Turnover of ribosomal RNA in mouse fibroblasts (3T3) in culture.

Growing and confluent cultures of mouse fibroblasts were labeled with ³H-uridine and chased with an excess of nonradioactive uridine to investigate the turnover of ribosomal RNA. Growing cultures did not turn over their 18S and 28S ribosomal RNA; however, confluent cultures did show ribosomal RNA (rRNA) turnover. If the cells were labeled while growing, and chased when confluent, 18S RNA displayed a two-component decay curve, while 28S RNA showed only single-component decay, similar in lifetime to the first component of the 18S RNA decay curve. If the cells were labeled while confluent, both the 18S and 28S RNA showed single-component decay curves with a lifetime possibly only slightly longer than the lifetime of the first component of the 18S RNA and the single component of the 28S RNA of the cultures labeled while growing.     

Species specificity and developmental patterns of expression of the beta amyloid precursor protein (APP) gene in brain, liver and choroid plexus in birds.

1. Human APP cDNA hybridized to a 3.5 kb mRNA in liver and brain RNA from chickens, pigeons, quail and ducks as well as in RNA from choroid plexus of chicken and quail. In contrast to all other species hitherto examined a 1.6 kb mRNA hybridizing to APP cDNA was found in abundant amounts in RNA from chicken and quail livers. 2. In the chicken, before hatching, the levels of APP mRNA in total RNA from liver and choroid plexus were higher than those in RNA from liver and choroid plexus of adults. However, RNA from the rest of the brain of chicken embryos contained less APP mRNA than RNA from brain of adults. 3. In the chicken, between 10 and 40 days after hatching, APP mRNA levels in RNA from liver were higher than adult levels, APP mRNA levels in RNA from choroid plexus were similar to adult levels and APP mRNA levels in RNA from the rest of brain were below the adult levels.     

Effect of Chloramphenicol on the Synthesis and Stability of Ribonucleic Acid in Bacillus subtilis

The effect of chloramphenicol on the synthesis and accumulation of ribonucleic acid (RNA) in Bacillus subtilis was studied. In the presence of chloramphenicol, transfer RNA and ribosomal RNA were synthesized as rapidly 2 to 3 hr after challenge as they were just prior to the addition of the antibiotic. However, under the same conditions, net RNA accumulation ceased after only 30 to 45 min. The failure to accumulate RNA after this time resulted from a rapid degradation of ribosomal RNA synthesized in the presence of chloramphenicol and a slow degradation of mature ribosomes. Since transfer RNA was not appreciably degraded, the ratio of transfer RNA to total RNA increased during the challenge.     

RNA recombination in brome mosaic virus: effects of strand-specific stem-loop inserts

A model system of a single-stranded trisegment Brome mosaic bromovirus (BMV) was used to analyze the mechanism of homologous RNA recombination. Elements capable of forming strand-specific stem-loop structures were inserted at the modified 3' noncoding regions of BMV RNA3 and RNA2 in either positive or negative orientations, and various combinations of parental RNAs were tested for patterns of the accumulating recombinant RNA3 components. The structured negative-strand stem-loops that were inserted in both RNA3 and RNA2 reduced the accumulation of RNA3-RNA2 recombinants to a much higher extent than those in positive strands or the unstructured stem-loop inserts in either positive or negative strands. The use of only one parental RNA carrying the stem-loop insert reduced the accumulation of RNA3-RNA2 recombinants even further, but only when the stem-loops were in negative strands of RNA2. We assume that the presence of a stable stem-loop downstream of the landing site on the acceptor strand (negative RNA2) hampers the reattachment and reinitiation processes. Besides RNA3-RNA2 recombinants, the accumulation of nontargeted RNA3-RNA1 and RNA3-RNA3 recombinants were observed. Our results provide experimental evidence that homologous recombination between BMV RNAs more likely occurs during positive- rather than negative-strand synthesis.     

Mutant prohead RNAs in the in vitro packaging of bacteriophage phi 29 DNA-gp3.

