Benzoylated DEAE-cellulose Chromatography Research Articles

Sizing of single-stranded regions in double-stranded DNA by preparative benzoylated DEAE-cellulose chromatography

The concentration of caffeine required to elute wholly single-stranded DNA from benzoylated DEAE-cellulose is proportional to the polynucleotide length. The use of benzoylated DEAE-cellulose chromatography for isolating and sizing single-stranded regions in double-stranded DNA has been examined using a series of hybrid molecules. Restriction fragments of the replicating form of bacteriophage ϕX174 were hybridized to the intact ‘plus’ strand, thereby forming hybrids having single- and/or double-stranded regions in the kilobase range. A series of such hybrid preparations were subject to caffeine concentration gradient elution from benzoylated DEAE-cellulose. After logarithmic transformation, a linear relationship ( R = 0.94) could be demonstrated between eluting caffeine concentration and single-stranded length, irrespective of the length of associated double-stranded regions or the location, within a given fragment, of unpaired nucleotides. Benzoylated DEAE-cellulose chromatography may therefore be used to separate and characterize, on a preparative scale, double-stranded DNA containing single-stranded regions.

Persistent and heritable structural damage induced in heterochromatic DNA from rat liver by N-nitrosodimethylamine.

Analysis, by benzoylated DEAE-cellulose chromatography, has been made of structural change in eu- and heterochromatic DNA from rat liver following administration of the carcinogen N-nitrosodimethylamine (10 mg/kg body weight). Either hepatic DNA was prelabeled with [3H]thymidine administered 2-3 weeks before injection of the carcinogen or the labeled precursor was given during regenerative hyperplasia in rats treated earlier with N-nitrosodimethylamine. Following phenol extraction of either whole liver homogenate or nuclease-fractionated eu- and heterochromatin, carcinogen-modified DNA was examined by stepwise or caffeine gradient elution from benzoylated DEAE-cellulose. In whole DNA, nitrosamine-induced single-stranded character was maximal 4-24 h after treatment, declining rapidly thereafter; gradient elution of these DNA preparations also provided short-term evidence of structural change. Following incubation of purified nuclei with micrococcal nuclease, 10-12% of labeled DNA was solubilized (eu-chromatin) by 1.0 unit of micrococcal nuclease (5 mg of DNA)-1 mL-1 after 9 min. In prelabeled animals, administration of N-nitrosodimethylamine caused a marked fall in the specific radioactivity of solubilized DNA, while that of sedimenting DNA was not affected. Caffeine gradient chromatography suggested short-term nitrosamine-induced structural change in euchromatic DNA, while increased binding of heterochromatic DNA was evident for up to 3 months after carcinogen treatment. Preparations of newly synthesized heterochromatic DNA from animals subjected to hepatectomy up to 2 months after carcinogen treatment provided evidence of heritable structural damage. Carcinogen-induced binding of heterochromatic DNA to benzoylated DEAE-cellulose was indicative of specific structural lesions whose affinity equalled that of single-stranded DNA up to 1.0 kilobase in length.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-stage incorporation of thymidine triphosphate into mammalian DNA as indicated by chromatography on benzoylated DEAE-cellulose

Replicating DNA contains single-stranded regions as indicated by the extent of its recovery in the final fraction, following stepwise elution of sheared preparations from benzoylated DEAE-cellulose using 0.3 M sodium chloride, 1.0 M sodium chloride and caffeine solutions, respectively. In preparations of hepatic DNA, isolated up to 60 min after administration of [ 3H]thymidine to rats subjected earlier to partial hepatectomy, the proportion of radioactivity contained in the caffeine-eluted fraction progressively decreased. This change was attributable to migration of incorporated radioactivity from replicating forms to mature (double-stranded) DNA, the latter being recovered from the column in 1.0 M sodium chloride. In terms of the same chromatograms, the relative size of the 0.3 M sodium chloride-eluted fraction also decreased, this fraction of radioactivity co-eluting from benzoylated DEAE-cellulose with thymidine triphosphate. The precursor—intermediate—product relationship between the three products separated by benzoylated DEAE-cellulose chromatography was confirmed using DNA isolated from cultured mammalian cells. Early eluting ratioactivity co-chromatographed with thymidine triphosphate on thin-layer cellulose, whilst the intermediate status of caffeine-eluted radioactivity was confirmed following pulse-chase labelling procedures. Utilizing stepwise chromatography of such DNA on benzoylated DEAE-cellulose, the effect of three inhibitors could thus be described as affecting either an early or late stage of DNA synthesis. Such an approach offers a simple quantitative method of monitoring influences on DNA synthesis.

