Biosynthesis of Deoxyribonucleotides in Ochromonas malhamensis

SYNOPSIS. Uniformly ¹⁴C-labeled pyrimidine ribonucleosides and orotic-6-¹⁴C acid were fed to growing cultures of Ochromonas malhamensis and the radioactivity appearing in RNA and DNA was determined. The carbon skeletons of uridine and cytidine were incorporated intact into all of the pyrimidine nucleotides from RNA and DNA. Incorporation of radioactivity into the purine nucleotides was negligible. The evidence supports the conclusion that deoxyribonucleotide biosynthesis in this organism proceeds via a pathway involving the direct reduction of the corresponding ribonucleosides or ribonucleotides. An important role for a trans-N-deoxyribosylase in deoxyribonucleotide biosynthesis here appears to be ruled out.     

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.     

Bindings of RNAse to denatured and native deoxyribonucleic acids

The binding of pancreatic ribonuclease-A by denatured DNA, native DNA, poly-dA, and poly-dT, has been studied by a gel filtration method. With denatured DNA at pH 7.5, ionic strength 0.053M, there is one binding site per 12 nucleotides and the equilibrium binding constant per site is 9.7 × 10⁴ l./mole. The binding constant increases by a factor of 8 as the pH is decreased from 8 to 7. The strength of the binding of denatured DNA increases with decreasing ionic strength. At pH 7.5, native DNA binds about ? as strongly as does denatured DNA. The binding affinity increases in the order poly-dA, denatured DNA, and poly-dT. These results support the view that the binding of denatured DNA involves both electrostatic interactions between the negatively charged polynucleotide and the positively charged protein, and an interaction of the protein with a pyrimidine residue of the denatured DNA, and thus that the binding is basically similar to that between RNAse and its substrate RNA.     

The size of poly(A)-containing RNAs in Drosophila melanogaster embryos

The size range of poly(A)-containing RNA from Drosophila melanogaster embryos has been estimated by hybridization with ³H-labeled poly(U) and subsequent fractionation on sucrose gradients. The median size of nuclear poly(A)-containing RNA is about 30 S (6000 nucleotides), and the median size of cytoplasmic poly(A)-containing RNA is about 17 S (1800 nucleotides). The relationship of these sizes to messenger RNA needed to code for protein and to the length of DNA contained in a chromomere is discussed.Research grant support was provided by NIH (6M35558; HD-00266) and NSF (GB-30600).     

Modification of 50S ribosomal subunits withN-bromosuccinimide

The 50S subunits ofEscherichia coli ribosomes were modified with the tryptophan reagentN-bromosuccinimide, and the sulfhydryl groups, the modification of which is accompanied by stimulation of polypeptide synthesis (López-Rivas, A. et al. (1978) Eur. J. Biochem. 92, 121), were regenerated by incubation with simple thiols. This treatment inactivates poly(U)-dependent polyphenylalanine synthesis, peptidyl transferase and elongation factor G-dependent GTPase. Incubation with proteins from untreated 70S ribosomes produces partial reactivation of polyphenylalanine synthesis and GTPase activity. Modification is accompanied by loss of 4–5 tryptophan residues per subunit.Abbreviation SucNBr N-bromosuccinimide     

Probing Conformational Isomerizations of Double-Stranded Poly(dA-dT) by a Substitution of Minor Amounts of the Thymine Methyls with Bulky Hydrophobic Isopropyl Groups

Abstract

We probed conformational polymorphism of a synthetic DNA poly(dA-dT) by introducing various small amounts of bulky spherical hydrophobic isopropyl groups into the polynucleotide primary structure. For this purpose, three mixed copolymers of poly(dA-dT, ip⁵dU) were synthesized in which 2.6 %, 8.6 % or 14.2 % of the polynucleotide pyrimidine bases had the isopropyl group in position 5. The isopropyls made the formation of both A-form and X-form incomplete, and this effect increased with the increasing isopropyl amount in the polynucleotide. However, the polynucleotide isomerization into the A-form was hindered by the isopropyls while the isomerization into the X-form was rather promoted. This observation indicates that, unlike the A-form, the X-form has the base pairs shifted towards the double helix major groove. Z-form was also promoted by the lowest concentration of the isopropyl groups while the most isopropylated poly(dA-dT) aggregated under the Z-form inducing conditions.     

