The relationship between frameshift mutagenicity and DNA-binding affinity in a series of acridine-substituted derivatives of the experimental antitumour drug 4′-(9-acridinylamino)methanesulphonanilide (AMSA)

The mutagenicity and DNA-binding affinity of members of a series of acridine-substituted derivatives of 4′-(9-acridinylamino)methanesulphonanilide (AMSA) have been compared. The series includes compounds ranging from highly active to inactive in the L1210 murine leukaemia. Binding to DNA was measured by an ethidium displacement technique, with a correction being made for acridine-induced quenching of ethidium. Mutagenicity was assessed by measuring the reversion frequencies of the frameshift tester strain Salmonella typhimurium TA1537 in liquid culture. The results indicate that maximum mutagenicity is found in a “window” of DNA-binding affinities between 10⁶ and 5 × 10⁶ M^?1 (determined at 0.01 ionic strength). Compounds with binding affinities below 10⁶ M^?1 generally lacked both antitumour and mutagenic activity, whereas those with affinities above 5 × 10⁶ M^?1 were active against L1210 leukaemia but virtually inactive in inducing frameshift mutations.     

A characterization of alpha-bungarotoxin binding in the brain of the horseshoe crab, Limulus polyphemus

The binding of ¹²⁵I-labeled α-bungarotoxin to membrane fragments prepared from Limulus brain tissue has been investigated. Toxin binding approaches saturation in the range of 30 to 40 nm, with maximum binding of 2 to 6 pmol/mg of protein. The saturation kinetics and the rate of displacement of bound toxin are consistent with multiple toxin binding sites. Pharmacological studies show that binding is inhibited by both cholinergic agonists and antagonists, I₅₀′s for inhibition by d-tubocurarine, nicotine, decamethonium, carbachol, and atropine are 2 × 10^?6, 7 × 10^?6, 2 × 10^?5, 6 × 10^?4, and 3 × 10^?4m, respectively. Nicotinic ligands inhibited binding much more effectively than muscarinic ligands. Toxin binding activity was solubilized with Triton X-100. Velocity sedimentation analysis of the solubilized activity revealed three separate components. Seventy to eighty percent of the binding activity had a sedimentation coefficient of 8.6 S. The remaining activity was composed of two components with sedimentation coefficients of 15.1 and 17 S.     

Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31?kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K_D) for binding of MerR were: binding site I, 8.5?×?10^?9?M; binding site II, 1.2?×?10^?8?M; and for the complete promoter/operator region 1?×?10^?8?M. The half-life of the MerR-DNA complex was 19.4?min and 18.8?min for binding site I and binding site II, respectively. The K_D value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1?×?10^?7?M.     

Purification,characterization and docking studies of the HIN domain of human myeloid nuclear differentiation antigen (MNDA)

The HIN domain of myeloid nuclear differentiation antigen (MNDA) was expressed and purified as a monomer using E. coli JM109 as host. The protein interacted with double-stranded DNA at a K_d of 3.15 μM and did not recognize the termini of double-stranded DNA. Isothermal titration calorimetry indicated that the interaction between the protein and double-stranded DNA is mainly mediated by electrostatic attractions and hydrogen bonding. We developed a model to analyze the potential DNA binding site of the MNDA HIN domain. Based on the model, molecular docking and mutation studies suggest that the double-stranded DNA binding site of the protein is different from other HIN–DNA structures. This work facilitates the design of specific drugs against pathogens detected by human MNDA.     

Interaction of the regulatory gene product with the operator site in the l-arabinose operon of Escherichia coli

The DNA binding properties of the araC protein in the absence of l-arabinose have been studied in Escherichia coli using the nitrocellulose membrane filter technique. Equilibrium competition experiments demonstrate that the araC protein binds specifically to the ara operator. The apparent K_m of the interaction is 1 × 10^?12m at 20 °C. The rates of association and dissociation of the complex have also been determined. A k_a of 2 × 10⁹m^?1 s^?1at 20 °C is calculated assuming binding to a single site. The half-life of the complex is three minutes. The equilibrium constant calculated from the ratio of k_a to k_d is 2.8 × 10^?12m at 20 °C. The good agreement between the equilibrium and kinetic determinations of the equilibrium constant suggest that the kinetic studies are providing true rate constants. It is calculated that about 1% of the purified araC protein is active with respect to operator binding activity.     

Properties of α-bungarotoxin binding sites in foetal human brain

Maximum levels of binding of α-bungarotoxin to foetal human brain membranes were found to remain essentially constant at 30–50 fmol/mg protein (1.1–1.5 pmol/g wet weight in whole brain) between gestational ages of 10 and 24 weeks. Equilibrium binding of α-bungarotoxin to both membranes and to detergent extracts showed saturable specific binding to a single class of sites with K_d (app) values of 3.5 × 10^?9 M and 2.4 × 10^?9 M respectively. Association rate constants, determined from time courses of binding of α-bungarotoxin to membranes and detergent extracts, were 2.3 × 10⁵ M^?1 sec^?1 and 2.6 × 10⁵ M^?1 sec^?1 respectively. Dissociation of α-bungarotoxin from both membrane and detergent extracts showed a rapid initial rate with $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx 15 min which, in the case of the detergent extract, was followed by a slower dissociation accounting for the remaining 20% of the bound ligand. Competition studies with a number of cholinergic ligands indicated that the α-bungarotoxin-binding sites in foetal brain display a predominantly nicotinic profile.     

