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PubMed1988

Implications for the formation of abasic sites following modification of polydeoxycytidylic acid by acrolein in vitro.

Polydeoxycytidylic acid (poly dC) was incubated with excess acrolein. A Nensorb 20 nucleic acid purification cartridge was used to bind the polymeric material in the poly dC/acrolein reaction mixture. The non-polymeric material eluted from this column had a UV absorbance four times higher than that of the control. The fluorescence spectrum of the eluted material did not correspond to that of unmodified cytosine. Separate aliquots of the reaction mixture were digested to deoxynucleotide 3'-monophosphates by incubation with micrococcal nuclease and spleen phosphodiesterase. The products were converted to 32P-labeled deoxynucleotide 3',5'-bisphosphates by incubation with T4 polynucleotide kinase and excess [gamma-32P]ATP. The 3'-monophosphate was selectively removed by incubation with nuclease P1. Two-dimensional thin-layer chromatography (TLC) on polyethyleneimine cellulose (PEI)-cellulose and detection of 32P-labeled deoxynucleotide 5'-monophosphates by autoradiography failed to provide evidence for the formation of an acrolein adduct of deoxycytidine 5'-monophosphate. When acrolein-modified deoxycytidine 3'-monophosphate was 32P post-labeled, a new product, which co-chromatographed with UV markers synthesized by reaction of acrolein with deoxycytidine 5'-monophosphate, was detected. These data show that acrolein-modified deoxycytidine 3'-monophosphates are substrates for 32P labeling by T4 polynucleotide kinase and are stable under the assay conditions employed. The inability to detect the acrolein-modified nucleotides after reaction with poly dC in vitro suggests that the modified bases are lost from poly dC by cleavage of the N-glycosyl bond resulting in the formation of an abasic site.

Acrolein↗
PubMed1976

Protein-polynucleotide scattering centers as a protein structure probe.

When certain basic globular proteins are mixed with nucleic acids near a critical concentration ratio, large, low density scattering centers of about 10(9) particle weight are created. Scattering from these complexes is altered when thermally inactivated proteins are substituted for enzymes in their native, globular conformation. Scattering data from heat-treated ribonuclease and lysozyme mixed with four different synthetic homopolyribonucleotides are reported. The concentration of nucleic acid necessary to produce maximum scattering from a heat-treated protein sample is shown to be a direct indication of the amount of enzyme that remains biologically active after being heated.

Light↗
PubMed1983

Deuterium NMR in the solid-state and in solution of the molecular motion of the bases in poly(I) and poly(I) . poly(C).

To provide information regarding the conformational flexibility of nucleic acids, in particular the rate and amplitude of base motions, we have observed the deuterium NMR from single-stranded and double-stranded polynucleotides. Poly(I) was deuterated at the 8-position of the base, and the deuterium NMR was examined in solution (at 23.0 and 55.4 MHz) and for hydrated and dry fibers (at 23.0 MHz). In the solid state, the deuterium signal of dry poly(I) exhibits a powder pattern with the maximal expected quadrupolar splitting, while the relatively short spin-lattice relaxation time indicates the presence of a rapid internal reorientation of the C-D bond with an amplitude of that motion of at least +/- 2.4 degrees. Hydrating the poly(I) fibers to the extent of eight molecules of water per nucleotide results in the disappearance of the deuterium signal, apparently due to a decreased spin-spin relaxation time shorter than the instrumental dead-time (even using the quadrupolar echo technique); this could occur if conformational fluctuations are occurring at a rate comparable to the deuterium quadrupole interaction strength, i.e., 175 kHz. In solution, a theoretical fit to the measured Lorentzian linewidths and spin-lattice relaxation times necessitates the inclusion of at least two motional correlation times, with a subnanosecond internal motion. Double-stranded poly(I) . poly(C) yielded a solid state spectrum similar to poly(I), albeit with a longer T1, which reduced the lower limit for the amplitude of an internal motion to +/- 1.9 degrees. The 2H signal from the poly(I) . poly(C), hydrated to a degree of approx. eight molecules of water per base pair, retained its solid-state lineshape (with a reduced T1 value, indicating increased internal mobility of the bases with a lower limit on amplitude of +/- 4.7 degrees). In solution, however, the 2H-NMR signal from poly(I) . poly(C) became virtually undetectable, even in solid-echo experiments, when the echo was observed after 52 microseconds. This indicates that the spin-spin relaxation time of the deuterium nucleus must be close to its theoretical minimum of about 9 microseconds, and the correlation time for an isotropic reorientation of the C-D vector can be estimated to be between 0.2 and 200 microseconds.

Magnetic Resonance Spectroscopy↗
PubMedDate not supplied

The cloning and analysis of the aroD gene of E. coli K-12.