Isolation of a replication-complex from eukaryotes

Treatment of the eukaryotic organism $\text{Tetrahymena pyriformis}$ with low concentrations of Ethidium Bromide causes accumulation of a protein-nucleic acid complex consisting of a DNA polymerase, a RNA polymerase, a deoxyribo-nuclease and a RNA linked DNA fragment. The length of the RNA is about 30 nucleotides, while the DNA part is around 200 nucleotides long. Degradations with ribonucleases and deoxyribonucleases strongly indicate that the RNA exists in a non-hybrid structure with a homogenous base composition and that the DNA is single-stranded. The complex is purified 1100 fold from whole cells and sodium dodecyl sulphate acrylamide gel electrophoresis gives 9 defined bands. The polynucleotide in the isolated complex accounts for only 10^?4 of the total cellular DNA.As the complex contains some of the enzymes essential for discontinous DNA replication, in addition to a RNA linked Okazaki fragment, it is concluded that a highly purified $\text{replication complex}$ has been isolated.     

Interaction of poly-L-lysine with nucleic acids. II. Poly(A+U), poly(A+2U), and rice dwarf virus RNA

The optical density–temperature profile of double-stranded poly(A + U), triple stranded poly(A + 2U), and double-stranded RNA from rice dwarf virus in solutions with and without poly-L -lysine has been examined. When poly-L -lysine is added, more than one melting temperature T_m is observed for poly(A + U) and poly(A + 2U). One of them is considered to correspond to the melting of the polynucleotide molecule free from poly-L -lysine, and another to the melting of a polynucleotide–poly-L -lysine complex. For rice dwarf virus RNA, the T_m assignable to the complex is not found to be lower than 99°C. In every case, however, the hyperchromicity observed at the T_m of the free poly-nucleotide molecule is lowered linearly as the amount of poly-L -lysine added to the solution increases. This fact is taken as indicating that there is a stoichiometric complex formed. The stoichiometric ratio lysine/nucleotide in each complex is determined by examining the relation between the amount of poly-L -lysine added to the solution and the percentage of hyperchromicity remaining at T_m of the free polynucleotide molecule. The ratio is found to be 2/3 for all of the three complexes. A discussion is given on the molecular conformations of four types of polynucleotide–polylysine complex hitherto found: (A) double-stranded DNA plus poly-L -lysine in which the lyslne/nucleotide ratio is 1, (B) three-stranded RNA [poly(A + 2U)] plus poly-L -lysine in which the ratio is 2/3, (C) double-stranded RNA [poly (A + U) or rice dwarf virus RNA] plus poly-L -lysine in which the ratio is 2/3, and (D) double-stranded RNA [poly(I + C)] plus poly-L -lysine in which the ratio is 1/2.     

Mechanism of DNA chain growth. XI. Structure of RNA-linked DNA fragments of Escherichia coli