The 174-base prohead RNA encoded by bacteriophage phi 29 of Bacillus subtilis, essential for packaging of the DNA-gp3 (DNA-gene product 3) complex, was expressed efficiently from the cloned gene. Computer programs for RNA structure analysis were used to fold hypothetical RNA mutants and thus to target mutagenesis of the RNA for studies of structure and function. Five mutants of the RNA were then produced by oligonucleotide-directed mutagenesis that were altered in the primary sequence at selected sites; two of these mutants were predicted to be altered in secondary structure from a model established previously by a phylogenetic analysis. The binding of the 32P end-labeled mutant RNAs to RNA-free proheads was comparable with that of the wild-type RNA. However, the capability of the mutant RNAs to reconstitute RNA-free proheads for DNA-gp3 packaging in the defined in vitro system and for assembly of phage in RNA-free extracts was variable, depending upon the alteration. Changes of highly conserved bases that retained the predicted secondary structure of the RNA model were tolerated to a much greater extent than changes predicted to alter the RNA secondary structure.     

UV-crosslinking of E1 small nucleolar RNA to proteins in frog oocytes

E1/U17 small nucleolar RNA (snoRNA) is a box H/ACA snoRNA. To detect protein bands that UV-crosslink to E1 RNA primarily at uridines, frog oocytes were injected with [alpha-32P]UTP-labeled E1 RNA and incubated, isolated nuclei were UV irradiated, and nuclear contents were digested with RNase A. Wild-type E1 RNA specifically UV-crosslinked to several protein bands. To identify E1 RNA sites involved in these interactions, we tested 21 E1 RNA mutants, each consisting of substitutions in a conserved sequence or structure. UV-crosslinking of different protein bands to E1 RNA depended on one of the following sets of conserved E1 RNA segments: two 5' end RNA sites; five 5' half RNA sites; two 3' half RNA sites; or 14 sites located throughout E1 RNA. Of these conserved E1 RNA sites, UV-crosslinking apparently depended on sequences at 11 sites, and structures at 2 sites. Gel electrophoresis with and without RNA competition detected protein bands that are not common to all of the box H/ACA snoRNAs.     

酵母发酵法制备核糖核酸研究进展

核糖核酸（RNA）是一种遗传物质,参与细胞蛋白质合成和免疫调节等生理活动。RNA及其降解物在药物开发、保健品和食品添加剂等领域具有良好的应用前景。利用富含RNA的酵母发酵提取RNA是生产RNA的有效途径。概述了酵母发酵法制备RNA的进展及其应用。     

Synthesis of ribonucleic acid in normal bone in vitro

1. The incorporation of [2-(14)C]uridine into nucleic acids of bone cells was studied in rat and pig trabecular-bone fragments surviving in vitro. 2. The rapid uptake of uridine into trichloroacetic acid-soluble material, and its subsequent incorporation into a crude nucleic acid fraction of bone or purified RNA extracted from isolated bone cells, was proportional to uridine concentration in the incubation medium over a range 0.5-20.0mum. 3. During continued exposure to radioactive uridine, bulk RNA became labelled in a curvilinear fashion. Radioactivity rapidly entered nuclear RNA, which approached its maximum specific activity by 2hr. of incubation; cytoplasmic RNA, and particularly microsomal RNA, was more slowly labelled. The kinetics of labelling and rapid decline of the nuclear/microsomal specific activity ratio were consistent with a precursor-product relationship. 4. Bulk RNA preparations were resolved by zonal centrifugation in sucrose density gradients into components with approximate sedimentation coefficients 28s, 18s and 4s. 5. Rapidly labelled RNA, predominantly nuclear in location, demonstrated a polydisperse sedimentation pattern that did not conform to the major types of stable cellular RNA. Material of highest specific activity, sedimenting in the 4-18s region and insoluble in 10% (w/v) sodium chloride, rapidly achieved its maximum activity during continued exposure to radioactive precursor and decayed equally rapidly during ;chase' incubation, exhibiting an average half-life of 4.3hr. 6. Ribosomal 28s and 18s RNA were of lower specific activity, which increased linearly for at least 6hr. in the continued presence of radioactive uridine. There was persistent but variable incorporation into ribosomal RNA during ;chase' incubation despite rapid decline in total radioactivity of the acid-soluble pool containing RNA precursors.     