A 5.6-kb PstI fragment containing the structural gene (aroD) for 5-dehydroquinate hydrolyase (DHQase) of Escherichia coli K-12 has been cloned into recombinant plasmid pJKK12. The bacterial fragment contains two Bg/II, one HpaII, one SalI and one XhoI site, but no EcoRI, HindIII or BamHI sites. the DHQase activity extracted from strains harboring pJKK12 had properties identical to those of the enzyme isolated from wild-type E. coli. The native protein appears to be a dimer composed of two 31 500 dalton subunits. aroD6 strains transformed with pJKK12 had an 11-fold and 34-fold increase in activity compared to untransformed wild-type controls grown on L broth and minimal medium, respectively. No increase of dehydroquinase activity was found in polynucleotide phosphorylase deficient strains of E. coli. At least four constitutively expressed genes are encoded on the fragment.

Amino Acids↗
PubMed1983

Cloning and physical analysis of the pyrF gene (coding for orotidine-5'-phosphate decarboxylase) from Escherichia coli K-12.

The structural gene (pyrF) for orotidine-5'-phosphate decarboxylase (OMPase, EC 4.1.1.23) of Escherichia coli K-12 has been cloned as part of two PvuII fragments (1.2 and 0.9 kb) to form the recombinant plasmid pDK26. Extracts of E. coli [pDK26] had 80-fold higher levels of OMPase activity than wild-type strains without the plasmid. Maxicell analysis showed that pDK26 encoded two proteins of Mr 27 000 [pyrF(OMPase)] and 15 000 (Z) in addition to the ampicillin-resistance determinant. The approximate initiation site and direction of transcription of the pyrF gene have been determined. Extracts of strains that were deficient in polynucleotide phosphorylase (PNPase) had higher levels of OMPase activity than isogenic PNPase+ strains when one or two copies of the pyrF gene were present per cell either in the chromosome or on a low copy number plasmid. However, no significant difference in OMPase activity was seen in PNPase- strains that contained the pyrF gene cloned in a multicopy plasmid. Southern hybridization experiments showed that the yeast gene for OMPase (URA3) and the E. coli pyrF gene had less than 70% DNA sequence homology.

Base Sequence↗
PubMed1985

Promoter activity and transcript mapping in the regulatory region for genes encoding ribosomal protein S15 and polynucleotide phosphorylase of Escherichia coli.

The genes encoding ribosomal protein S15 (rpsO) and polynucleotide phosphorylase (pnp) occupy adjacent positions and are oriented in the same direction on the Escherichia coli chromosomes. The nucleotide sequence of the region controlling the expression of these two genes has been determined. Two in-phase gene fusions between pnp and lacZ were constructed. The fusions define the translational reading frame of the pnp gene and indicate that the expression of pnp is independent of the upstream rpsO gene. Transcript mapping with nuclease S1 demonstrated that the two genes are transcribed from separate promoters and that the rpsO-pnp intergenic space contains a strong transcriptional terminator. The transcriptional start points have been localized.

Amino Acid Sequence↗
PubMed1992

Translational initiation factors IF-1 and eIF-2 alpha share an RNA-binding motif with prokaryotic ribosomal protein S1 and polynucleotide phosphorylase.

Initiation of translation is a complicated process involving numerous accessory factors whose functions remain incompletely understood. Bacterial ribosomal protein S1 is known to contain a repeated sequence motif (S1-RM), also found in polynucleotide phosphorylase, that is thought to be involved in binding to RNA. Using the technique of profile analysis, the S1-RM can also be found in bacterial and chloroplast translation initiation factor IF-1 sequences, and in the sequences of eukaryotic translation initiation factor eIF-2 alpha chains. The significance of the similarity of the sequences is very high suggesting that the occurrence of the S1-RM in these diverse proteins represents homology. The similarity of S1 to IF-1 further suggests that S1 has evolved from an IF-1 like ancestor, and therefore that the two proteins have a similar or competitive function. The most obvious common function of the proteins containing the S1-RM seems to be RNA binding, suggesting that IF-1 and eIF-2 alpha may bind to RNA.

Amino Acid Sequence↗
PubMed1994

Analysis of the yeast NSR1 gene and protein domain comparison between Nsr1 and human hnRNP type A1.

