A microprocedure for extracting tissue nucleotides for analysis by high-performance liquid chromatography.

A method is described for the preparation of concentrated tissue extracts for nucleotideanalysis by high-performance liquid chromatography (hplc). Ten to one hundred milligrams of tissue was extracted in a combined weighing-homogenizing-centrifuge tube using a trichloracetic acid (TCA)-methanol extractant containing a radioactive internal standard. This extractant eliminated nucleotide interconversion which was found to occur when TCA alone was employed. High ATP/ADP and ATP/AMP ratios were observed and recoveries of greater than 97% were obtained with exogenous radioactive nucleotides. The method has been applied successfully in studies on muscle, heart, liver, kidney, lung, brain, and subcellular fractions.     

A study of histone-histone interactions by affinity chromatography

Homologous whole histone from calf thymus was adsorbed on Sepharose 4B columns with covalently coupled histone fractions H2a, H2b, H3 or H4 in 0.01 M phosphate buffer, pH 6.7–1 M NaCl. The adsorbed histones were eluted from the columns with 5 M urea in the same buffer. Electrophoretic analysis has shown that the different columns exhibit selective affinity to the histone fractions: the H2b column to histone H2b and H2a (with only weak affinity to histones H3 and H4), the H2a column to histones H2b and H3 (moderate affinity to histones H2a and H4), the H3 column to histones H3, H4, H2a (moderate affinity to histone H2b), and the H4 column to histone H3, H4 and H2b (weak affinity to histone H2a). Histone H1 displayed no fixation by either of the columns tested.     

The inhibition of RNA polymerase by daunomycin

The effect of daunomycin on the in vitro activity of Escherichia coli DNA-dependent RNA polymerase has been studied under a variety of experimental conditions. The inhibition of RNA synthesis by this DNA-binding antibiotic is overcome by an increase in the DNA concentration but is unaffected by an increase in the concentration of the RNA polymerase. It is concluded that, under conditions used, the inhibition is predominantly due to the interaction of the drug with the template DNA. At the concentration used (20 μM), daunomycin is able to inhibit RNA polymerization even after its initiation. However, the possibility remains that other steps are sensitive to daunomycin. A comparison of the effect of daunomycin on RNA synthesis using different DNAs as templates suggests that the extent of inhibition depends on base composition and on the secondary structure of the DNA. The effect of base composition on the melting temperature of antibiotic-DNA complexes is consistent with the inhibiting effect on RNA synthesis.     

The relationship between the turnover of UTP and RNA synthesis in cultured cells treated with aflatoxin B1.

Methods have been adapted to measure the specific activity of UTP in cells in monolayer culture. In HeLa cells labelled with [3H]uridine and treated with aflatoxin B1 there was reduced radioactivity in crude acid extracts, but the toxin did not affect the radioactive incorporation into UTP. Using cells in which the UTP was pre-labelled, the subsequent addition of aflatoxin B1 inhibited UTP incorporation into RNA. Accordingly aflatoxin B1 did not inhibit the uptake of uridine or the latter's conversion to UTP but inhibited the incorporation of UTP into RNA.     

Effects of growth phase and repair capacity on rejoining of ethyl methanesulfonate-induced DNA breaks in Escherichia coli

The effects of growth phase and DNA repair capacity on the production and rejoining of ethyl methanesulfonate (EMS)-induced single-strand breaks were studied in 4 strains of E. coli. DNAs from logarithmic and stationary phase cells of the DNA polymerase I deficient mutant, P₃₄₇8 polA, a recombination deficient mutant, DZ₄₁₇recA, and from the respective parental strains, W₃₁₁₀pol⁺ and AB₂₅₃rec⁺ were examined by sedimentation in alkaline sucrose gradients.In both parental strains, stationary phase cells exhibited enhanced strand rejoining. In the mutants, alkylated DNA was repaired to some extent in both growth phases, but it contained a greater proportion of small DNA fragments compared to the parental strains. Some DNA breakdown occured in all four strains but this was most extensive in stationary phase cells of the repair-deficient mutants.These results indicate that the four strains can rejoin EMS-induced DNA strand breaks with varying efficiency depending on the physiological state and the genetic capacity for repair.     

