The binding of Mg++ ions to polyadenylate, polyuridylate, and their complexes

The binding of Mg ⁺⁺ to polyadenylate (poly A), Polyuridylate(poly U), and their complexes, poly (A + U) and poly (A + 2U), was studied by means of a technique in which the dye eriochrome black T is used to measure the concentration of free Mg^?. The apparent binding constant K_X = [MgN]/[Mg⁺⁺][N], N = site for Mg⁺⁺ binding (the phosphate group of the nucleotide), was found to decrease rapidly as the extent of binding increased and, at low extents of binding, as the concentration of Na^? increased in poly A, poly (A + U), and poly (A + 2U), and somewhat less so in poly U. K_x is generally in the range 10⁴ > K_X > 10². The cause of these dependences is apparently, primarily, the displacement of Na⁺ by Mg⁺⁺ in poly U and poly (A + U) on the basis of the similarity of extents of displacement measured in this work and those measured potentiometrically. was calculated and was found to approach zero as the concentration of Na⁺ increased. In poly U, poly (A + U), and poly (A + 2U) at low ΔH′ v.H. > 0, about + 2 kcal/“mole.” In poly A, also at low salt, ΔH′ v.H. ≈ ?4 kcal/“mol” for the initial binding of Mg⁺⁺, and increases to +2 kcal/“mol” at saturation. This enthalpic variation probably accounts for the anticooperativity in the binding of Mg⁺⁺ not ascribable to the displacement of Na⁺⁺.     

Immunologic-mediated Protection of Trypanosoma congolense-Infected Mice by Polyribonucleotides

SYNOPSIS. When the synthetic polyribonucleotides polyinosinic acid-polycytidylic acid (poly I poly C) and polyadenylic acid-polyuridylic acid (poly A poly U) were tested against mice infected with varying numbers of Trypanosoma congolense the results varied with the method of passage of trypanosomes in mice. Thus, when 100 flagellates were passaged every 7th day and experiments were initiated with these trypanosomes from mice on the 7th day of their infection, the protective effects of poly I poly C and poly A poly U apparently varied independently of each other as assayed by the mean parasitemias and cumulative mortalities of infected mice. Poly I poly C-resistant and poly I poly C-susceptible variants (“R” and “S”, respectively) were isolated and maintained in mice by passage of 10⁶ trypanosomes every 4th day. Mice infected with these variants responded consistently to poly I poly C and poly A poly U injections in that mice infected with the “R” variant showed no response to either polyribonucleotide but those infected with the “S” variant consistently had a decrease in mean parasitemias and cumulative mortality when treated with poly I poly C, but not with poly A poly U. Using mice infected with the “S” variant, the protective effect of poly I poly C was dose-dependent and best protection was afforded when 1st injections of poly I poly C were given around the time of infection of the mice. The protective effects of the synthetic polyribonucleotides used in these experiments are probably due to their immunologic enhancing capacities and not to interferon. To support this, injections of Newcastle disease virus, a strong inducer of interferon in mice, did not protect against T. congolense in mice. It was not possible to determine whether serum from poly I poly C-treated mice had a greater neutralizing effect upon trypanosomes in vitro than serum from saline-treated mice since any effect of antibody was masked by a naturally occurring inhibitor in normal mouse serum which was reduced during infection. The protective effects of poly I poly C in T. congolense-infected mice were reversed by treatment with cyclophosphamide. This strong immunosuppressant, however, could not reverse the protective effects of poly I poly C against mice infected with Semliki Forest virus, strongly suggesting that the protective mechanisms stimulated by poly I poly C in these 2 infections were different. The response of mice infected with the “R” and “S” variants of T. congolense to synthetic polyribonucleotides is discussed in relation to antigenic variation of trypanosomes.     

The synthesis and turnover of virus-specific polyadenylated RNA in polyoma-infected cells.

