Poliovirus RNA-Dependent RNA Polymerase Synthesizes Full-Length Copies of Poliovirion RNA, Cellular mRNA, and Several Plant Virus RNAs In Vitro

The poliovirus RNA-dependent RNA polymerase was active on synthetic homopolymeric RNA templates as well as on every natural RNA tested. The polymerase copied polyadenylate. oligouridylate [oligo(U)], polycytidylate . oligoinosinate, and polyinosinate. oligocytidylate templates to about the same extent. The observed activity on polyuridylate. oligoadenylate was about fourfold less. Full-length copies of both poliovirion RNA and a wide variety of other polyadenylated RNAs were synthesized by the polymerase in the presence of oligo(U). Polymerase elongation rates on poliovirion RNA and a heterologous RNA (squash mosaic virus RNA) were about the same. Changes in the Mg(2+) concentration affected the elongation rates on both RNAs to the same extent. With two non-polyadenylated RNAs (tobacco mosaic virus RNA and brome mosaic virus RNA3), the results were different. The purified polymerase synthesized a subgenomic-sized product RNA on brome mosaic virus RNA3 in the presence of oligo(U). This product RNA appeared to initiate on oligo(U) hybridized to an internal oligoadenylate sequence in brome mosaic virus RNA3. No oligo(U)-primed product was synthesized on tobacco mosaic virus RNA. When partially purified polymerase was used in place of the completely purified enzyme, some oligo(U)-independent activity was observed on the brome mosaic virus and tobacco mosaic virus RNAs. The size of the product RNA from these reactions suggested that at least some of the product RNA was full-sized and covalently linked to the template RNA. Thus, the polymerase was found to copy many different types of RNA and to make full-length copies of the RNAs tested.     

The separation of 9 S RNA from avian immature red blood cells into f2c-histone mRNA and globin mRNA.

9 S RNA from avian immature red blood cells was isolated from polysome-released ribonucleoprotein particles by sucrose-gradient techniques. Translation of this RNA in an Ehrlich ascites cell-free system and product analysis revealed that globin mRNA was contaminated by f2c-histone mRNA. When 9 S RNA was applied to oligo(dT)-cellulose columns a partial separation could be achieved. Poly (A)-containing globin mRNA did not contain f2c-histon mRNA, whereas the RNA which was not absorbed to oligo(dT)-cellulose contained all the f2c-histone mRNA besides substantial amounts of globin mRNA.     

Sequence content of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells

Oligo(uridylic acid)-containing [oligo(U+)] RNA was isolated from poly(adenylic acid)-containing [poly(A+)] mRNA from HeLa cells by using either formaldehyde pretreatment or poly(A) removal, both of which resulted in increased accessibility of oligo(U)-rich sequences to a poly(A)-agarose affinity column. In this report, we compared the sequence content of oligo(U+) RNA with that of molecules lacking oligo(U) [oligo(U-) RNA] by their relative hybridization to cDNA reverse-transcribed from poly(A+) mRNA and by comparison of their in vitro translation products synthesized in a rabbit reticulocyte lysate. Formaldehyde-modified poly(A+) RNA, treated to remove the formol adjuncts, was inactive as a template for in vitro protein synthesis; consequently, only depolyadenylated RNA, which retains its translatability, could be used in the translation studies. The hybridization kinetic experiments revealed that oligo(U+) RNA contained most of the sequence information present in oligo(U-) RNA but at a reduced level (ca. 25%), the majority of the oligo(U+) RNA sequences being poorly represented in the cDNA. This result was supported by one- and two-dimensional gel analysis of their in vitro translation products which showed that oligo(U+) RNA, although less effective as a template for translation than oligo(U-) RNA, coded for proteins, the most abundant of which were encoded by rare messages not highly represented in oligo(U-) RNA or the total poly(A+) RNA. Although some minor products were synthesized by both oligo(U+) and oligo(U-) RNA, at least 33 proteins were unique to or highly enriched in the pattern of products directed by oligo(U+) RNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Translational properties of a non-polyadenylated oligo(U)-containing RNA fraction from wheat embryos