Dose-dependent persistence of alkylation-induced single stranded regions in rat liver DNA in vivo

Single stranded regions in DNA, presumed to be indicative of DNA repair, may be readily detected in rat liver DNA following injections of nonnecrotizing single doses of methyl methanesulfonate, dimethylnitrosamine and diethylnitrosamine. The present study concerns persistence of such structural defects in vivo, as determined by benzoylated DEAE-cellulose chromatography, in relation to the dose of alkylating agent. For all these agents, the period during which structural damage may be detected is markedly dependent upon dose: prolonged persistence only occurs after the highest dose. The findings, in relation to other data, implicate reaction processes involving the final stages of repair as being critical to prolongation of this type of genomic damage in vivo.

Patterns of structural change in DNA during tissue necrosis indicated by benzoylated DEAE-cellulose chromatography

Structural change in liver DNA, isolated following administration of a necrogenic dose of carbon tetrachloride to rats, was examined by benzoylated DEAE-cellulose (BD-cellulose) chromatography. Greatest increase in the amount of DNA exhibiting single stranded character was detected 48 h after treatment, at which time massive necrosis was evident histologically. By 72 h after dosing, normal hepatic architecture had been restored and gross structural change in DNA could no longer be detected. The extent of single stranded regions in DNA isolated at various times after carbon tetrachloride intoxication was assessed by caffeine gradient elution of respective samples from BD-cellulose. By this criterion, structural damage to DNA was again most marked 48 h after treatment at which time single stranded regions extending over several kilobases were apparent. At earlier times such lesions were considerably shorter. Single stranded regions of discrete length detected 72 h after administration of the hepatotoxin appeared to be associated with cell division. Structural damage to DNA coincident with toxic injury was further characterized using BD-cellulose, by caffeine gradient elution of preparations subjected to denaturation before chromatography. The data indicate that production of single stranded regions is a major feature of DNA degradation during tissue necrosis. Whilst such degradation might otherwise confound interpretation of structural analyses based only upon strand breakage, the size of these regions suggests a basis for distinguishing between them and lesions specifically associated with repair, replication and transcription of DNA.

Sizing of DNA fragments by preparative, benzoylated DEAE-cellulose chromatography

Sizing of DNA fragments by preparative, benzoylated DEAE-cellulose chromatography

Isolation and partial characterization of Escherichia coli valine transfer RNA with uridine and uridine-derived residues replaced by 5-fluorouridine

To examine the role of minor bases in transfer RNA, valine 1 transfer RNA of Escherichia coli with the majority of its uridine and uridine-derived residues replaced by fluorouridine, was purified by benzoylated DEAE-cellulose chromatography. 87% replacement of pseudouridine, 95% replacement of ribothymidine, dihydrouridine, 4-thiouridine, and uridine and 74% replacement of the uridine-5-oxyacetic acid were achieved. 6% of the cytidine was replaced by 5-fluorocytidine. Comparison of the ultraviolet absorbance—temperature profiles and circular dichroism spectra of normal and analogue-containing tRNA indicated that this extensive base substitution had no major effects on the secondary structure of the molecule. The minor bases do not appear to be essential for proper recognition by synthetase since in the aminoacylation reaction with partially purified valyl-tRNA synthetase, normal and 5-fluorouridine-containing valine tRNA had virtually the same Km, 1.5 to 1.6 × 10−7m.

