of RNAs gave a series of more than thirty spots. RNase T1 fingerprints of these spots were identical except for differences in 3'-terminal oligonucleotides, which were mainly due to different numbers of uridylic acid residues, larger RNA-molecules containing poly(U)sequences at their 3'-termini. This series of RNAs is also associated with poly(A)-containing nuclear and cytoplasmic RNAs from SFFV-infected cells.     

大鼠4.5S RNAs的cDNA克隆与序列分析

利用ＲＴ－ＰＣＲ方法,首次从大鼠肝脏细胞总ＲＮＡ中扩增出４．５Ｓ ＲＮＡｓ的ｃＤＮＡ。该ｃＤＮＡ被克隆到ｐＧＥＭ３Ｚｆ（＋）质粒上,经酶切电泳鉴定,然后测序。与报道的小鼠和仓鼠４．５Ｓ ＲＮＡｓ序列进行了比较研究,并对该分子的结构特点进行了初步分析。     

Low-molecular weight nuclear RNA components in human lymphocytes cultured without or with phytohaemagglutinin

Purified human lymphocytes were cultured without or with phytohaemagglutinin (PHA) in the presence of radioactive RNA precursors. RNA was extracted with phenol at 0°, 40° or 62°C and separated on polyacrylamide gels. RNA extracted with phenol either in presence or absence of the RNAse inhibitor diethylpyrocarbonate showed no sign of degradation when separated on 2.6 or 3% polyacrylamide gels. Ten percent gel profiles of whole cell or nuclear RNA showed a a number of small mol. wt RNA components (K, L, M, N, A, B, C, D, F) apart from tRNA, 5 S RNA and 5.5 S RNA. Profiles of cytoplasmic RNA showed only components K and L apart from tRNA, 5 S RNA and 5.5 S RNA. L, C, D and F have an electrophoretic mobility similar to the corresponding components in various ascites cells, while M, N and B may be unique for human cells.The low-molecular wt nuclear RNA components (snRNA) are found in non-stimulated as well as in PHA-stimulated cells and the relative amounts of the snRNA components are not changed during PHA-induced transformation. It is therefore concluded that the relative amounts of the different snRNA components are not related to the dynamic state of the cell.