A non-polyadenylated oligo(U)-containing RNA (poly(A)- . oligo(U)+ RNA) fraction was isolated from wheat embryo cytoplasm and its properties were compared with those of polyadenylated RNA (poly(A)+ RNA) from the same source. Both RNA preparations were highly heterogeneous and effectively stimulated [14C]leucine incorporation in a wheat germ cell-free translation system. Electrophoretic patterns of the translation products appearing in the non-polyadenylated RNA- and polyadenylated RNA-supplemented translation assays, respectively, differed from each other. The non-polyadenylated RNA-specific translation products included, in particular, a series of high molecular weight polypeptides. It is concluded that a specific class of non-polyadenylated oligo(U)-containing mRNA species (other than histone mRNAs) occurs in the wheat embryo cells.     

A method for isolation of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells

When cytoplasmic polyadenylated ribonucleic acid [poly(A+)RNA] from HeLa cells was treated with ribonuclease H (RNase H) and oligodeoxythymidylate [oligo(dT)] to remove its 3'-poly(A) tail, an increased binding to poly(A)-agarose was observed. The bound material, which comprised 4-6% of the initial RNA, contained 65-80% of the oligo(uridylic acid) [oligo(U)] sequences generated by RNase T1 digestion. Oligo(U) isolated from the bound fraction was shown to be 83% U and to have a U/G ratio of 33. In contrast, oligo(U) from the unbound material was 77% U and had a U/G ratio of 13, suggesting that it is shorter and less U rich than the oligo(U) in the bound fraction. On sucrose gradients, oligo(U+)RNA consistently sedimented with a larger s value than oligo(U-) RNA. The oligo(U) content of oligo(U+) RNA suggests one oligo(U) tract of 33 nucleotides per RNA molecule of 2000-3000 residues.     

DNA Polymerase of Murine Sarcoma-Leukemia Virus: Lack of Detectable RNase H and Low Activity With Viral RNA and Natural DNA Templates

Kirsten murine sarcoma-leukemia virus (Ki-MSV[MLV]) was found to contain less RNase H per unit of viral DNA polymerase than avian Rous sarcoma virus (RSV). Upon purification by chromatography on Sephadex G-200 and subsequent glycerol gradient sedimentation the avian DNA polymerase was obtained in association with a constant amount of RNase H. By contrast, equally purified DNA polymerase of Ki-MSV(MLV) and Moloney [Mo-MSV(MLV)] lacked detectable RNase H if assayed with two homopolymer and phage fd DNA-RNA hybrids as substrates. On the basis of picomoles of nucleotides turned over, the ratio of RNase H to purified avian DNA polymerase was 1:20 and that of RNase H to purified murine DNA polymerase ranged between <1:2,800 and 5,000. Based on the same activity with poly (A).oligo(dT) the activity of the murine DNA polymerase was 6 to 60 times lower than that of the avian enzyme with denatured salmon DNA template or with avian or murine viral RNA templates assayed under various conditions (native, heat-dissociated, with or without oligo(dT) and oligo(dC) and at different template enzyme ratios). The template activities of Ki-MSV(MLV) RNA and RSV RNA were enhanced uniformly by oligo(dT) but oligo(dC) was much less efficient in enhancing the activity of MSV(MLV) RNA than that of RSV RNA. It was concluded that the purified DNA polymerase of Ki-MSV(MLV) differs from that of Rous sarcoma virus in its lack of detectable RNase H and in its low capacity to transcribe viral RNA and denatured salmon DNA. Some aspects of these results are discussed.     

Quantitation of labeled globin messenger RNA by hybridization with excess complementary DNA covalently bound to cellulose.

A method is described to quantitate labeled globin mRNA by hybridization with excess cDNA which was enzymatically polymerized on oligo(dT)-cellulose. In a large excess of cDNA-cellulose the rate of RNA hybridization was dependent on DNA concentration and not on RNA concentration. Nonhybridized RNA can be digested by RNase and washed from the cDNA which is covalently bound to cellulose. This enables the detection of labeled globin mRNA even when present in a porportion as low as 0.02-0.03% of the total RNA.     