Mouse embryo cells infected with the 3049 strain of polyoma virus contain several fold more virus-specific, polyadenylated RNA beginning between 4 and 8 hours after the onset of viral DNA synthesis than do cells infected with wild-type virus (lpS). Following infection with either virus strain, there is an identical small but significant enhancement of the level of total polyadenylated RNA measured by binding of ¹²⁵I-labeled RNA to poly(dT)cellulose. The polyadenylation of “early” virus-specific RNA is inhibited 85–90% by cordycepin resulting in an “early” RNA preparation which competes fully with polyadenylated “early” virus-specific RNA in the ternary complex assay. Utilizing the nonpolyadenylated “early” RNA, competition hybridization demonstrated that approximately 78% of the enlarged pool of “late” 3049 polyadenylated RNA and 72% of the “late” lpS pool consisted of sequences unique to the “late” period. No significant difference in the rate of decay of 3049 and lpS-specific, “late” polyadenylated RNA following actinomycin D block was found. Infection by either strain of polyoma virus did not alter the rate of decay of total polyadenylated RNA.     

Differential accumulation of two size classes of poly(A) associated with messenger RNA during oogenesis in Xenopus laevis

The RNA of full-grown oocytes of Xenopus laevis contains two distinct size classes of poly(A), designated poly(A)_S and poly(A)_L, which contain 15–30 (mean = 20) and 40–80 (mean = 61) A residues, respectively. Both poly(A)_L and poly(A)_S are associated with RNA which is heterogeneous in size. The two classes of poly(A)⁺ RNA can be separated by affinity chromatography: Only poly(A)_L⁺ RNA binds to oligo(dT)-cellulose under appropriate conditions, but up to 50% of the poly(A)_S⁺ RNA can be isolated from the void fraction by binding to poly(U)-Sepharose. Both classes of poly(A)⁺ RNA are active as messenger RNA in an in vitro system and yield identical patterns of in vitro protein products. Previtellogenic oocytes contain almost exclusively poly(A)_L, which accumulates up to vitellogenesis but remains almost constant in amount (molecules/oocyte) during vitellogenesis and in the full-grown oocyte. Poly(A)_S accumulates (molecules/oocyte) from early vitellogenesis up to the full-grown oocyte. The total number of poly(A)⁺ RNA molecules per oocyte increases throughout oogenesis from 2 × 10¹⁰/previtellogenic oocyte [80–90% poly(A)_L] to 20 × 10¹⁰/full-grown oocyte (25–40% poly(A)_L). It is argued that poly(A)_S is protected from degradation in the oocyte, thus stabilizing the “maternal” poly(A)⁺ mRNA.     

Methacrylate polymerization by AzoRNA: potential usefulness for chromosomal localization of genes

An azo pyrimidine nucleotide has been prepared and enzymatically attached to oligo(A) primers. The nucleotide's azo pyrimidine group has previously been shown to initiate polymerization of methacrylate esters designed to bind marker groups for visualization by microscopy. When attached to RNA molecules complementary to a chromosomal DNA segment, these nucleotides may allow localization of the DNA segment following in situ hybridization of the probe, methacrylate polymerization, and marker attachment. Since mRNA molecules of potential interest as probes bear a 3′-poly(A) tail, the modified nucleotides were added to oligo(A) primers as models. First, N⁴-ureidocytosine nucleotides were enzymatically added to ApApA, (Ap)₉A, or [5′-³²P]-(pA)₁₀, using the modified cytidine 5′-diphosphate and “primer-dependent” polynucleotide phosphorylase (M. luteus). In the case of the ApApA-primed reaction, the N⁴-ureidocytosine nucleotides in the product polynucleotide were converted into azo nucleotides by oxidation with N-bromosuccinimide. The other two primers were employed to study the time course of polynucleotide formation and to verify that primer was indeed being utilized by the enzyme. The suitability of the modified nucleotide for in situ hybridization studies was examined. Poly(N⁴-ureidocytidylic acid) was prepared from poly(C) and semicarbazide by the bisulfite-catalyzed transamination reaction. It was found that 95% of the N⁴-ureidocytosine nucleotides in this polynucleotide survive the elevated temperatures typically required for DNA:DNA denaturation and RNA:DNA annealing. When poly(N⁴-ureidocytidylic acid) was mixed with poly(I) in buffered aqueous salt solutions, no evidence for hybridization was found, so binding of the probe RNA to the denatured chromosomal DNA molecule via the modified nucleotides is not expected. Upon oxidation of poly(N⁴-ureidocytidylic acid) with N-bromosuccinimide, the azo nucleotides were formed, as judged by the appearance of a characteristic peak at approximately 350 nm in the uv-absorption spectrum of the yellow-orange product, azoRNA. The azo nucleotides in azoRNA exhibited the expected acid lability, which is known to be accompanied by 1-glyceryl methacrylate polymerization in the case of the simple azo pyrimidine. Because 1-glyceryl metharcylate bears substituent glycol groups for attaching heavy atoms or fluorescent markers, it is possible that probe RNA molecules bearing azo nucleotides may be useful for localizing low-multiplicity genes along eukaryotic chromosomes.     