Phenylalanine transfer RNA species in early development of barley

Nine phenylalanine tRNA species were identified during early development in barley, eight of which were detected by reversed-phase 2 chromatography when germ, root, green and etiolated leaf, and chloroplast tRNAs were compared. Benzoylated DEAE-cellulose chromatography further resolved one of the eight species into two forms. Of the five species present in the 4.5-day-old leaves, two were found in chloroplasts and these were aminoacylated specifically by the purified phenylalanyl-tRNA synthetases from Escherichia coli and from Euglena chloroplasts. The barley chloroplast synthetase preparation showed limited specificity in this respect. Two patterns of fluorescence were detected in leaf phenylalanine tRNA fractionated by benzoylated DEAE-cellulose. The ethanol fraction, which contained two cytoplasmic species, had a maximum fluorescence emission at 420 nm when excited at 312 nm. The emission spectrum was similar to that of wheat germ tRNA, which contains a fluorescent Y-type base in a phenylalanine tRNA. One of the two fractions that eluted with the salt gradient was fluorescent, exhibiting an emission maximum at 375 nm when excited at 312 nm. This fraction contained the two chloroplast species.

Patterns of Base Modification in tRNA

WE have previously demonstrated that the N-hydroxy-succinimide ester of phenoxyacetic acid (HSP) reacts with certain species of rat liver tRNA1. The reactive species were identified by an alteration in their elution pattern on benzoylated DEAE-cellulose chromatography. Those species that reacted with HSP were not eluted from the column unless ethanol was included in the eluting solvent.

Isoaccepting transfer rna species in differing morphogenetic states of Rhodopseudomonas spheroides

1. Benzoylated DEAE-cellulose chromatography was employed to compare isoaccepting tRNA species from aerobic and anaerobic photosynthetic cultures of Rhodopseudomonas spheroides. 2. Variation in isoaccepting species were observed with phenylalanine and tryptophan tRNAs which were independent of the source of aminoacyl synthetase. 3. Tryptophanyl-tRNA formed in vivo was chromatographed and found to show similar variations to those observed with in vitro charged tRNA.

Purification of an Escherichia coli Leucine Suppressor Transfer Ribonucleic Acid and Its Aminoacylation by the Homologous Leucyl-Transfer Ribonucleic Acid Synthetase

Abstract The leucine amber suppressor tRNA of an Escherichia coli strain carrying the Su6+ gene has been purified completely by benzoylated DEAE-cellulose chromatography. This tRNA mediates in vitro polyleucine formation with poly r(U-A-G). In the aminoacylation reaction with the pure Escherichia coli leucyl-tRNA synthetase it possesses the same Km (5 x 10-8 m) as the other tRNAleu species.

A specific hepatic transfer RNA for phosphoserine.

Radioactive O-phosphoryl-L-serine was detected after alkaline deacylation of rat and rooster liver [(3)H]seryl-tRNA acylated in vitro with homologous synthetases. Ribonuclease treatment of this tRNA yielded a compound with the properties of phosphoseryl-adenosine. Benzoylated DEAE-cellulose chromatography of seryl-tRNA yielded four distinct peaks, only one of which contained phosphoserine. A unique fraction for phosphoserine was also found on chromatography of nonacylated tRNA. In ribosome binding studies, this fraction responded very slightly with poly(U,C), but not with any of the known serine trinucleotide codons. Substantial incorporation of [(3)H]-serine into protein from this tRNA species was observed in an aminoacyl-tRNA dependent polysomal system derived from chick oviducts. No phosphoserine was found in Escherichia coli or yeast seryl-tRNA acylated with homologous enzymes, nor in E. coli seryl-tRNA acylated with liver synthetase. In the absence of tRNA, free phosphoserine was not formed in reaction mixtures, which suggests that phosphoseryl-tRNA arises by phosphorylation of the unique seryl-tRNA species. These results demonstrate a discrete tRNA(Ser) species in rat and rooster liver containing phosphoserine and suggest that this tRNA is involved in ribosomal polypeptide synthesis.

Purification of Escherichia coli pulse-labeled RNA by benzoylated DEAE-cellulose chromatography

The rapidly-labeled RNA fraction of Escherichia coli has been purified approximately 15-fold on benzoylated DEAE-cellulose columns. It is metabolically unstable (as shown by a pulse/chase experiment), and is considered to represent messenger RNA. The yield of pulse-labeled RNA is about 70% and comprises 4 to 5% of the RNA of the cell. The true size distribution of this RNA, determined by sedimentation in a denaturing solvent (99% dimethyl sulfoxide), does not change during purification. This result indicates that neither degradation nor selection for molecules of a particular size has occurred. Upon sedimentation of the final preparation in dimethyl sulfoxide, the distribution of pulse label is the same as that of RNA mass, indicating nearly complete separation from longer-lived RNA components.