The measurement of plant polyadenylic acid by hybridisation with radioactive polyuridylic acid

Summary Saturation hybridisation of polyadenylic acid with [³H]polyuridylic acid is described. Under conditions of [³H]poly(U) excess, poly(A) is detected in the RNA of a number of higher plants. The ribonuclease resistant hybrids melt sharply when subjected to thermal denaturation. Plant RNA which contains poly(A) sequences detected by [³H]poly(U) hybridisation is polydisperse in molecular weight. Data presented shows that the amount of poly(A) in plant RNA is variable. This technique is useful for the qualitative and quantitative detection of poly(A) sequences in higher plant RNA.Abbreviations A.R. Analar Reagent - Poly(A) Polyadenylic acid - Poly(U) Polyuridylic acid - Oligo(dT)-cellulose oligo(deoxythymidylate)-cellulose - Tm melting temperature - SSC standard saline citrate     

Poly(A) and synthesis of polyadenylated RNA in the preimplantation mouse embryo

Investigations were conducted to quantitate polyadenylic acid and estimate the synthesis of polyadenylated RNA in mouse embryos at several stages of preimplantation development. Poly(A) was assayed by molecular hybridization of total embryonic RNA with [³H]polyuridylic acid. The mean values of poly(A) in the ovulated oocytes and in the one-cell, two-cell, and blastocyst stages of the embryo were 1.9, 1.6, 0.68, and 3.8 pg, respectively. Synthesis of polyadenylated RNA was estimated by affinity chromatography of [³H]uridine-labeled embryo RNA on oligo(dT)-cellulose. The proportions of newly synthesized RNA bound by oligo(dT)-cellulose at the 2-cell, 8- to 16-cell, and blastocyst stages were 6.7, 3.5, and 3.3%, respectively. These results suggest that significant quantities of maternal mRNA are present during early development of the mouse, but that polyadenylation of RNA transcribed from the embryonic genome occurs as early as the two-cell stage.     

Extensive homology between poly(A)-containing mRNA and purified nominal poly(A)-lacking mRNA in mouse kidney

To investigate poly(A)-lacking mRNA in mouse kidney, we studied a fraction of renal mRNA that does not bind to oligo(dT)-cellulose but can be purified by benzoylated cellulose chromatography. Nominal poly(A)-lacking mRNA and poly(A)-containing mRNA have complete nucleotide sequence homology, suggesting that kidney does not contain mRNAs that are not represented in the polyadenylated RNA fraction. Translation products directed by nominal poly(A)-lacking mRNA and poly(A)-containing mRNA are qualitatively and quantitatively similar in one-dimensional polyacrylamide gels. [3H]cDNA transcribed from poly(A)-containing mRNA hybridizes with its template and with nominal poly(A)-lacking mRNA to the same extent (95%) and with the same kinetics; reaction of [3H]cDNA to nominal poly(A)-lacking mRNA with the two mRNA populations gives the same result. The extensive homology these two mRNA populations share is important to the interpretation of mRNA lifetime and to the analysis of authentic poly(A)-lacking mRNAs.     

Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H.

A model RNA template-primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP, and dATP, 3'-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3' end of the RNA template. With this model RNA template-primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) is directing the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) is acting as template for poly(dA) synthesis. Furthermore, selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anti-complementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primer for poly(dA) synthesis was investigated. By use of formamide--poly-acrylamide slab gel electrophoresis, it is shown that poly(dT) is not acting as both template and primer for poly(dA) synthesis since no poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2',3'-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [alpha-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only is RNase H responsible for the degradation of poly(rA) following formation of a poly(rA)-poly(dT) hybrid but also the poly(rA)fragments generated are serving as primers for initiation of synthesis of the second strand of the double-stranded DNA.     

Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus.