Synthesis and conformational study of poly(N delta-carbobenzoxy, N delta-benzyl-L-ornithine) and poly(N delta-benzyl-L-ornithine)

Poly(N^δ-carbobenzoxy, N^δ-benzyl-L -ornithine) (PCBLO) was prepared by the standard NCA method. PCBLO was converted into poly(N^δ-benzyl-L -ornithine) (PBLO) through decarbobenzoxylation with hydrogen bromide. The monomer N^δ-benzyl-L -ornithine was synthesized by reacting L -ornithine with benzaldehyde, followed by hydrogenation. The conformation of the two polypeptides was studied by optical rotatory dispersion and circular dichroism. PCBLO forms a right-handed helix in helix-promoting solvents. In mixed solvents of chloroform and dichloroacetic acid (DCA) it undergoes a sharp helix–coil transition at 12% (v/v) DCA at 25°C, as compared with 36% for poly(N^δ-carbobenzoxy-L -ornithine) (PCLO). Like PCLO, the helix–coil transition is “inverse,” that is, high temperature favors the helical form. PBLO is soluble in water at pH below 7 and has a “coiled” conformation. In 88% (v/v) 1-propanol above pH (apparent) 9.6 it is completely helical. In 50% 1-propanol the transition pH (apparent) is about 7.4; this compares with a pH_tr of about 10 for poly-L -ornithine in the same solvent.     

A cloned unique gene of drosophila melanogaster contains a repetitive 3′ exon whose sequence is present at the 3′ ends of many different mRNAs

We have started to study a cloned genomic DNA fragment ～7 kb long (denoted as H55) from the 7B3-4 region in the X chromosome of Drosophila melanogaster. The major part of the fragment is a single-copy sequence. It directs the synthesis of mRNA that makes up ～0.1% of the cytoplasmic poly(A)⁺ RNA from Drosophila embryos. The H55 gene is split by an intervening sequence, yielding a large single-copy exon and a small repetitive 3′ exon represented by hundreds of copies in the genome. This repetitive sequence (“suffix”) is also present at the 3′ ends of ～2% of all cytoplasmic poly(A)⁺ RNA chains.     

A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis

Summary Nuclear poly(A)⁺ and polysomal poly(A)⁺ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)⁺ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)⁺ RNA hybridization reactions suggest the existence of polysomal poly(A)⁺ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)^– RNA it could be shown that most of the poly(A)⁺ containing RNA sequences transcribed into cDNA were also present within the poly(A)^– RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)^– polysomal RNA and 3–6 more abundant within the poly(A)^– nuclear RNA as compared to the poly(A)⁺ RNAs.     

