Hydrolysis of nucleic acids by sepharose-micrococcal endonuclease

Initial reaction rates for the hydrolysis of nucleic acids with micrococcal endonuclease (EC 3.1.31.1) insolubilized on Sepharose are strongly influenced by diffusional limitations. Although the absolute values are low, they can be increased substantially by changing particle and pore size of the support, or enzyme concentration in the insoluble derivative. As a result of steric and diffusional limitations, the course of the reaction and selectivity to hydrolysis products for the insoluble derivatives are different to those of the native enzyme; the former produces mainly large and small fragments but few of intermediate size. Because of these differences in course and selectivity of the reaction, diffusional limitations become less important when high initial reaction rates are not required.     

Changes in nucleic acids, protein and ribonuclease activity during maturation of peanut seeds

Changes in weight, protein, RNA, DNA and ribonuclease activityduring maturation of the peanut seed have been studied. Theinvestigation period began with fruits harvested four weeksafter the pegs turned at right angles in the soil and continuedthrough the fourteenth week. Fresh weights of both cotyledonsand embryonic axes increased rapidly during early maturation(8 weeks) and continued in the cotyledons through 12 weeks.The water content at maturity was still adequate for enzymaticprocesses at 45% and 39%, respectively, for embryonic axes andcotyledons. The levels of DNA and RNA also increased rapidlyduring early maturation of both cotyledons and embryonic axes.DNA content decreased during the remainder of maturation whileRNA decreased from 8 to 11 weeks in the cotyledons and thenincreased during the after-ripening period to a value near itsprevious maximum at 8 weeks. RNA levels in the embryonic axesunderwent a gradual increase throughout maturation. The largedecrease in cotyledonary RNA during weeks 8 through 11 couldbe related to increased RNase activity during the period. ¹ Paper number 3370 of the Journal Series of the North CarolinaState University Agricultural Experiment Station, Raleigh, NorthCarolina 27607, U. S. A. ² Present address: Department of Botany, Arts and Sciences,University of the Phillippines, Diliman, Q. C., Phillipines. (Received December 22, 1971; )     

Phosphoryl amino acids: Common origin for nucleic acids and protein

A series of compounds (DAP-AA) composed of an amino acid (AA) and a dialkyl phosphoryl group (DAP) is the basic elements of life chemistry. Self-catalysis of DAP-AA gives the self-assembly oligopeptides, even in aqueous medium at 38°C. The oligo-nucleotides could also be assembled from nucleosides' phosphorylation by DAP-AA. DAP-AA acts as the energy source as well as the phosphoryl donor for the synthesis of nuclic Acids and protein. A general expression for the self assembly system is proposed.     

Watersoluble synthetic nucleic acid analogs--polyethyleneimine derivatives containing nucleic acid bases--conformation and interactionwith nucleic acids

Water soluble polyethyleneimine derivatives containing nucleic acid bases were found to interact with polynucleotides, DNA, RNA. The conformational change by formation of complex was observed by CD spectra and was discussed with the hypochromicity in UV spectra. The rates of interactions between nucleic acid bases in polymers were slow as shown by UV spectra, but the conformational changes of the polynucleotides were fast as shown by CD spectra. In the case of the uracil derivative (PEI-Hse-Ura), high value of CD spectra [theta] 2.80 = -8.0 x 10(-4) for the complex with DNA might be caused by psi type conformation of DNA.     

Affinity of protein HU for different nucleic acids

The binding of protein HU from Escherichia coli to nucleic acids was investigated by affinity chromatography under various conditions, by a nitrocellulose retention assay and by isopycnic centrifugations in metrizamide gradients. The results indicate that HU has a preference for binding to RNA and single-stranded DNA over double-stranded DNA. The affinity of HU for supercoiled DNA was also less than that of the corresponding relaxed DNA.     

