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.     

DNA ligase activity in crude extracts of fibroblasts and lymphocytes

DNA ligase activity was determined in crude cell extracts using a new assay which measures the retention of double stranded circular phage λ DNA on nitrocellulose filters, and allows accurate determinations of the enzyme activity with cell concentration corresponding to 0.1 μg of proteins. Using this assay, we show that the DNA ligase activity varies greatly among mammalian cell lines. The higher activity is found in actively growing fibroblasts where it is stimulated by dimethyl sulfate pretreatment of the cells, whereas the low activity measured in resting lymphocytes is not modified by dimethyl sulfate. The DNA ligase activity correlates with the cells sensitivity towards ionizing radiations.     

The repair of x-ray-induced chromosome aberrations in stimulated and unstimulated human lymphocytes

The repair time for chromosome breaks induced by X-irradiation of unstimulated (G₀) and stimulated (G₁) human lymphocytes has been determined by dose fractionation studies. In both types of cells repair time was approx. 4–5 h. Treatment with hydroxyurea, a DNA synthesis inhibitor, did not prevent or delay the rejoining of broken chromosomes, whereas treatment with cycloheximide, a potent protein synthesis inhibitor, did. Thus, the repair of radiation-induced chromosome breaks in human lymphocytes is similar to the repair observed with plant cells.     

Formylation of enzyme-bound valine and stepwise elongation of intermediate peptides of gramicidin A by a cell-free enzyme system.

A protein, tentatively named apo-Q-protein I, with molecular weight of 15,000¹ from the reconstitutively active cytochrome $\text{b-c}{}_1$ complex has been identified as being responsible for electron transfer between succinate dehydrogenase and ubiquinone. The identification was based on the chemical modification, proteolytic enzyme digestion, and isolation and purification of the protein to nearly pure form.     

Serum albumin biosynthesis and secretion by resting and lectin stimulated human lymphocytes

Normal human peripheral lymphocytes, cultured in serum-deprived medium, synthetized and released serum albumin and some glycoproteins into the culture supernatant. With the use of [3H]leucine, it was shown that this biosynthetic activity was increased about 2-3 times when the mitogenic lectin from Robinia pseudo acacia was added to the lymphocyte culture medium.     

Physical map of DNA from a new cauliflower mosaic virus strain

The restriction enzymes AluI, BamHI, BglII, EcoRI, HindIII, and SalI have been used to characterize and map a new cauliflower mosaic virus strain (Cabb-S). These fragments have been ordered by examining their overlapping regions after double enzymatic digestion. The single SalI cleavage site was chosen as the point of origin. We compare this strain with those already described.     

DNA distortion and specificity in a sequence-specific endonuclease

Five new structures of the Q138F HincII enzyme bound to a total of three different DNA sequences and three different metal ions (Ca²⁺, Mg²⁺, and Mn²⁺) are presented. While previous structures were produced from soaking Ca²⁺ into preformed Q138F HincII/DNA crystals, the new structures are derived from cocrystallization with Ca²⁺, Mg²⁺, or Mn²⁺. The Mn²⁺-bound structure provides the first view of a product complex of Q138F HincII with cleaved DNA. Binding studies and a crystal structure show how Ca²⁺ allows trapping of a Q138F HincII complex with noncognate DNA in a catalytically incompetent conformation. Many Q138F HincII/DNA structures show asymmetry, despite the binding of a symmetric substrate by a symmetric enzyme. The various complexes are fit into a model describing the different conformations of the DNA-bound enzyme and show how DNA conformational energetics determine DNA-cleavage rates by the Q138F HincII enzyme.     

Solubilization and hydrophobic immobilization assay of a cAMP binding protein from Dictyostelium discoideum plasma membranes

A cAMP binding site present on isolated plasma membranes of aggregation-competent $\text{D.}\text{discoideum}$ cells has been solubilized with the nonionic detergent Emulphogene BC-720. An assay has been developed based on the principle of hydrophobic chromatography, in which the detergent solubilized cAMP binding protein is immobilized on alkyl-agarose beads at low detergent concentration. This allows the necessary rapid separation of bound and free [³H]-cAMP by filtration of the beads. The kinetics and nucleotide specificity of the detergent solubilized cAMP binding protein are comparable to those of the cAMP chemotactic receptor on intact cells and plasma membranes. The alkyl-agarose bead assay may have general utility for the assay of detergent solubilized membrane receptors.     