TRANSCRIPTIONAL CONTROL OF PROTEIN SYNTHISIS IN THE DEVELOPING OVARIES OF BOMBYX MORI*

Amino acid incorporation was studied with cell-free extracts and ribosomes prepared from pupal ovaries at different ages of Bombyx mori. Poly(U)-directed ³H-phenylalanine incorporation attained a maximum rate at a certain stage of development, but soon dropped to a low level and was replaced by ³H-leucine incorporation, which was due to endogenous mRNA. The latter incorporation occurred at the stage when actual protein synthesis takes place in the ovaries. “Run-off” of the ribosomes which had a high endogenous activity resulted in an enhancement of the poly(U)-dependent activity. The results indicate that the protein synthesis in the ovary is mainly controlled at the level of mRNA. This was further supported by the fact that the relative amount of an ovarian poly(A)-containing “mRNA” fraction increased in parallel with the endogenous activity.     

Spatial distribution of mRNA during pre-fertilization ovule development in Capsella bursa-pastoris

Summary In situ hybridization of sections of the ovary and ovule of Capsella bursa-pastoris with ³H-polyuridylic acid [³H-poly(U)] showed the presence of polyadenylic acid-containing RNA [poly(A) + RNA] in the cells of the placenta and nucellus. During megasporogenesis there was a decrease in ³H-poly(U) binding activity of the nucellar cells concomitant with the appearance of poly(A) + RNA in the integuments. As the typical eight-nucleate embryo sac was formed, ³H-poly(U) binding was not apparent around the quartet of nuclei at the chalazal end, while it persisted at the micropylar end. Both the egg and synergids as well as the chalazal proliferating tissue showed high concentrations of poly(A) + RNA in their cytoplasm. The results suggest a role for transient localizations of poly(A) + RNA during female sporogenesis and gametogenesis in C. bursa-pastoris.     

5'-terminal structures of poly(A)+ cytoplasmic messenger RNA and of poly(A)+ and poly(A)- heterogeneous nuclear RNA of cells of the dipteran Drosophila melanogaster.

Structures at the 5′ terminus of poly (A)-containing cytoplasmic RNA and heterogeneous nuclear RNA containing and lacking poly(A) have been examined in RNA extracted from both normal and heat-shocked Drosophila cells. ³²P-labeled RNA was digested with ribonucleases T₂, T₁ and A and the products fractionated by a fingerprinting procedure which separates both unblocked 5′ phosphorylated termini and the blocked, methylated, “capped” termini, known to be present in the messenger RNA of most eukaryotes.Approximately 80% of the 5′-terminal structures recovered from digests of poly(A)-containing Drosophila mRNA are cap structures of the general form m⁷G^5′ppp^5′X(m)pY(m)pZp. With respect to the extent of ribose methylation and the base distribution, the 5′-terminal sequences of Drosophila capped mRNA appear to be intermediate between those of unicellular eukaryotes and those of mammals. Drosophila is the first organism known in which type 0 (no ribose methylations), type 1 (one ribose methylation), and type 2 (two ribose methylations) caps are all present. In contrast to mammalian cells, the caps of Drosophila never contain the doubly methylated nucleoside N⁶,2′-O-dimethyladenosine. Both purines and pyrimidines can be found as the penultimate nucleoside of Drosophila caps and there is a wide variety of X-Y base combinations. The relative frequencies of these different base combinations, and the extent of ribose methylation, vary with the duration of labeling. The large majority of poly(A)-containing cytoplasmic RNA molecules from heat-shocked Drosophila cells are also capped, but these caps are unusual in having almost exclusively purines as the penultimate X base.Greater than 75% of the 5′ termini of heterogeneous nuclear RNA (hnRNA) containing poly(A) and greater than 50% of the termini of hnRNA lacking poly (A) are also capped. Triphosphorylated nucleotides, common as the 5′ nucleotides of mammalian hnRNA, are rare in the poly(A)-containing hnRNA of Drosophila. The frequency of the various type 0 and type 1 cap sequences of cytoplasmic and nuclear poly (A)-containing RNA are almost identical. The caps of hnRNA lacking poly(A) are also quite similar to those of poly-adenylated hnRNA, but are somewhat lower in their content of penultimate pyrimidine nucleosides, suggesting that these two populations of molecules are not identical.     

Electrophoretic analysis of newly synthesized and stored maternal RNA during oogenesis ofCalliphora erythrocephala (Dipt.)