The size of RNA attached to nascent DNA fragments of Escherichia coli with a chain length of 400 to 2000 nucleotides is estimated to be about 50 to 100 nucleotides from: (a) the density of the molecules of known sizes; (b) the decrease of the molecular size produced by hydrolysis with RNases or alkali; and (c) the size of RNA released by DNase treatment. Only a small decrease in molecular size is produced by RNase or alkali treatment, excluding the possibility that the RNA is located in the middle of the fragment or that ribonucleotide sequences are scattered in the molecule. The RNA is not located at the 3′ end of the molecule either, since the DNA is degraded by 3′ → 5′ exonuclease action of bacteriophage T4 DNA polymerase which has neither RNase nor DNA endonuclease activity. Positive evidence for the covalent attachment of the RNA to the 5′ end of the DNA is provided by the finding that one 5′-OH terminus of DNA is created from each RNA-linked DNA fragment by alkaline hydrolysis. The quantitative production of the 5′-OH group at the 5′ end of DNA is also found upon hydrolysis with pancreatic RNase, indicating that the 3′-terminal base of the RNA segment of the fragments is a pyrimidine. On the other hand, when the RNA-linked DNA fragments hydrolysed with alkali or pancreatic RNase are incubated with [γ-³²P]ATP and polynucleotide kinase and the DNA thus labelled is degraded to constituent 5′-mononucleotides, the ³²P is found only in dCMP. Therefore, C is the specific 5′-terminal base of the DNA segment of the RNA-linked DNA fragments, and the RNA-DNA junction has the structure … p(rPy)p(dC)p …     

Synthesis of a novel flourescent polyriboadenylic acid analog by polynucleotide phosphorylase

Poly(1,N⁶-etheno-2-aza-adenylic acid) [poly(2-aza-εA)] was synthesized from 1,N⁶-etheno-2-aza-adenosine 5′-diphosphate (2-aza-εADP) and Escherichia coli polynucleotide phosphorylase. The values K_m = 1.02 mM, V = 1.06 μmol hr^?1 enzyme unit^?1 were found for the polymerization reaction. In contrast to polyadenylic acid, this novel fluorescent polymer has a random structure in solution. The application of the 2-aza-εADP for localization of polynucleotide phosphorylase was also described.     

Free energy of imperfect nucleic acid helices. II. Small hairpin loops

Physical studies of enzymically synthesized oligonucleotides of defined sequence are used to evaluate quantitatively the stability of small RNA hairpin loops and helices. The series (Ap)₄G(pC) _N(pU)₄, N = 4, 5 or 6, exists as monomolecular hairpin helices when N ≥ 5, and as imperfect dimer helices when N ≤ 4. In this size range, hairpin loops become more favorable (less destabilizing thermodynamically) as they increase in size from 3 to 4 to 5 unbonded nucleotides. Very small hairpin loops are particularly destabilizing; molecules whose base sequence would imply a hairpin loop of three nucleotides will generally exist with a loop of five, including a broken terminal base pair.Thermodynamic parameters for base pair and loop formation are calculated by a method which makes unnecessary the use of measured enthalpies of polynucleotide melting. Literature data on oligonucleotide double helices yield estimates of the free energy contribution from each of the six types of stacking interactions between three possible neighboring base pairs. The advantage of this approach is that the properties of oligonucleotides are used in predicting the stability of small RNA helices, avoiding the long extrapolation from the properties of high polymers.We provide Tables of temperature-dependent free energies that allow one to predict the stability and thermal transition temperature of many simple RNA secondary structures (applicable to ~1 m-Na⁺ concentration). As an example, we apply the rules to an isolated fragment of tRNA^Ser (yeast) (Coutts, 1971), whose properties were not used in calculating the free-energy parameters. The experimental melting temperature of 88 °C is predicted with an error margin of 5 deg. C.     

SYNTHESIS OF A NEW 5′-O-TRIPHOSPHATE ANALOG OF 5-METHYL 2′-O-DEOXYCYTIDINE. PRELIMINARY IN VITRO LABELING FOR NON-RADIOACTIVE DETECTION OF DNA

We report the synthesis of the triphosphate of 5-methyl 4-N-[6-(p-bromobenzamido)hex-1-yl]-2′-O-deoxycytidine 3A . We also analyzed the formation of intramolecular H-bonds of 5-methyl 4-N-{n-[6-(p-bromobenzamido) caproyl amino]alk-1-yl}-2′-deoxycytidine compounds, and confirmed their presence by ¹H-NMR studies. In vitro DNA labeling with modified nucleotides is preliminarily evaluated.     

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.     