The Fate of RNA Degradation Products in Starved Cultures of Tetrahymena pyriformis W.*

The RNA content of a population of Tetrahymena pyriformis W was followed during the growth phases of the culture. The cellular RNA levels were found to reach a maximum in early log phase and to decrease throughout the remainder of the log and deceleration phases. There was a 25% decrease in RNA amount when cells in late stationary phase were compared to those in deceleration. This loss of RNA was mimicked when cells from the deceleration phase were suspended in a non-nutrient buffered medium. Procedures were established to determine RNA content and the intra- and extracellular distribution of RNA degradation products, namely purine and pyrimidine bases and orthophosphate. Balance sheets are presented to show that the decrease in RNA levels was accompanied by an equivalent increase in purine and pyrimidine bases and phosphorus derivatives. The validity of the procedures employed was demonstrated. The influence of magnesium, cholesterol and glucose on the cells suspended in a non-nutrient buffer was examined. Each was found to affect the ultimate distribution of RNA products in a characteristic fashion suggesting that each compound acts by a different mode of action.     

条斑紫菜丝状体总RNA提取方法比较

目的:为了获得质量较高的条斑紫菜丝状体总RNA,对几种常用提取方法进行研究。方法:以条斑紫菜自由丝状体为材料,比较了用异硫氰酸胍法、CTAB法、SDS/酚法、TRIzol法、RNAplant法提取的RNA的质量和纯度。结果:异硫氰酸胍法提取RNA的成本低,但纯度不高;CTAB法产率较小,且不能完全去除多糖或蛋白质;SDS/酚法未能获得完整的RNA;TRIzol法未能见到5SrRNA条带,且带有杂带;而RNAplant法提取RNA的质量好、纯度高、提取效率高,其D260nm/D280nm值为1.836,经逆转录得到的双链cDNA扩增产物长度在200bp以上。结论:实验结果表明RNAplant法更适于条斑紫菜丝状体总RNA的提取。     

The role of structural elements in dimerization of RNA fragments in avian retroviruses

The slipped loop structure, earlier identified as an unusual DNA structure, was found to be a possible element of the RNA folding. In order to experimentally test this suggestion, model oligoribonucleotides capable of forming the SLS were synthesized. Treatment of the oligoribonucleotides with nuclease S1 and RNases specific for single- and double-stranded RNA demonstrated the steric possibility of SLS formation. To determine the possible functional role of SLS-RNA, various naturally occurring RNAs were screened in silico. Among the most interesting findings were dimerization initiation sites of avian retroviral genomic RNAs. Analysis of RNA from 31 viruses showed that formation of the intermolecular SLS during RNA dimerization is theoretically possible, competing with the formation of an alternative hairpin structure. Identification of the secondary structure of selected RNA dimers employing nuclease digestion techniques as well as covariance analysis of the retroviral RNA dimerization initiation site sequences were used to show that the alternative conformation (loop-loop interaction of two hairpins) is the most preferred. Alternative structures and conformational transitions in RNA dimerization mechanisms in avian retroviruses are discussed.     

Degradation of pre-messenger RNA in bovine thyroid slices; effect of thyrotropin

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     

Alterations of protein synthesis in arbovirus-infected L cells

Lust, George (Fort Detrick, Frederick, Md.). Alterations of protein synthesis in arbovirus-infected L cells. J. Bacteriol. 91:1612-1617. 1966.-Cellular protein synthesis and ribonucleic acid (RNA) synthesis in mouse L cells were markedly depressed 1 hr after infection with Venezuelan equine encephalomyelitis virus. Host RNA and protein synthesis were inhibited more rapidly by the virus infection than by actinomycin D. In cells infected 4 hr, a cytoplasmic RNA polymerase was demonstrated which was absent in uninfected cells. At this time, deoxyribonucleic acid-directed RNA synthesis catalyzed by the nuclear RNA polymerase was inhibited in vitro in enzyme preparations from nuclei of virus-infected cells. For optimal activity, the cytoplasmic RNA polymerase required the four nucleoside triphosphates, Mg(++), and RNA. The enzyme was insensitive to actinomycin D and deoxyribonuclease, indicating that it catalyzed RNA-directed RNA synthesis. Attempts to purify the induced polymerase further were unsuccessful. Fresh preparations had to be used because the enzymatic activity was unstable.     