Modulation of carcinogen chromatin-DNA interaction by polyamines

The methylation of rat liver chromatin DNA has been studied in vitro by the direct-acting carcinogen N-methyl N-nitrosourea. It is shown that spermine inhibits the methylation of chromatin DNA at the N⁷ and O⁶ positions of guanine and the N³ position of adenine. However, spermine does not inhibit the methylation of 2-deoxy-5′-guanilic acid included as an internal control in the reaction. Under the experimental conditions, spermine exerts no influence on the degradation of N-methyl N-nitrosourea. The study has revealed that compounds like spermine or spermidine which bind tightly to DNA can modulate carcinogen-DNA interaction either by altering the net charge and/or the conformation of DNA.     

Enzymatic repair of O-alkylated thymidine residues in DNA: involvement of a O4-methylthymine-DNA methyltransferase and a O2-methylthymine DNA glycosylase

The distribution and intracellular translocation of AFB1 in various subcellular fractions was investigated in isolated hepatocytes by pulse-chase experiments. After labeling the hepatocytes with [3H]-AFB1 (14.5 nM) for 15 min, the highest concentration of [3H]-AFB1 was found in the cytosolic fraction where 66% was bound noncovalently and 1.5% covalently. The lowest concentration of [3H]-AFB1 was found in the nuclear fraction; 36% and 4.9% were bound noncovalently and covalently respectively. When the [3H]-AFB1 loaded cells were chased with unlabeled AFB1 (1 microM), the radioactivity of [3H]-AFB1 in the cell lysate and cytosolic fraction decreased in time with an apparent rate of elimination (t1/2) of 93 min and 66 min, respectively. The levels of covalently bound AFB1 increased with time and reached a maximum at 60 min in nuclei (270%), and at 120 min in mitochondria (220%) and cytosol (430%) as compared to the zero time. Only in the microsomal fraction was there no significant increase with time in covalently bound AFB1. These results suggest that the toxin after activation by the microsomal mixed function oxidases was either detoxified or transported to other cellular organelles where covalent binding of macromolecules occurred.     

Studies of the possibility that RNA polymerase participates in mutagenesis in bacteriophage T4

An increased rate of mutagenesis of phage T₄ by base analogues was observed in Escherichia coli strains resistant to both rifampicin and streptolydigin and shown to have defective RNA polymerase. The results suggest that RNA polymerase may be involved in the production of mutations by errors of replication.     

Screening of protein-ligand interactions by affinity chromatography

This paper examines affinity chromatography (AC) as an alternative tool for the determination of protein-ligand interactions for the particular case in which the ligand is the same protein. The methodology is less labor-intensive and more sample-efficient than traditional methods used to measure the second virial coefficient (B(22)), a parameter commonly used to evaluate protein-protein interactions. The chromatographic capacity factor (k') was studied for lysozyme and equine serum albumin for a wide range of experimental solution conditions such as crystallizing agent concentration, protein concentration and pH. Parallel experiments using AC to determine k' and static light scattering (SLS) to determine B(22) showed that the two parameters were highly correlated. Two different column volumes ( approximately 1 and approximately 0.1 mL) were tested and gave essentially the same values for k', showing the feasibility of miniaturization.     

Low-molecular weight nuclear RNA components in human lymphocytes cultured without or with phytohaemagglutinin

Purified human lymphocytes were cultured without or with phytohaemagglutinin (PHA) in the presence of radioactive RNA precursors. RNA was extracted with phenol at 0°, 40° or 62°C and separated on polyacrylamide gels. RNA extracted with phenol either in presence or absence of the RNAse inhibitor diethylpyrocarbonate showed no sign of degradation when separated on 2.6 or 3% polyacrylamide gels. Ten percent gel profiles of whole cell or nuclear RNA showed a a number of small mol. wt RNA components (K, L, M, N, A, B, C, D, F) apart from tRNA, 5 S RNA and 5.5 S RNA. Profiles of cytoplasmic RNA showed only components K and L apart from tRNA, 5 S RNA and 5.5 S RNA. L, C, D and F have an electrophoretic mobility similar to the corresponding components in various ascites cells, while M, N and B may be unique for human cells.The low-molecular wt nuclear RNA components (snRNA) are found in non-stimulated as well as in PHA-stimulated cells and the relative amounts of the snRNA components are not changed during PHA-induced transformation. It is therefore concluded that the relative amounts of the different snRNA components are not related to the dynamic state of the cell.     