Minimal methylation of yeast messenger RNA

Most, if not all, yeast mRNAs are capped at their 5-terminus by m⁷G. Apart from m⁷G no other methylated nucleotides could be detected in poly (A)⁺ mRNA isolated from yeast polysomes.Abbreviations used poly (A)⁺ mRNA messenger RNA containing poly (A) - poly (A)^- RNA RNA lacking poly (A) - m⁷G N₇-methyl guanosine - N_m any 2-0 methylated nucleoside - mN any basemethylated nucleoside     

Enzymatic analysis of isomeric trithymidylates containing ultraviolet light-induced cyclobutane pyrimidine dimers. II. Phosphorylation by phage T4 polynucleotide kinase

Phage T4 polynucleotide kinase (EC 2.7.1.78) proved incapable of catalyzing the phosphorylation of thymidylyl-(3'----5')-thymidine containing either a cis-syn-cyclobutane pyrimidine dimer (d-T less than p greater than T) or a 6-4'-[pyrimidin-2'-one]pyrimidine photoproduct (d-T[p]-T), and similarly the UV-modified compounds of (dT)3 bearing either photoproduct at their 5'-end (d-T less than p greater than TpT and d-T[p]TpT). In contrast, the 3'-structural isomers of these trinucleotides (d-TpT less than p greater than T and d-TpT[p]T) were phosphorylated at the same rate as the parent compound. These phosphorylatable lesion-containing oligonucleotides are quantitatively released from UV-irradiated poly(dA):poly(dT) by enzymatic hydrolysis with snake venom phosphodiesterase and alkaline phosphatase (Liuzzi, M., Weinfeld, M., and Paterson, M. C. (1989) J. Biol. Chem. 264, 6355-6363). By combining this digestion regimen with phosphorylation by polynucleotide kinase and [gamma-32P]ATP, pyrimidine dimers were quantitated at the fmol level following exposure of poly(dA):poly(dT) and herring sperm DNA to biologically relevant UV fluences. The rate of dimer induction in the synthetic polymer, approximately 10 dimers/10(6) nucleotides/Jm-2, was in close agreement with that obtained by conventional methods. Dimers were induced at one-fourth of this rate in the natural DNA. Further treatment of the phosphorylated oligonucleotides derived from irradiated herring sperm DNA with nuclease P1 released the labeled 5'-nucleotide, thus permitting analysis of the nearest-neighbor bases 5' to the lesions. We observed a ratio for pyrimidine-to-purine bases of almost 6:1, implicating tripyrimidine stretches as hotspots for UV-induced DNA damage.     

Complexity and characterization of polyadenylated RNA in the mouse brain

The complexity of nuclear RNA, poly(A)hnRNA, poly(A)mRNA, and total poly(A)RNA from mouse brain has been measured by saturation hybridization with nonrepeated DNA. These DNA populations were complementary, respectively, to 21, 13.5, 3.8, and 13.3% of the DNA. From the RNA Cot required to achieve half-sturation, it was estimated that about 2.5–3% of the mass of total nuclear RNA constituted most of the complexity. Similarly, complexity driver molecules constituted 6–7% of the mass of the poly(A)hnRNA. 75–80% of the poly(A)mRNA diversity is contained in an estimated 4–5% of the mass of this mRNA. Poly(A)hnRNA constituted about 20% of the mass of nuclear RNA and was comprised of molecules which sedimented in DMSO-sucrose gradients largely between 16S and 60S. The number average size of poly(A)hnRNA determined by sedimentation, electron microscopy, or poly(A) content was 4200–4800 nucleotides. Poly(A)mRNA constituted about 2% of the total polysomal RNA, and the number average size was 1100–1400 nucleotides. The complexity of whole cell poly(A)RNA, which contains both poly(A)hnRNA and poly(A)mRNA populations, was the same as poly(A)hnRNA. This implies that cytoplasmic polyadenylation does not occur to any apparent qualitative extent and that poly(A)mRNA is a subset of the poly(A)hnRNA population. The complexity of poly(A)hnRNA and poly(A)mRNA in kilobases was 5 × 10⁵ and 1.4 × 10⁵, respectively. DNA which hybridized with poly(A)mRNA renatures in the presence of excess total DNA at the same rate as nonrepetitive tracer DNA. Hence saturation values are due to hybridization with nonrepeated DNA and are therefore a direct measure of the sequence complexity of poly(A)mRNA. These results indicate that the nonrepeated sequence complexity of the poly(A)mRNA population is equal to about one fourth that observed for poly(A)hnRNA.     