Two RNase H (RNA-DNA hybrid ribonucleotidohydrolase, EC 3.1.4.34) activities separable by Sephadex G-100 gel filtration were identified in lysates of Moloney murine sarcoma-leukemia virus (MSV). The larger enzyme, which we have called RNase H-I, represented about 10% of the RNase H activity in the virion. RNase H-I (i) copurified with RNA-directed DNA polymerase from the virus, (ii) had a sedimentation coefficient of 4.4S (corresponds to an apparent mol wt of 70,000), (iii) required Mn-2+ (2 mM optimum) for activity with a [3-h]poly(A)-poly(dT) substrate, (iv) eluted from phosphocellulose at 0.2 M KC1, and (v) degraded [3-H]poly(A)-poly(dT) and [3-H]poly(C)-poly(dG) at approximately equal rates. The smaller enzyme, designated RNase H-II, which represented the majority of the RNase H activity in the virus preparation, was shown to be different since it (i) had no detectable, associated DNA polymerase activity, (ii) had a sedmimentation coefficient of 2.6S (corresponds to an apparent mol wt of 30,000), (iii) preferred Mg-2+ (10 to 15 mM optimum) over Mn-2+ (5 to 10 mM optimum) 2.5-fold for the degradation of [3-H]poly(A)-poly(dT), and (iv) degraded [3-H]poly(A)-poly(dT) 6 and 60 times faster than [3-H]poly(C)-poly(dG) in the presence of Mn-2+ and Mg-2+, respectively. Moloney MSV DNA polymerase (RNase H-I), purified by Sephadex G-100 gel filtration followed by phosphocellulose, poly(A)-oligo(dT)-cellulose, and DEAE-cellulose chromatography, transcribed heteropolymeric regions of avian myeloblastosis virus 70S RNA at a rate comparable to avian myeloblastosis virus DNA polymerase purified by the same procedure.     

CHARACTERIZATION OF POLY(A) SEQUENCES IN BRAIN RNA

—Nuclear and polysomal brain RNA from the rabbit bind to Millipore filters and oligo(dT)-cellulose suggesting the presence of poly(A) sequences. The residual polynucleotide produced after RNase digestion of ³²P pulse-labelled brain RNA is 95% adenylic acid and 200-250 nucleotides in length. After longer isotope pulses the polysomal poly(A) sequence appears heterodisperse in size and shorter than the nuclear poly (A). Poly(A) sequences of brain RNA are located at the 3′-OH termini as determined by the periodate-[³H]NaBH₄ labelling technique. Cordycepin interferes with the processing of brain mRNA as it inhibits in vivo poly(A) synthesis by about 80% and decreases the appearance of rapidly labelled RNA in polysomes by about 45%. A small poly(A) molecule 10-30 nucleotides in length is present in rapidly labelled RNA. It appears to be less sensitive to cordycepin than the larger poly(A) and is not found in polysomal RNA.     

Binding properties of avian myeloblastosis virus DNA polymerases to nucleic acid affinity columns.

A new method for the analysis and purification of the RNA-directed DNA polymerase of RNA tumor viruses has been developed. This nucleic acid affinity chromatography system utilizes an immobilized oligo (dT) moiety annealed with poly (A). The alpha and alphabeta DNA polymerases of avain myeloblastosis virus bound effectively to poly (A) oligo (dT)-cellulose. Alpha DNA polymerase did not bind effectively to poly (A) oligo (dT)-cellulose, poly (A)-cellulose, or to cellulose. Alphabeta bound to oligo (dT)-cellulose and cellulose at the same extent (approximately 30%), indicating that this enzyme did not bind specifically to the oligo (DT) moiety only. However, alphabeta bound to poly (A)-cellulose two to three times better than to cellulose itself, showing that alphabeta could bind to poly (A) without a primer. Alphabeta DNA polymerase also bound to poly (C)-cellulose, whereas alpha did not. These data show that the alpha DNA polymerase is defective in binding to nucleic acids if the beta subunit is not present. Data is presented which demonstrates that the alphabeta DNA polymerase bound tighter to poly (A). oligo (DT)-cellulose and to calf thymus DNA-cellulose than the alpha DNA polymerase, suggesting that the beta subunit or, at least part of it is responsible for this tighter binding. In addition, alphabeta DNA polymerase is able to reversibly transcribe avian myeloblastosis virus 70S RNA approximately fivefold faster than alpha DNA polymerase in the presence of Mg2+ and equally efficient in the presence of Mn2+. alpha DNA polymerase transcribed 9S globin m RNA slightly better than alphabeta with either metal ion.     