Prediction of affinity and kinetics in biomolecular interactions by affinity chromatography

Computer simulation of affinity chromatography is a valuable tool for accurate prediction of column performance. In our study affinity pairs based on lectin and antibody interactions with carbohydrates have been used as model systems. In this well-characterized system we have demonstrated the usefulness of the simulation approach for determination of affinity and kinetics. These properties are typically difficult to obtain for many weakly interacting molecular species (i.e., when dissociation constants (K(D)) are greater than 10(-5) M). The influence of affinity and kinetics on peak broadening in affinity chromatography has also been investigated.     

Analysis of ribonuclease-nucleotide interactions by quantitative affinity chromatography.

Quantitative affinity chromatography on uridine-5'-(Sepharose-4-aminophenylphosphoryl)-2'(3')-phosphate was developed for the study of binding of ribonuclease species to nucleotide ligands. Elution of the native species ribonuclease-A and -S on the afffinity matrix in 0.4 M ammonium acetate, pH 5.2, containing various amounts of the soluble competing ligand 2'-cytidine monophosphate, reveals an inverse response of elution volume to concentration of soluble ligand. This response conforms to behavior expected for the competing binding equilibria enzyme-soluble ligand and enzyme-insoluble ligand. A-NALYSIS OF ELUTION DATA ALLOWS CALCULATION OF KI and KIM, the dissociation constants, respectively, for the soluble and insoluble protein-ligand complexes. The values of these chromatographically derived constants are similar to values of dissocation constants determined in solution by kinetics of inhibition by 2'-cytidine monophosphate and uridine-5'-(j-aminophenylphosphoryl)-2'(3')-phosphate. Successful competitive elution experiments with [p-F-Phe8]semisynthetic ribonuclease-S' and individual elution trials for [4-F-His12]semisynthetic ribonuclease-S' indicate the utility of the quantitative affinity chromatographic technique for determination of ligand binding properties of ribonuclease derivatives, including inactive species. Nonbiospecific aspects of the interaction of ribonuclease with the affinity matrix in ammonium acetate buffers of concentrations 0.1 M and below were noted, delinating limits of conditions allowing the biospecificity needed for ligand-binding analyses by competitive elution. The dependence of ribonuclease competitive elution behavior on the amount of protein eluted also was examined and related to theoretical considerations in the quantitative application of affinity chromatography.     

A method of DNA quantitation for localization of DNA in metrizamide gradients.

A quantitative assay for DNA is described that involves the binding of the cytologicalstain, methyl green, to DNA. At pH 7.9, solutions containing free methyl green undergo complete fading whereas solutions containing DNA-bound methyl green retain color in proportion to the amount of DNA present. The procedure allows for the quantitation of DNA in the presence of urea, sucrose, EDTA, protein, dithiothreitol, metrizamide, and low-concentration salts. This method is also applicable to the quantitation of DNA in chromatin.     

Further results on lipase-colipase interactions studied by affinity chromatography

Affinity chromatography of lipase on a colipase-coupled gel was studied in the present paper. The elution volume of the associable lipase increased when the loaded amount decreased. A KD value of 1.9 X 10(-6) M at pH 6.2 was thus deduced. A minimum value of 1.5 X 10(-6) M was obtained at pH 5.1-5.3. Mixed micelles associated with coupled colipase, but no modifications of lipase-colipase interactions took place when mixed micelles were added to the elution buffer. DMMA-modified coupled colipase failed to interact with lipase, owing to the specific orientation of the modified cofactor in the gel.     