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.     

Fluorescent 2-aza-epsilon-adenosine derivatives of polyadenylic acid, polyuridylic acid, and polyinosinic acid.

A new fluorescent analog of adenosine, 1,N⁶-etheno-2-aza-adenosine, has been incorporated into polynucleotides by polynucleotide phosphorylase polymerization of 1,N⁶-etheno-2-aza-adenosine-5′-diphosphate and adenosine-5′-diphosphate, uridine-5′-diphosphate, or inosine-5′-diphosphate. These new oligonucletides possess high fluorescence when excited at 358 nm and emit at 495 nm. The ratio of the fluorescent and nonfluorescent portions of the copolymer can be controlled by the initial composition of the 2-aza-ε-adenosine-diphosphate and the corresponding nucleoside diphosphate. Fluorescent copolymers with a ratio varying from 1.6 to 35 have thus been synthesized. The physicochemical study of copolymers containing less than 10% of the 1,N⁶-etheno-2-aza-adenosine moiety showed that they are similar to poly(A), poly(U), or poly(I). Therefore, fluorescence and polarization study of the 1,N⁶-etheno-2-aza-adenosine residues that have been incorporated into the copolymer provides a sensitive indicator for the structure of the copolymer. Potentially these new copolymers may provide unique roles in probing the structure of poly(C) and poly(A) in cellular mRNA.     

New Wrinkles on Polynucleotide Duplexes

Abstract

Most fibrous polynucleotides of general sequence exhibit secondary structures that are described adequately by regular helices with a repeated motif of only one nucleotide. Such helices exploit the fact that A:T, T:A, G:C, and C:G pairs are essentially isomorphous and have dyadically-related glycosylic bonds. Polynucleotides with regularly repeated base-sequences sometimes assume secondary structures with larger repeated motifs which reflect these base-sequences. The dinucleotide units of the Z-like forms of poly d(As⁴T):poly d(As⁴T), poly d(AC):poly d(GT) and poly d(GC):poly d(GC) are dramatic instances of this phenomenon. The wrinkled B and D forms of poly d(GC):poly d(GC) and poly d(AT):poly d(AT) are just as significant but more subtle examples. It is possible also to trap more exotic secondary structures in which the molecular asymmetric unit is even larger. There is, for example, a tetragonal form of poly d(AT):poly d(AT) which has unit cell dimensions a = b = 1.71nm, c= 7.40nm, γ = 90°. The C dimension corresponds to the pitch of a molecular helix which accommodates 24 successive nucleotide pairs arranged as a 4₃ helix of hexanucleotide duplexes. The great variety of nucleotide conformations which occur in these large asymmetric units has prompted us to describe them as pleiomeric, a term used in botany to describe whorls having more than the usual number of structures. Pleiomeric DNAs need not contain nucleotide conformations that are very different from one another. On the other hand, DNAs carrying nucleotides of very different conformation must be pleiomeric. This is because 4 nucleotides of different conformation are needed to join patches of secondary structure which are as different as A or B or Z. Differences in nucleotide structures may occur also between chains rather than within chains. In poly d(A):poly d(T), the purine nucleotides all contain Ci'-endo furanose rings and the pyrimidine nucleotides C2 '-endo rings. Analogous heteronomous structures may exist in DNA-RNA hybrids although these duplexes are also found to have symmetrical A-type conformations.