Relative amounts of newly synthesized poly(A)+ and poly(A)- messenger RNA during development of Xenopus laevis

The relative amounts of newly synthesized poly(A)⁺ and poly(A)^? mRNA have been determined in developing embryos of the frog Xenopus laevis. Polysomal RNA was isolated and fractionated into poly(A)⁺ and poly(A)^? RNA fractions with oligo(dT)-cellulose. In normal embryos the newly synthesized polysomal poly(A)⁺ RNA has a heterodisperse size distribution as expected of mRNA. The labeled poly(A)^? RNA of polysomes is composed mainly of rRNA and 4S RNA. The amount of poly(A)^? mRNA in this fraction cannot be quantitated because it represents a very small proportion of the labeled poly(A)^? RNA. By using the anucleolate mutants of Xenopus which do not synthesize rRNA, it is possible to estimate the percentage of mRNA which contains poly(A) and lacks poly(A). All labeled polysomal RNA larger than 4S RNA which does not bind to oligo(dT)-cellulose in the anucleolate mutants is considered presumptive poly(A)^? mRNA. The results indicate that about 80% of the mRNA lacks a poly(A) segment long enough to bind to oligo(dT). The poly(A)⁺ and poly(A)^? mRNA populations have a similar size distribution with a modal molecular weight of about 7 × 10⁵. The poly(A) segment of poly(A)⁺ mRNA is about 125 nucleotides long. Analysis of the poly(A)^? mRNA fraction has shown that it lacks poly(A)₁₂₅.     

Characteristics of rabbit globin mRNA purification by oligo(dT) cellulose chromatography

We have purified rabbit globin mRNA using oligo(dT)-cellulose and sucrose gradient centrifugation. Both α- and β-globulin mRNA molecules behave heterogeneously with respect to their elution properties during chromatography on oligo(dT)-cellulose. Those fractions eluted at the lowest ionic strength are most active in directing cell-free globin biosynthesis. By making use of hybridization with synthetic [³H]DNA complementary to globin mRNA, we have shown that this technique can be used to quantitate the extent of mRNA purification. Thus, globin mRNA is approximately 90-fold purified from reticulocyte polysomal RNA and originally constituted slightly more than 1% of the polysomal RNA. Since more than 98% of the globin mRNA sequences are bound to oligo(dT)-cellulose, we suggest that most polysomal globin mRNAs contain a poly (A)-rich region and that this region is not of uniform length nor preponderately associated with either the α- or β-globin mRNAs. In addition, we observe that the 9S globin mRNA most resistant to dissociation from oligo (dT)-cellulose is most active in directing globin biosynthesis.     

Activation of ribosomal and messenger RNA synthesis in excised Jerusalem artichoke tuber slices

RNA synthesis was studied in Jerusalem artichoke (Helianthus tuberosus L.) tuber slices immediately following excision and during the early period of aging in water. Incorporation of [³H]adenosine into RNA was detected as early as 20 min after excision. Measurement of the specific activities of RNA (cpm/g) and of ATP showed that RNA synthesis proceeded at a constant rate for the first several hours of aging and then increased moderately. [³H]adenosine was incorporated into polysomes throughout the aging period examined. Sucrose gradient fractionation of EDTA-dissociated polysomes showed that during the first 2 h of aging most of this incorporation was not into ribosome subunits but into presumed mRNA. Autoradiographic analysis of [³H]adenosine labelled nuclei showed that this was caused, at least in part, by a delay in the onset of rRNA synthesis synthesized during this time chromatographed as poly(A)-RNA on oligo(dT)-cellulose, indicating that a large part of the mRNA was not polyadenylated.     

Minor groove binding of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various duplex and triplex polynucleotides

The binding site and the geometry of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) complexed with double helical poly(dA)·poly(dT) and poly(dG)·poly(dC), and with triple helical poly(dA)·[poly(dT)]₂ and poly(dC)·poly(dG)·poly(dC)⁺ were investigated by circular and linear dichroism (CD and LD). The appearance of monomeric positive CD at a low [porphyrin]/[DNA] ratio and bisignate CD at a high ratio of the CoTMPyP-poly(dA)·poly(dT) complex is almost identical with its triplex counterpart. Similarity in the CD spectra was also observed for the CoTMPyP-poly(dG)·poly(dC) and -poly(dC)·poly(dG)·poly(dC)⁺ complex. This observation indicates that both monomeric binding and stacking of CoTMPyP to these polynucleotides occur at the minor groove. However, different binding geometry of CoTMPyP, when bind to AT- and GC-rich polynucleotide, was observed by LD spectrum. The difference in the binding geometry may be attributed to the difference in the interaction between polynucleotides and CoTMPyP: in the GC polynucleotide case, amine group protrude into the minor groove while it is not present in the AT polynucleotide.     