The yeast nucleolar protein-encoding gene NSR1 was isolated by low-stringency screening of a yeast genomic library with the human heterogeneous nuclear ribonucleoprotein type A1 (hnRNP A1) cDNA probe, and was mapped to chromosome VII. RNA abundance was determined and the transcription start point and polyadenylation site were mapped. A comparison between the Nsr1 and hnRNP A1 proteins, based on homopolymer RNA binding to their structural domains in vitro, revealed a striking biochemical similarity. When the N-terminal, lysine- and arginine-rich domain of Nsr1 was removed, the truncated protein behaved similarly to hnRNP A1; furthermore, the two RRM (RNA recognition motif) domains of Nsr1 behaved in the same manner as the two RRM domains of hnRNP A1. The biochemical data, therefore, would support the hypothesis that the two RRM domains in hnRNP A1 and Nsr1 interact with RNA in a similar manner in both mammalian and yeast cells, respectively.

Base Sequence↗
PubMed1988

Binding of Ru(bpy)3(2+) and Ru(phen)3(2+) to polynucleotides and DNA: effect to added salts on the absorption and luminescence properties.

The interactions of tris(2,2'-bipyridyl)ruthenium(II) chloride and tris-(1,10-phenanthroline)ruthenium(II) chloride, Ru(bpy)3Cl2 and Ru(phen)3Cl2 respectively, with nucleic acids were studied by means of absorption spectroscopy and time-resolved and steady state luminescence techniques in unbuffered aqueous solution at room temperature as a function of added salt, oxygen and the [nucleotide]/[sensitizer] ratio (N/S). The hypochromicity of the visible absorption band of Ru(ligand)3(2+) and the changes in the luminescence intensity and luminescence decay kinetics are considerably larger in the presence of double-stranded calf thymus DNA (dsDNA) than in the presence of single-stranded DNA and polynucleotides. This is suggested to be the result of partial intercalation of the ruthenium complex into the dsDNA rather than just its higher charge density with respect to ssDNA. Spectral changes in the presence of dsDNA increase with increasing N/S ratio (maximum changes reached at N/S = 10-12, half-value 3-4). This is postulated to be due to a transition from mainly electrostatic binding to a binding in which partial intercalation plays an increased role. Addition of alkali or alkaline earth salts at very low concentrations stabilizes partial intercalation whereas higher salt concentrations lead to a release of the ruthenium complex from the strand. This effect of the salt cation increases in the order Cs less than Rb less than K less than Na less than Li less than Ba less than Sr less than Ca less than Mg less than Be. For Ru(bpy)3(2+) the presence of 0.5 mM Mg(ClO4)2 or 6 mM NaClO4 are sufficient to release 50% of the ruthenium complexes which are bound to the dsDNA (N/S = 10); the corresponding half-concentrations for Ru(phen)3(2+) are 0.8 mM and 40 mM respectively. The half-concentrations for release increase with increasing N/S ratio and decrease with the ionic radius of the added salt.

2,2'-Dipyridyl↗
PubMed1990

Phosphorescence of nucleic acids and DNA components at 77 K.

The emission spectra of nucleic acids, pyrimidine and purine nucleotides, nucleosides and bases and a series of pyrimidine derivatives were obtained using UV light excitation in glasses (ethanol and 2:1 mixtures of ethylene glycol and water (EG-H2O); also partly in butyronitrile and 2-methyltetrahydrofuran) at 77 K. The quantum yields of fluorescence phi f and phosphorescence phi p of some 30 compounds are presented; for several substituted uracils they are reported for the first time. The values cover a range from phi f = 0.0002 and phi p = 0.001 for uracil in ethanol to phi f = 0.50 for guanosine in acidic ethanol and phi p = 0.095 for guanosine-5'-monophosphate in EG-H2O (pH 6-7). The phosphorescence lifetime tau p at 77 K ranges from about 0.3 s (uracil moiety) to 3 s (adenine moiety). The measured tau p, phi f and phi p values are compared with those available in the literature.

DNA↗
PubMed1993

Photoinduced interaction of Ru(bpy)3 2+ with nucleotides and nucleic acids in the presence of S2O8 2-; a transient conductivity study.

The photochemical reactions of Ru(bpy)3(2+) with single- and double-stranded DNA, polynucleotides and purine-containing nucleotides in argon-saturated aqueous solution in the presence of S2O8(2-) were studied using time-resolved absorption and conductivity methods. The conversion of Ru(bpy(3(3+) to Ru(bpy)3(2+), monitored spectroscopically either after rapid mixing with substrate or after laser flash excitation (lambda exc = 353 nm) is quantitative at nucleotide-to-sensitizer ratios [N]/[S] of 1-2 for DNA and other guanine-containing compounds. Conductivity measurements following the laser pulse revealed a fast conductivity increase (rise time, less than 0.1 ms) due to the formation of protons and, to a lesser degree, to charged species of much lower ion mobility. A slower component in the 0.01-1 s range was observed for nucleic acids; its amplitude is markedly reduced at pH 6-9. In buffered neutral solution the signal is replaced by a slight decrease in conductivity. Electronically excited Ru(bpy)3(2+) bound to DNA reacts with S2O8(2-) to form Ru(bpy)3(3+) and SO4(.-) as primary oxidizing species both of which react with bases. The resulting base radicals react subsequently with Ru(bpy)3(3+) and Ru(bpy)3(2+) or the ligands in the ruthenium complex, producing protons which give rise to the slower conductivity increase. The formation of single-strand breaks and the ensuing release of condensed counterions does not appear to contribute significantly to the slow component. The transient conductivity behaviour is sensitive to the single- or double-stranded nature of DNA.