Protein and RNA synthesis in the skeletal muscle of hereditary dystrophic mouse.

Protein and RNA contents in muscle of normal and hereditary dystrophic mice C57BL/6J-dy/dy were reexamined on the basis of DNA. It was observed that protein and RNA contents in dystrophic muscle decreased at the early stage of the disease, in disagreement with the reported results on a wet weight basis, in which RNA content in dystrophic muscle had been found to increase. Rates of protein and RNA systhesis in the early stage of the disease were also determined with a concomitant check of the specific activities of free amino acids and free nucleotides. The rates of both protein and RNA synthesis (i.e., specific activities of protein and RNA) were higher in the dystrophic muscle, but when they were expressed on a DNA basis, the total protein synthesis per cell was the same as that of normal muscle and the total RNA synthesis per cell showed a smaller increase in dystrophic muscle. These apparent increases of protein and RNA synthesis were discussed in connection with the decreased protein and RNA contents in the cells of dystrophic muscle. The synthesized RNAs seemed to contain mRNA on the basis of sedimentation character and Millipore filter binding ability. However, no particular RNA was mainly synthesized in dystrophic muscle.     

Reinitiation of synthesis of small cytoplasmic RNA species K and L in isolated HeLa cell nuclei in vitro.

Isolated HeLa cell nuclei were used to synthesize low molecular weight RNA species in-vitro. The labelled RNA released from the nuclei during the incubation mainly consists of 5S RNA, pre-tRNA and small cytoplasmic RNA species K and L. All these low molecular weight RNA species are synthesized by RNA polymerase C (or III). The polyanion heparin was applied to study the reinitiation of these RNA molecules in-vitro. A comparison of the kinetics of RNA synthesis in the absence and in the presence of this inhibitor demonstrates a highly efficient in-vitro reinitiation of scRNA species K and L as well as 5S and pre-tRNA by RNA polymerase C. These results indicate a general competence of this enzyme to catalyze the de-novo formation of specific gene products in-vitro.     

Effect of growth rate on the amounts of ribosomal and transfer ribonucleic acids in yeast.

The steady-state growth rate of Saccharomyces cerevisiae was varied by growing the cells in different media. The total amount of ribonucleic acid (RNA) per cell was found to decrease as a nonlinear function of decreasing growh rate. The RNA from cells growing in different media was analyzed by polyacrylamide gel electrophoresis. Although the amounts of both ribosomal RNA and transfer RNA decreased with decreasing growth rate, the ratio of ribosomal to transfer RNA was not constant. As the growth rate was reduced the ribosomal RNA fraction decreased slightly, whereas the transfer RNA fraction increased slightly. Thus the levels of ribosomal and transfer RNA were regulated to similar yet different extents. The levels of the different ribosomal RNA species were more closely coordinated. At all growth rates the ribosomal RNAs (including 5S RNA) were present in equimolar amounts. The rate of protein synthesis in yeast cells also decreased with decreasing growth rate. The low rates of protein synthesis did not appear to be due to limiting numbers of ribosomes or transfer RNA molecules.     

Frequent homologous recombination events between molecules of one RNA component in a multipartite RNA virus

Brome mosaic bromovirus (BMV), a tripartite plus-sense RNA virus, has been used as a model system to study homologous RNA recombination among molecules of the same RNA component. Pairs of BMV RNA3 variants carrying marker mutations at different locations were coinoculated on a local lesion host, and the progeny RNA3 in a large number of lesions was analyzed. The majority of doubly infected lesions accumulated the RNA3 recombinants. The distribution of the recombinant types was relatively even, indicating that both RNA3 counterparts could serve as donor or as acceptor molecules. The frequency of crossovers between one pair of RNA3 variants, which possessed closely located markers, was similar to that of another pair of RNA3 variants with more distant markers, suggesting the existence of an internal recombination hot spot. The majority of crossovers were precise, but some recombinants had minor sequence modifications, possibly marking the sites of imprecise homologous crossovers. Our results suggest discontinuous RNA replication, with the replicase changing among the homologous RNA templates and generating RNA diversity. This approach can be easily extended to other RNA viruses for identification of homologous recombination hot spots.