A micromethod for measuring the molar concentration of polyadenylated RNA in the presence of ribosomal RNA

A method has been developed for measuring the molar concentration of RNA and the mole fraction of polyadenylated RNA. Using known mixtures of globin mRNA and rRNA composed of 20 to 85% rRNA, the molar concentration of globin mRNA, a polyadenylated species, was determined in 45 min, with the consumption of less than 100 ng of total RNA. The technique is particularly well suited for determining the molar concentration of poly(A)+ RNA after chromatographic enrichment in columns of oligo(dT)-cellulose or poly(U)-Sepharose. The method makes possible the adoption of a molar standard.     

有限延伸法检测端粒酶活性

人端粒酶是一种核蛋白体,通过其内含的RNA模板与端粒末端配对把重复端粒片段添加在端粒3＇末端|因此,端粒酶活性与细胞凋亡、衰老、永生化有密切关系,是癌症临床预测诊断的一个生物标签．现有的端粒酶活性检测方法,存在灵敏度低和不易定量等问题．本研究采用错配有限延伸法检测端粒酶活性：在人端粒酶延伸人工合成的游离端粒酶底物时,只加入dATP和dGTP,端粒酶只能把底物延伸4个脱氧核糖核苷酸AGGG．然后加入dNTP,让端粒酶延伸的产物和一条长的引物配对从而延伸出PCR模板|再加入引物进行热启动PCR．PCR后进行非变性PAGE (polyacrylamide gel electrophoresis),得到希望的唯一1条目标带．同时,用不同的端粒酶浓度梯度进行优化,发现有限延伸法检测端粒酶活性的下限达到250个HeLa细胞．     

Hybridization of Oligo(dT) to RNA on nitrocellulose

Quantitation of mRNA immobilized on nitrocellulose filters is an essential aspect of some studies in molecular biology. Hybridization of oligo(dT)₁₈ to the poly(A) tails of mRNA can be used to measure filter-bound mRNA and thus provides a basis for comparing abundance of specific mRNAs. Hybridization rate of ³²P-labeled oligo(dT)₁₈ in 0.75 M NaCl, 75 mM sodium citrate, pH 7 (5 × SSC) to immobilized RNA was maximal at 25°C. Filters were fully hybridized under these conditions within 1 hr when the oligo(dT)₁₈ concentration was 10 pmol/ml or higher. Salt dependence of the dissociation temperature (T_d) of oligo(dT)₁₈:RNA duplex on filters was described by the equation T_d = 42 − 20log₁₀[molar Na⁺] (°C). With stringent washing of the duplex (four 5-min washes in 2 × SSC at room temperature), oligo(dT)₁₈ gave no signal with plasmid DNA, rRNA, or tRNA. We have found that olig(dT)₁₈ can be used to normalize signal strengths rapidly and conveniently from total or oligo(dT)-selected eukaryotic RNA.     

Selection of oligonucleotide probes for protein coding sequences

MOTIVATION: Large arrays of oligonucleotide probes have become popular tools for analyzing RNA expression. However to date most oligo collections contain poorly validated sequences or are biased toward untranslated regions (UTRs). Here we present a strategy for picking oligos for microarrays that focus on a design universe consisting exclusively of protein coding regions. We describe the constraints in oligo design that are imposed by this strategy, as well as a software tool that allows the strategy to be applied broadly. RESULT: In this work we sequentially apply a variety of simple filters to candidate sequences for oligo probes. The primary filter is a rejection of probes that contain contiguous identity with any other sequence in the sample universe that exceeds a pre-established threshold length. We find that rejection of oligos that contain 15 bases of perfect match with other sequences in the design universe is a feasible strategy for oligo selection for probe arrays designed to interrogate mammalian RNA populations. Filters to remove sequences with low complexity and predicted poor probe accessibility narrow the candidate probe space only slightly. Rejection based on global sequence alignment is performed as a secondary, rather than primary, test, leading to an algorithm that is computationally efficient. Splice isoforms pose unique challenges and we find that isoform prevalence will for the most part have to be determined by analysis of the patterns of hybridization of partially redundant oligonucleotides. AVAILABILITY: The oligo design program OligoPicker and its source code are freely available at our website.     