Reactive oligonucleotide derivatives and sequence-specific modification of nucleic acids

Selective modification of nucleic acid base sequences can be achieved by complementary oligonucleotides carrying reactive groups. Different types of reactive groups are briefly presented.     

Reduction of the nucleic acid content of single-cell protein concentrates

Methods for reducing the content of nucleic acid in protein concentrates from disintegrated yeast and microalgae were investigated. Protein concentrates were prepared by acid precipitation of extracted protein after cell wall separation. The influence of alkaline protein extraction on the content of RNA in isoelectrically precipitated protein concentrates was studied. It was found that when a strong decrease in the RNA content was obtained, this was followed by a decrease in the yield of protein concentrate. Protein concentrates were also prepared without cell wall separation by precipitation with different agents after cell disintegration. In the precipitates from microalgae, a RNA reduction was obtained. Precipitation of yeast, protein gave no essential reduction with the precipitants used. Precipitation of yeast protein by heating at an alkaline pH gave a protein concentrate with a low content of RNA. A slightly lower RNA content was obtained when the precipitation was performed in the presence of NaCl. The yield of amino acid nitrogen was 70–80% and the RNA content was 1–2%. A process with precipitation at alkaline pH for the production of microbial protein concentrates with a low content of nucleic acid is suggested.     

Exploring the impact of nucleic acids on protein stability in bacterial cell lysate

- Addition of salt enhanced thermal stability of model substrate proteins by reducing electrostatic repulsion between protein molecules.- However, the opposite effect was observed with bacterial cell lysate, indicating that certain molecules within the lysate could enhance protein stability via electrostatic interactions.- Such molecules present in cell lysate were found to be nucleic acids known to have a potent anti-aggregation activity toward proteins involving electrostatic interactions.- Nucleic acids showed chaperone activity in physiological salt concentration within cells and in buffer or medium commonly used in experiments.- The chaperone activity of nucleic acids should be taken into account when performing various in vitro assays using cell lysate or samples containing nucleic acids.     

Convergent synthesis of ribonuclease L-active 2',5'-oligoadenylate-peptide nucleic acids

2-5A was conjugated to N-(2-aminoethyl)-glycyl PNA by periodate oxidization, followed by coupling with amino-derivatized PNA and final cyanoborohydride reduction. An adduct of 2-5A pentamer with tetrameric thymine PNA activated RNase L with the same potency as earlier versions of 2-5A-PNA or 2-5A-DNA.     

Evolution of nucleic acids coding for ribonucleases: the mRNA sequence of mouse pancreatic ribonuclease

The cDNA of mouse pancreatic mRNA has been cloned. After the library was screened with a rat ribonuclease cDNA probe, the positive clones were isolated and sequenced. There were no differences from the previously determined protein sequence. The mRNA codes for a preribonuclease of 149 amino acid residues including a signal peptide of 25 amino acids. The 3' noncoding region has a length of 260 bp, and the total mRNA length is approximately 940 bp. Comparison with the rat pancreatic ribonuclease sequence showed a high rate of nucleotide substitution. Within the coding region, nonsynonymous and synonymous substitution rates are 4.3 X 10(-9) and 15 X 10(-9) nucleotide substitutions/site/year, respectively. The latter value is one of the highest rates observed in the molecular evolution of mammalian nuclear genes. In the signal sequences the synonymous substitution rate is much lower and about the same as the nonsynonymous rate. Signal sequences of other mouse and rat proteins also exhibit little difference between synonymous and nonsynonymous rates. The sequences of rat and mouse pancreatic ribonuclease messengers were compared with those of bovine pancreatic, seminal, and brain ribonuclease. While the 3' noncoding regions of rat and mouse are very similar, as are those of the three bovine messengers, there is no significant similarity between both rodent and the three bovine messengers for the greater part of these regions. There is a duplication of approximately 50 nucleotides in the 3' noncoding region of the bovine messengers, with a region rich in A and C in between. The presence of this structural feature may be correlated with recent gene duplications that have occurred in the bovine genome.     