Evidence of DNA repair in the guinea pig pancreas, in vivo and in vitro, following exposure to N-methyl-N-nitrosourethane

The nature of DNA damage induced by N-methyl-N-nitrosourethane (NMUT) in the guinea pig pancreas, both in vitro and in vivo, and subsequent repair was investigated by alkaline sucrose density gradient analysis, using a non-radioactive fluorimetric procedure for DNA determination in gradient fractions. In vitro exposure of pancreatic slices to 20 mM NMUT for 30 min damaged DNA to less than 2.24 . 10(6) dalton fragments. However, incubation of NMUT-treated slices for 3 h in a fresh medium resulted in the repair of most of DNA damage, as indicated by the conversion of low molecular weight DNA fragments into heavy DNA of molecular weight comparable to DNA from control slices. Additionally, a single administration of NMUT (30 mg/kg, i.p.) to guinea pigs induced extensive DNA damage, to less than 2.24 . 10(6) dalton fragments in the pancreas within 4 h; similar DNA damage was observed in the liver. However, in the pancreas and liver of guinea pigs sacrificed at increasing intervals after NMUT administration, there was a gradual conversion of shortened DNA fragments to heavy high molecular weight DNA, indicating repair of DNA damage. It appears that most of DNA damage in the pancreas and liver was repaired by 14 and 7 days, respectively, following NMUT administration.     

Nuclear thyroid hormone receptors: evidence for association with nucleolar chromatin.

When hypothyroid rat liver nuclei labeled $\text{in vivo}$ with [125 I]L-triiodothyronine are incubated with micrococcal nuclease, the nuclear chromatin is digested and chromatin particles are released into the medium. The nuclease-treated nuclei contain intact nucleoli and a residual chromatin fraction. When this residual chromatin is purified, it contains only a small percentage of the initial nuclear DNA but is strikingly enriched in [125 I]L-triiodothyronine. This chromatin fraction has many of the characteristics of nucleolar chromatin including a high protein to DNA ratio, an abundance of nonhistone proteins, and a relatively high RNA to DNA ratio. An association of thyroid hormone receptors with a nucleolar component implicates this organelle in the early events of thyroid hormone action.     

Use of protein-protein interactions in affinity chromatography.

Biospecific recognition between proteins is a phenomenon that can be exploited for designing affinity-chromatographic purification systems for proteins. In principle, the approach is straightforward, and there are usually many alternative ways, since a protein can be always found which binds specifically enough to the desired protein. Routine immunoaffinity chromatography utilizes the recognition of antigenic epitopes by antibodies. However, forces involved in protein-protein interactions as well the forces keeping the three-dimensional structures of proteins intact are complicated, and proteins are easily unfolded by various factors with unpredictable results. Because of this and because of the generally high association strength between proteins, the correct adjustment of binding forces between an immobilized protein and the protein to be purified as well as the release of bound proteins in biologically active form from affinity complexes are the main problem. Affinity systems involving interactions like enzyme-enzyme, subunit-oligomer, protein-antibody, protein-chaperone and the specific features involved in each case are presented as examples. This article also aims to sketch prospects for further development of the use of protein-protein interactions for the purification of proteins.     

Repair and replication of DNA in rat and mouse tissues in relation to cancer induction by N-nitroso-N-alkyl-ureas

The high susceptibility of certain organs, for example rat brain, to induction of cancer by N-nitroso-N-alkyl-ureas, has been related to a low ability to remove O⁶-alkylguanine (O⁶AG) from DNA. It is therefore reasonable to ask why mouse brain, in which there is also a slow disappearance of O⁶AG from DNA after treatment with nitroso-alkyl-ureas, is not susceptible and why, in mice, thymus and lung are the main target organs. The explanation of the species difference could lie in the fact that replication of alkylated DNA is an essential event in initiation. If nitroso-alkyl-ureas had a greater inhibitory effect in some organs than in others, replication might be inhibited until after the O⁶AG had been removed, so preventing replication of DNA while still alkylated. This concept was tested by comparing the effect of N-nitroso-N-methyl-urea (NMU) on incorporation of [³H]TdR into DNA of relevant organs in Wistar rats and C57BL mice, and by determining ability to remove O⁶AG from DNA by measuring the alkyl acceptor protein (AAP) concentrations in these organs. No evidence was obtained that the AAP content was lower or inhibition of replication was less extensive in the organ of the species more susceptible to carcinogenesis than in the same organ of the less susceptible species.