Serum, bradykinin and vasopressin stimulate release of inositol phosphates from human fibroblasts

The mitogens serum, vasopressin and bradykinin stimulate a significant rise in the inositol phosphate content of cultured human fibroblasts within 10 seconds, while serum- and bradykinin-stimulated arachidonic acid release does not occur until after 30 seconds. The release of inositol phosphates is not secondary to a rise in Ca activity since the Ca ionophore ionomycin does not stimulate release of inositol phosphates. Moreover, we show that phospholipase C in human fibroblasts is activated by these mitogens at resting Ca levels since TMB-8, which blocks the mitogen-induced rise in Ca activity, does not affect the serum-stimulated accumulation of inositol phosphates.     

A DNA dependent ATPase from HeLa cells

The purification and the properties of the major DNA dependent ATPase from HeLa cells are described. This enzyme is present in the nucleus and in the cytoplasm in approximately equal amounts. It has a Mr of about 110000 dalton and it hydrolyzes ATP (and dATP) to ADP+Pi only in the presence of single-stranded DNA. The enzyme shows an ATP dependent unwinding activity on DNA duplex, with a 3′ to 5′ polarity of the unwound strand. Under certain conditions the enzyme is able to stimulate the activity of DNA polymeraseα on appropriate DNA templates. Such stimulation is synergistic with that exerted by a DNA binding protein from calf thymus.     

The role of protein kinases in ACTH-stimulated steroidogenesis.

The effect of highly purified bovine cytosolic adrenal cortical protein kinase isozyme II catalytic subunit (ATP: Protein Phosphotransferase EC 2.7.1.37) on the formation of pregnenolone from cholesterol in rat and bovine adrenal mitochondrial extracts has been investigated. No stimulation was observed although a low level incorporation of [³²P] from [³²P]-ATP into a component or components of the extract was detected. The mitochondrial extracts contained alkaline phosphatase activity that was inhibited by L-cysteine and dithiothreitol. It is concluded that the acute stimulation by ACTH of corticoid production in the rat adrenal does not involve protein kinase mediated phosphorylation of a component or components of the cholesterol sidechain cleavage mixed-function oxygenase system.     

The effect of trans-stilbene oxide and other structurally related inducers of drug-metabolizing enzymes on glucuronidation

Administration of trans-stilbene oxide, and new type of inducer of drug-metabolizing enzymes, to rats was found to increase hepatic microsomal UDP-glucuronyl transferase activity with both p-nitrophenol and chloramphenicol as substrate. In Triton X-100 activated microsomes the increase with p-nitrophenol as substrate was to approx. 250% of the control value, while the corresponding value for chloramphenicol was about 600%. These observations indicate that trans-stilbene oxide causes a mixed type 'induction' of UDP-glucuronyl transferase(s), i.e., changes in activity which resemble both those seen after induction with phenobarbital and after treatment with 3-methylcholanthrene. We have also shown that the activity of UDP-glucose dehydrogenase, the enzyme which produces UDP-glucuronic acid, is increased to about 300% of the control after administration of trans-stilbene oxide. The time course of this increase and of the return to control activity after cessation of treatment, the dose-response of this increase and the structural features of the trans-stilbene oxide molecule which are essential for the increase have all been examined. The other two enzymes involved in the conversion of glucose 6-phosphate to UDP-glucuronic acid, namely, phosphoglucomutase and UDP-glucose pyrophosphorylase, were found to be only slightly affected (a 30-60% increase) by treatment with trans-stilbene oxide. After induction with trans-stilbene oxide the hepatic level of UDP-glucuronic acid was unchanged.     

Apurinic and/or apyrimidinic endonuclease activity in ataxia telangiectasia cell extracts.

The possibility that the increased sensitivity of ataxia telangiectasia towards ionizing radiation is related to a DNA-repair deficiency has been examined further. When compared to unaffected controls, 6 lines of fibroblast cells derived from ataxia patients demonstrated a slightly reduced endonucleolytic activity (165 +/- 12 units vs. 214 +/- 28 units) towards apurinic and/or apyrimidinic sites as determined in a "nicking" assay.     

Lumcorin: A leucine-rich repeat 9-derived peptide from human lumican inhibiting melanoma cell migration

We previously showed that lumican decreases melanoma progression. The aim of the present study was to determine the active sequence of the lumican core protein responsible for the inhibition of melanoma cell migration. Using different recombinant and synthetic peptides derived from lumican, we localized an active site in the leucine-rich repeat 9 domain of the lumican core protein. We propose the name lumcorin (fragment of lumican core protein) for the active peptide derived from this site. Lumcorin was able to inhibit melanoma cell migration in vitro.     