Oligodeoxynucleotide base recognition by steroid hormone receptors

Oligodeoxynucleotides covalently linked to cellulose were used as probes of the DNA-binding domains of mouse steroid holoreceptors. With uterine cytosol estrogen receptor (E₂R) the relative binding order, in prior studies, was oligo(dG) > oligo(dT) ≧ oligo(dC) > > oligo(dA) > oligo(dI). The binding reactions were salt-sensitive with an optimal KCl concentration of 0.1–0.2 M. There was no enhancement of binding by activation, either temperature- or salt-induced. In the present study, using the oligomer ligands at a lower concentration, oligo(dT) binding was greater than that to oligo(dC). Quantitative differences in oligodeoxynucleotide binding were elicited by a number of inhibitors. These differences are again seen by exposure of E₂R to chaotropic salts such as SCN^?, ClO₄^? and NO₃^? as well as to putative modifiers of receptor amino acids, ie, iodoacetamide, 1,2 cyclohexanedione, and Rose Bengal. These results, and the quantitative differences following heat and purification, led to a designation of two types of subsites within the DNA-binding domain of uterine E₂R. These are stable G sites, which interact with oligo(dG); and labile N sites, which bind to oligo(dT), oligo(dC) and oligo(dA). Stimulation of binding to N sites and stabilization of the holoreceptor was effected by histones H2A and H2B. However, the differential response to incubation at 37°C was not altered by addition of H2B. Treatment of uterine E₂R by limited proteolysis also eliminated the stimulatory response to H2B. The above data, as well as prior studies, indicate that steroid holoreceptors can discriminate between the structural features of deoxynucleotide bases and this recognition process can be modulated by accessory proteins.     

Binding geometries of triple helix selective benzopyrido [4,3-b]indole ligands complexed with double- and triple-helical polynucleotides

The binding modes of three benzopyrido [4,3-b]indole derivatives (and one benzo[-f]pyrido [4-3b] quinoxaline derivative) with respect to double helical poly(dA) · poly(dT) and poly[d(A-T)]₂ and triple-helical poly(dA) · 2poly(dT) have been investigated using linear dichroism (LD) and CD: (I) 3-methoxy-11-amino-BePI where BePI = (7H-8-methyl-benzo[e]pyrido [4,3-b]indole), (II) 3-methoxy-11-[(3′-amino) propylamino]-BePI, (III) 3-methoxy-7-[(3′-diethylamino)propylamino] BgPI where BgPI = (benzo[g]pyrido[4,3-b]indole), and (IV) 3-methoxy-11-[(3′-amino)propylamino] B f P Q where B f P Q = {benzo[-f]pyrido[4-3b]quinoxaline}. The magnitudes of the reduced LD of the electronic transitions of the polynucleotide bases and of the bound ligands are generally very similar, suggesting an orientation of the plane of the ligands' fused-ring systems preferentially perpendicular to the helix axis. The LD results suggest that all of the ligands are intercalated for all three polynucleotides. The induced CD spectrum of the BePI chromophore in the (II-BePI)-poly[d(A-T)]₂ complex is almost a mirror image of that for the (I-BePI)-poly(dA) · poly(dT) and (I-BePI)-poly(dA) · 2poly(dT) complexes, suggesting an antisymmetric orientation of the BePI moiety upon intercalation in poly[d(A-T)]₂ compared to the other polynucleotides. The induced CD of I-BePI bound to poly(dA) · 2poly(dT) suggests a geometry that is intermediate between that of its other two complexes. The concluded intercalative binding as well as the conformational variations between the different BePI complexes are of interest in relation to the fact that BePI derivatives are triplex stabilizers. © 1997 John Wiley & Sons, Inc. Biopoly 42: 101–111, 1997     

Effective solvent systems for separation and an improved detection method for amino acids by paper chromatoghraphy

Binding of poly(A)-containing RNP to oligo(dT)-cellulose has been investigated as a function of mono- and divalent ion concentration. 80–90% binding was obtained either in high (500 mM) or in moderate NaCl concentrations in the presence of 5 mM MgCl₂. At 40 mM NaCl and 5 mM MgCl₂ poly(A)⁺-RNP exhibit approximately t he same stability as poly(A)⁺-RNA in binding to oligo(dT)-cellulose with a melting temperature of 41 and 45°C, respectively, indicating that the protein moeity has no effect on the ribonucleoprotein binding in these conditions. Differences were observed int he elution of poly(A)⁺-RNA and poly(A)⁺-RNP from oligo(dT)-cellulose in buffer without salts. Poly(A)⁺-RNA was completely removed at 4°C whereas the melting temperature of poly(A)⁺-RNP was only decreased to 34°C. The isolation of poly(A)⁺-RNP by thermal elution from oligo(dT)-cellulose is described.     