Summary Ovaries ofC. erythrocephala synthesize large amounts of poly(A)⁺ and poly(A)^– RNA during early and middle stages of oogenesis as shown by labelling with³H-uridine in vivo. After incubation for 1 h, a striking difference in the electrophoretic pattern of newly synthesized labelled poly(A)⁺ RNA and the poly(A)⁺ RNA present in sufficient amounts for optical density measurements (steady state poly(A)⁺ RNA) was observed. During early and mid-oogenesis, in the poly(A)^– RNA fraction, 4S predominantly mature rRNA, 5S RNA and tRNA were labelled. These fractions were no longer synthesized during late oogenesis, whereas poly(A)⁺ RNA was labelled continously During oogenesis stage specific differences in the size distribution of newly synthesized and steady state poly(A)⁺ RNA were not obvious. However, different sizes of labelled poly(A)⁺ RNA species were detected in 0–2h old preblastoderm embryos, after injection of³H-uridine into females either 3–4 days (stage 3–4 of oogenesis) or 24 h before oviposition (stage 5–6 of oogenesis). This difference in RNA synthesis was related to the presence of active nurse cell nuclei. The poly(A)⁺ RNA fraction represents about 2–3% of the total RNA in both ovaries and freshly laid eggs as judged by measurements of optical density and radioactivity bound to oligo(dT). The length of poly(A)-segments in ovarian poly(A)⁺ RNA varied from about 30 to 200 nucleotides.     

Nature of the ribosomal RNA transcribed from the X and Y chromosomes of Drosophila melanogaster

In Drosophila melanogaster there is one nucleolar organizer (NO) on each X and Y chromosome. Experiments were carried out to compare the ribosomal RNAs derived from the two nucleolar organizers. ³²PO₄-labelled ribosomal RNA was isolated from two strains of D. melanogaster, one containing only the X chromosome NO, the other containing only the Y chromosome NO. 28 S and 18 S RNA from the two strains were subjected to a variety of “fingerprinting” and sequencing procedures. Fingerprints of 28 S RNA were very different from those of 18 S RNA. Fingerprints of “X” and “Y” 28 S RNA were indistinguishable from each other, as also were fingerprints of “X” and “Y” 18 S RNA. In combined “T₁ plus pancreatic” RNAase fingerprints several distinctive products were characterized and quantitated. Identical products were obtained from X and Y RNA, and the molar yields of the products were indistinguishable. Together these findings imply that the rRNA sequences encoded by the X and Y NOs are closely similar and probably identical to each other.Two further findings were of interest in “T₁ plus pancreatic” RNAase fingerprints: (1) in 28 S (as well as in 18 S) fingerprints several distinctive products were recovered in approximately unimolar yields. This indicates that 28 S RNA does not consist of two identical half molecules, though it does consist of two non-identical half molecules together with a “5.8 S” fragment. (2) Several methylated components in Drosophila rRNA also occur in rRNA from HeLa cells and yeast. This suggests that certain features of rRNA structure involving methylated nucleotides may be highly conserved in eukaryotic evolution.     

Calcium protects DNase I from proteinase K: a new method for the removal of contaminating RNase from DNase I

DNase I and proteinase K are two enzymes commonly used in the purification of highly polymerized RNA. In the presence of EDTA DNase I is rapidly inactivated by proteinase K while in 10 mm Ca²⁺ DNase is totally immune to proteinase K inactivation even at protease concentrations of up to 1 mg/ml. RNase A, a common contaminant of “RNase-free” DNase was inactivated by proteinase K in the presence or absence of Ca²⁺. Treatment of DNase I with proteinase K in the presence of Ca²⁺ selectively removed RNase A activity as judged by rRNA and poly(A⁺ RNA ribosomal RNA degradation monitored by sucrose gradient centrifugation. These results suggest that (i) DNase A and proteinase K can be used together in the presence of Ca²⁺ to obtain better digestion of nucleoprotein complexes, and (ii) proteinase K treatment of Ca²⁺ DNase can be used to selectively remove contaminating RNase.