Novel Rath peptide for intracellular delivery of protein and nucleic acids

In the present study, a novel cell penetrating peptide (CPP) named as Rath, has been identified from the avian infectious bursal disease virus. It has the potential to penetrate and translocate cargo molecules into cells independent of temperature. Additionally, it can deliver oligonucleotide in 30 min and antibodies within an hour intracellular to chicken embryonic fibroblast primary cells. As an ideal delivery vehicle, it has the ability to protect the cargo molecules in the presence of serum, nucleases and has minimal or no cytotoxicity at even higher peptide concentrations studied. The biophysical characterizations showed that Rath has a dominant β structure with a small α helix and has remarkable binding ability with protein and DNA. Thus, the characterization of unique Rath peptide to deliver protein or nucleic acid into the cells with non-covalent interaction could be used as an effective delivery method for various cell based assays.     

Selenium modification of nucleic acids: preparation of phosphoroselenoate derivatives for crystallographic phasing of nucleic acid structures

This protocol describes a simplified means of introducing an anomalously scattering atom into oligonucleotides by conventional solid-phase synthesis. Replacement of a nonbridging phosphate oxygen in the backbone with selenium is practically suitable for any nucleic acid. The resulting oligonucleotide P-diastereomers can be separated using anion exchange HPLC to yield diastereomerically pure phosphoroselenoates (PSes). The total time for the synthesis and ion-exchange HPLC separation of pure PSe is approximately 60 h.     

Accurate ab initio prediction of NMR chemical shifts of nucleic acids and nucleic acids/protein complexes

NMR chemical shift predictions based on empirical methods are nowadays indispensable tools during resonance assignment and 3D structure calculation of proteins. However, owing to the very limited statistical data basis, such methods are still in their infancy in the field of nucleic acids, especially when non-canonical structures and nucleic acid complexes are considered. Here, we present an ab initio approach for predicting proton chemical shifts of arbitrary nucleic acid structures based on state-of-the-art fragment-based quantum chemical calculations. We tested our prediction method on a diverse set of nucleic acid structures including double-stranded DNA, hairpins, DNA/protein complexes and chemically-modified DNA. Overall, our quantum chemical calculations yield highly/very accurate predictions with mean absolute deviations of 0.3–0.6 ppm and correlation coefficients (r²) usually above 0.9. This will allow for identifying misassignments and validating 3D structures. Furthermore, our calculations reveal that chemical shifts of protons involved in hydrogen bonding are predicted significantly less accurately. This is in part caused by insufficient inclusion of solvation effects. However, it also points toward shortcomings of current force fields used for structure determination of nucleic acids. Our quantum chemical calculations could therefore provide input for force field optimization.     

Correlation among turnover of nucleic acids,ribonuclease activity and sporulation ability of Saccharomyces cerevisiae

The turnover of nucleic acids and changes in ribonuclease activity during sporulation of Saccharomyces cerevisiae were studied. In the sporulating strains, 37–58% of vegetatively synthesized RNA were degraded during the sporulation process. The degree of degradation of vegetative RNA was proportional to the sporulation ability. In the non-sporulating strains, the degradation of vegetative RNA was less than 28% in the sporulation medium. Accompanied by the degradation of vegetative RNA, a ribonuclease activity increased several times during sporulation. We have found a close relation among the sporulation rate, the degree of the degradation of vegetative RNA and the increase in ribonuclease activity in the sporulation medium, using cells of which sporulation ability was repressed by changing the age or carbon source in various degrees.     

Reaction of nucleic acids bases and their derivatives with peroxodisulfate ion

Reaction with peroxodisulfate ion was investigated, that is, reaction of 1,3-dimethyluracil, 1,3-dimethylthymine, and caffeine with carbon radicals formed from decarboxylation of carboxylic acids, oxidation of the methyl group at 5-position of thymines, and halogenation of nucleic acids bases and their derivatives with alkali halides.