Inhibition of reverse transcriptase activity of RNA-tumor viruses by fagaronine+.

Fagaronine, a benzophenanthridine alkaloid from roots of $\text{Fagara zanthoxyloides}$ (Rutaceae), has been reported to possess anti-leukemic activity. It inhibited RNA-directed DNA polymerase activity from avian myeloblastosis virus, Rauscher leukemia virus and simian sarcoma virus. With poly rA·oligo dT, the alkaloid concentration for 50% inhibition of the enzyme activity from these viruses was in the range of 6–12 μg (15 – 31 nmoles) per ml of reaction mixture. The enzyme reaction was also inhibited with activated DNA and 70S RNA as templates; however, with poly rC·oligo dG no inhibition of enzyme activity was obtained. These results suggest that fagaronine inhibits enzyme activity by interaction with the A:T templateprimer.     

Oligomeric sequences in the cytoplasmic RNA of sea urchin embryos

Oligomeric stretches of adenylate and uridylate and polymeric segments of adenylate have been shown to exist in sea urchin embryo hnRNA. It is demonstrated here that at least some oligo(U)-enriched sequences are conserved in sea urchin cytoplasmic RNA, whereas apparently few, if any, oligo(A) sequences are so conserved.     

A new class of antivirals: antisense oligonucleotides combined with a hydrophobic substituent effectively inhibit influenza virus reproduction and synthesis of virus-specific proteins in MDCK cells.

To enhance the penetration of oligonucleotide ('oligo') into cells, the oligo was combined with the hydrophobic undecyl residue. Using the 'DNA-synthesator', we synthesized oligo, complementary to the loop-forming site of the RNA, encoding polymerase 3 of the influenza virus (type A), and combined it with the undecyl residue added to the 5' terminal phosphate group. It was found that the modified oligo effectively suppresses the influenza A/PR8/34 (H1N1) virus reproduction and inhibits the synthesis of virus-specific proteins in MDCK cells. Under the same conditions, the non-modified antisense oligo and modified nonsense oligo did not affect the virus development.     

Human PABP binds AU-rich RNA via RNA-binding domains 3 and 4.

Poly(A) binding protein (PABP) binds mRNA poly(A) tails and affects mRNA stability and translation. We show here that there is little free PABP in NIH3T3 cells, with the vast majority complexed with RNA. We found that PABP in NIH3T3 cytoplasmic lysates and recombinant human PABP can bind to AU-rich RNA with high affinity. Human PABP bound an AU-rich RNA with Kd in the nm range, which was only sixfold weaker than the affinity for oligo(A) RNA. Truncated PABP containing RNA recognition motif domains 3 and 4 retained binding to both AU-rich and oligo(A) RNA, whereas a truncated PABP containing RNA recognition motif domains 1 and 2 was highly selective for oligo(A) RNA. The inducible PABP, iPABP, was found to be even less discriminating than PABP in RNA binding, with affinities for AU-rich and oligo(A) RNAs differing by only twofold. These data suggest that iPABP and PABP may in some situations interact with other RNA regions in addition to the poly(A) tail.     

Purification of oligo dG-tailed Okayama-Berg linker DNA fragments by oligo dC-cellulose chromatography

A simple procedure for preparation of oligo dG-tailed DNA fragments is presented. The fragments are first purified by ultracentrifugation through sucrose gradients at low salt concentration. Appropriate gradient fractions are then adjusted to 1 M NaCl and immediately applied to a column of oligo dC-cellulose equilibrated in buffered 1 M NaCl at 4 degrees C. Fragments are eluted with water at room temperature. Passage through the column achieves, in one step, the concentration and purification of oligo dG-tailed DNA fragments free from sucrose.