Characterization of poly(a)-protein complexes isolated from free and membrane-bound polyribosomes of Ehrlich ascites tumor cells

Proteins present in messenger ribonucleoprotein particles were labeled with [³⁵S]-methionine in Ehrlich ascites tumor cells in which synthesis of new ribosomes was inhibited. Poly(A)-protein complexes were isolated from free and membrane-bound polyribosomes by sucrose gradient centrifugation and affinity chromatography on oligo(dT)-cellulose. Both classes of Poly(A)-protein particles contain a poly(A) chain of about 70 adenyl residues and a protein with a molecular weight of 76000 attached to it.     

Ribonucleoprotein organization of polyadenylate sequences in HeLa cell heterogeneous nuclear RNA.

Ribonucleoprotein particles containing heterogeneous nuclear RNA (Pederson, 1974) were isolated from HeLa cells and digested with ribonucleases A and T₁ at high ionic strength. The nuclease-resistant material, comprising 9.4% of the initial acid-insoluble [³H]adenosine radioactivity, was further fractionated by poly(U)-Sepharose chromatography. The bound fraction eluted from the column with 50% formamide and banded in cesium sulfate gradients (without aldehyde fixation) at a buoyant density characteristic of ribonucleoprotein (1.45 g/cm³). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this material revealed two Coomassie blue-stained bands. The major polypeptide had a molecular weight of 74,000 a less prominent band had a molecular weight of 86,000. The RNA components contained 74.4 mol % AMP and 17.7 mol % UMP. Polyacrylamide gel electrophoresis of the RNA, labeled with [³H]adenosine, demonstrated the presence of molecules 150 to 200 nucleotides in length (poly(A)), as well as molecules 20 to 30 nucleotides long (oligo(A)). Both poly(A) and oligo(A) sequences have previously been identified in HeLa heterogeneous nuclear RNA. These data demonstrate that both the poly (A) and oligo(A) sequences in HeLa heterogeneous nuclear RNA exist in vivo tightly complexed with specific proteins.     

Methylated messenger RNA in mouse kidney.

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.     

Localization of host poly(A)+ mRNA in the ribosome profile of mengovirus-infected Ehrlich ascites tumor cells

The distribution of poly(A)⁺ mRNA among polysomes, monosomes, and ribosome-free supernatant fractions after mengovirus infection of Ehrlich ascites tumor (EAT) cells was investigated employing sucrose gradient centrifugation of their corresponding postnuclear supernatants. Poly(A)⁺ mRNA was isolated from sucrose gradient fractions and quantitated in a cell-free protein synthesizing system from uninfected EAT cells. It was also localized by annealing [³H]-poly(U) to the poly(A)-tracts of mRNA present in the sucrose gradient fractions. Both experiments revealed a gradual shift of host poly(A)⁺ mRNA from large to small polysomes and monosomes, respectively, with the time postinfection. The greatest part of host template RNA appears to remain ribosome-bound and only a fraction seems to be detached from the ribosomes in the course of mengovirus infection. At the end of the infectious cycle, 8 h postinfection, approximately 70% of the poly(A)⁺ mRNA detected in uninfected cells is still biologically active, but not translated in vivo, in agreement with data from the [³H] poly(U) hybridization experiment.     

Ribonuclease H activities associated with Herpes simplex virus

Ribonuclease H activities were detected in purified particles of herpes simplex virus type 1 and 2. The RNase H specifically degrades the ribo moiety of the synthetic homopolymet [³H]-poly rA·dT as well as the RNA part of the hybrid [³H]-RNA·DNA duplex. Single-stranded polymers were not degraded under the conditions used. p-Hydroxy-mercuribenzoate inhibits the RNase H activities which co-purify with HSV type 1 and 2.