2,2'-Dipyridyl↗
PubMed1994

Photochemistry of DNA and related biomolecules: quantum yields and consequences of photoionization.

The reactions of nucleic acids and constituents, which can be induced by laser UV irradiation, are described. Emphasis is placed on the quantum yields of various stable photoproducts of DNA and model compounds upon irradiation at 193, 248, 254 or 266 nm. In particular, those quantum yields and processes are discussed which involve photoionization as the initial step and occur in aqueous solution under well defined conditions, e.g. type of atmosphere. The efficiencies of some photoproducts, with respect to photoionization using irradiation at 193 or 248 nm, are presented. Radical cations of nucleobases are important sources of damage of biological substrates since they can cause lesions other than dimers and adducts, e.g. strand breakage, abasic sites, crosslinks or inactivation of plasmid and chromosomal DNA. While competing photoreactions, such as hydration, dimerization or adduct formation, diminish the selectivity of the photoionization method, a combination with model studies on pyrimidine- and purine-containing constituents of DNA has brought about an enhanced insight into the reaction mechanisms. The knowledge concerning the lethal events in plasmid and cellular DNA has been greatly improved by correlation with the chemical effects obtained by gamma-radiolysis, vacuum-UV (< 190 nm) and low-intensity irradiation at 254 nm.

Adenosine↗
PubMed2004

Polynucleotide phosphorylase-based photometric assay for inorganic phosphate.

Polynucleotide phosphorylase is a prokaryotic enzyme that catalyzes phosphorolysis of polynucleotides with release of nucleotide diphosphates. By taking advantage of this property, we developed a photometric assay for inorganic phosphate. In the presence of polyadenylic acid, phosphate is converted into adenosine 5'-diphosphate (ADP) by this enzyme. ADP then reacts with phosphoenolpyruvate in a pyruvate kinase-catalyzed reaction, thus giving rise to adenosine 5'-triphosphate and pyruvate. Finally, pyruvate oxidizes reduced nicotinamide adenine dinucleotide (NADH) through the action of L-lactate dehydrogenase, with concomitant decrease in absorbance at 340 nm. As expected, in this detection system 1 mol of NADH was oxidized per mole of phosphate. The assay showed an excellent reproducibility, as the standard deviations never exceeded 5%. It also was shown to be unaffected by several compounds that are regarded as major interferents of the traditional colorimetric assays. Absence of interference was also demonstrated when determining phosphate content in different biological samples, such as human serum and perchloric acid extracts from Escherichia coli, yeast, and bovine liver. An E. coli strain overexpressing His-tagged polynucleotide phosphorylase developed in our laboratories allowed quick and straightforward purification of enzyme, making the assay feasible and convenient. Since all other reagents required are inexpensive, the assay represents a cheaper alternative to commercially available phosphate assay kits.

Adenosine Diphosphate↗
PubMed2005

Conserved domains in polynucleotide phosphorylase among eubacteria.

Polynucleotide phosphorylase (PNPase) is a polynucleotide nucleotidyl transferase (E. C. 2.7.7.8) that is involved in mRNA degradation in prokaryotes. PNPase structure analysis has been performed in Streptomyces antibioticus; this revealed the presence of five domains: two ribonuclease PH (RPH)-like (pnp1 and pnp2), one alpha helical, one KH, and one S1 domains. The trimeric nature of this enzyme was also confirmed. In this work, we have investigated conserved domains or subdomains in bacterial PNPases (55), for this structure-based sequence homology analysis between predicted amino acid sequences from bacterial PNPases and that of S. antibioticus was performed. Our findings indicate that while pnp2 (% similarity average S = 84/% identity average I = 22), KH (S = 74.3%/I = 5.3%), S1 (S = 71.3%/I = 1.2%); and pnp1 (S = 52.8%/I = 0.3%) domain; structure and sequence are well conserved among different bacteria, alpha helical domain (S = 39.5%/I = 0) although conservation of the structure is somewhat maintained, the sequence is not conserved at all. Implications of such findings in PNPase activity will be discussed.

Amino Acid Sequence↗