A simple and sensitive apparatus for continuous monitoring of orthophosphate in the presence of acid-labile compounds

A new technique is described for obtaining reproducible transient-state isoelectric focusing patterns of human pancreatic secretory proteins. This method is excellent for preservation of the proteins for further analysis because of the avoidance of urea, the short running times, and low temperatures. The protein patterns were analyzed on a point-by-point basis by a computer using an extension of our previously published method (Allan, B. J., Kirk, J., and White, T. T., 1978, Biochem. Biophys. Res. Commun., 85, 1239–1246). It was possible with these techniques to subtract various types of backgrounds and to construct average densitometric tracings for statistical analysis.     

Progesterone receptor in the chick thymus

Specific, high affinity binding sites for progesterone and promegestone /R-5020/ have been shown to be present in the chick thymus measured by experiments with intact cells or under cell-free conditions. In isolated thymocytes most of the receptor-R-5020 complex is bound to the nucleus. Dissociation constants were determined in thymic cytosol by Scatchard plot analysis and were found to be 3.1 and 2.6 nM for progesterone and R-5020, respectively. On the basis of competition assays the binding sites seemed to be specific for progesterone and R-5020. Glucocorticoids bind only slightly and only at high concentrations. By gel-filtration experiments the thymic R-5020 binding site was shown to be a macromolecule. In vivo treatment of chicks with progesterone or R-5020 caused a significant increase in thymidine kinase activity of the thymus.     

Failure of 2 Hydroxyestradiol to interact with dopamine inhibition of human prolactin secretion in vitro and with dopamine receptors of prolactin-secreting adenomas

The ability of 2-Hydroxyestradiol, a catecholestrogen, and 17 beta Estradiol to interact with the dopamine inhibition of prolactin and with dopamine receptors has been tested on dispersed human prolactin-secreting cells obtained from ten pituitary adenomas. There is a 80% inhibition of prolactin secretion obtained by addition of dopamine in a superfusion system. This inhibition is not affected by preexposure to the steroids, or by their introduction into the perifusion medium. Moreover 2 Hydroxyestradiol and 17 beta Estradiol do not interact with the binding of 3H Domperidone to DA receptors.     

beta-Endorphin: evidence for the existence of opioid and non-opioid binding components for the tritiated human hormone in NG108-15 cells

Human beta-endorphin (beta h-EP) binding on neuroblastoma X glioma hybrid NG108-15 cells using tritiated human beta endorphin (3H-beta h-EP) as a primary ligand was found to have a component which was not displacable with [D-Ser2 )-Leu-enkephalin-Thr6 (DSLET). The beta h-EP binding on these cells after saturation of the delta opiate sites with 200 nM DSLET was further characterized with synthetic beta h-EP analogs. The nonopioid binding site appears to recognize beta h-EP-(6-31), beta h-EP-(21-31) and beta h-EP-(28-31). Under these conditions, these COOH-terminal segments fully displace the tritiated beta h-EP. However, beta h-EP-(1-27) does not further displace 3H-beta h-EP in the presence of DSLET. The fact that a combination of DSLET and beta h-EP-(6-31) results in a full displacement of 3H-beta h-EP provides direct evidence for the existence of two binding sites for beta h-EP in NG108-15 cells, one recognizing the NH2-terminal enkephalin sequence and the other the non-opioid COOH-terminal segment.     

Understanding apparent DNA flexibility enhancement by HU and HMGB architectural proteins

Understanding and predicting the mechanical properties of protein/DNA complexes are challenging problems in biophysics. Certain architectural proteins bind DNA without sequence specificity and strongly distort the double helix. These proteins rapidly bind and unbind, seemingly enhancing the flexibility of DNA as measured by cyclization kinetics. The ability of architectural proteins to overcome DNA stiffness has important biological consequences, but the detailed mechanism of apparent DNA flexibility enhancement by these proteins has not been clear. Here, we apply a novel Monte Carlo approach that incorporates the precise effects of protein on DNA structure to interpret new experimental data for the bacterial histone-like HU protein and two eukaryotic high-mobility group class B (HMGB) proteins binding to ∼ 200-bp DNA molecules. These data (experimental measurement of protein-induced increase in DNA cyclization) are compared with simulated cyclization propensities to deduce the global structure and binding characteristics of the closed protein/DNA assemblies. The simulations account for all observed (chain length and concentration dependent) effects of protein on DNA behavior, including how the experimental cyclization maxima, observed at DNA lengths that are not an integral helical repeat, reflect the deformation of DNA by the architectural proteins and how random DNA binding by different proteins enhances DNA cyclization to different levels. This combination of experiment and simulation provides a powerful new approach to resolve a long-standing problem in the biophysics of protein/DNA interactions.