Affinity and Conformation of the Estrogen Receptor When Bound to Single-Stranded vs. Double-Stranded Nonspecific DNA

Abstract

The affinity of the hormone-bound estrogen receptor for single-stranded and double-stranded DNA was compared using isocratic elution chromatography. The receptor bound single-stranded DNA with a two-fold higher affinity than double-stranded DNA (17.9 × 10⁴ M^?1 vs. 9.1 × 10⁴ M^?1) at 0.2 M KCl. The same number of ions were released when the receptor bound either single-stranded or double-stranded DNA (11.8 vs. 10.6, respectively). These results indicate the hormone-bound estrogen receptor has no strong preference for single-stranded vs. double-stranded nonspecific DNA, and has a similar conformation when bound to either form of DNA at physiological salt concentrations.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

Asymmetric crossing over in the spontaneous formation of large deletions in the tonB-trp region of the Escherichia coli K-12 chromosome

The chromosomal tonB gene of Escherichia coli was used as a target for the detection of spontaneous deletion mutations. The deletions were isolated in both recA ⁺ and recA ^? cells, and mutants carrying large deletions were identified because they also lacked part or all of the trp operon. The frequencies of tonB-trp deletion were 1.79?×?10^?9 and 1.09?×?10^?9 for recA ⁺ and recA ^? cells, respectively. We analyzed 12 deletions from recA ⁺ and 10 from recA ^? cells by cloning and direct sequencing. The deletions ranged in size from 5612?bp to 15142?bp for recA ⁺ and from 5428?bp to 13289 for recA ^? cells. Three deletions from recA ⁺ cells and five deletions from recA ^? cells were found to have occurred between short sequence repeats at the termini of the deletion, leaving one copy of the repeat in the mutant sequence. Seven deletions from recA ⁺ cells and three deletions from recA ^? cells did not have repeats at their termini; in these cases, the DNA sequences that are adjacent to the deletion termini in the wild-type are characterized by short (2–4?bp) repeats. From these results, a model is presented for the generation of deletion mutations which involves formation of an asymmetric crossover mediated by repeated sequences of 2- to 4-bp.     

Somatostatin binding to pituitary plasma membranes.

A method has been developed for the study of somatostatin binding to anterior pituitary plasma membranes. When 5×10^?9M [¹²⁵I]Tyr¹-somatostatin (SA 18 Ci/mmol) was incubated with isolated pituitary plasma membranes (protein = 100 μg), 13.6% of total radioactivity was bound excluding nonspecific binding. The Scatchard plot could be resolved into two distinct components and analyzed to yield: K₁diss = 3.3×10^?8M and K₂diss = 7.7×10^?6M. This binding was shown to be specific for somatostatin.     

Deciphering the role of the AT-rich interaction domain and the HMG-box domain of ARID-HMG proteins of Arabidopsis thaliana

ARID-HMG DNA-binding proteins represent a novel group of HMG-box containing protein family where the AT-rich interaction domain (ARID) is fused with the HMG-box domain in a single polypeptide chain. ARID-HMG proteins are highly plant specific with homologs found both in flowering plants as well as in moss such as Physcomitrella. The expression of these proteins is ubiquitous in plant tissues and primarily localises in the cell nucleus. HMGB proteins are involved in several nuclear processes, but the role of ARID-HMG proteins in plants remains poorly explored. Here, we performed DNA-protein interaction studies with Arabidopsis ARID-HMG protein HMGB11 (At1g55650) to understand the functionality of this protein and its individual domains. DNA binding assays revealed that AtHMGB11 can bind double-stranded DNA with a weaker affinity (K_d?=?475?±?17.9 nM) compared to Arabidopsis HMGB1 protein (K_d?=?39.8?±?2.68 nM). AtHMGB11 also prefers AT-rich DNA as a substrate and shows structural bias for supercoiled DNA. Molecular docking of the DNA-AtHMGB11 complex indicated that the protein interacts with the DNA major groove, mainly through its ARID domain and the junction region connecting the ARID and the HMG-box domain. Also, predicted by the docking model, mutation of Lys⁸⁵ from the ARID domain and Arg¹⁹⁹ & Lys²⁰² from the junction region affects the DNA binding affinity of AtHMGB11. In addition, AtHMGB11 and its truncated form containing the HMG-box domain can not only promote DNA mini-circle formation but are also capable of inducing negative supercoils into relaxed plasmid DNA suggesting the involvement of this protein in several nuclear events. Overall, the study signifies that both the ARID and the HMG-box domain contribute to the optimal functioning of ARID-HMG protein in vivo.     

Spectroscopic Studies on the Interaction of Fluorine Containing Triazole with Bovine Serum Albumin

The binding of one fluorine including triazole (C₁₀H₉FN₄S, FTZ) to bovine serum albumin (BSA) was studied by spectroscopic techniques including fluorescence spectroscopy, UV–Vis absorption, and circular dichroism (CD) spectroscopy under simulative physiological conditions. Fluorescence data revealed that the fluorescence quenching of BSA by FTZ was the result of forming a complex of BSA–FTZ, and the binding constants (K _a) at three different temperatures (298, 304, and 310 K) were 1.516?×?10⁴, 1.627?×?10⁴, and 1.711?×?10⁴?mol L^?1, respectively, according to the modified Stern–Volmer equation. The thermodynamic parameters ΔH and ΔS were estimated to be 7.752 kJ mol^?1 and 125.217 J?mol^?1?K^?1, respectively, indicating that hydrophobic interaction played a major role in stabilizing the BSA–FTZ complex. It was observed that site I was the main binding site for FTZ to BSA from the competitive experiments. The distance r between donor (BSA) and acceptor (FTZ) was calculated to be 7.42 nm based on the Förster theory of non-radioactive energy transfer. Furthermore, the analysis of fluorescence data and CD data revealed that the conformation of BSA changed upon the interaction with FTZ.     

DNA-binding protein from HeLa cells that binds preferentially to supercoiled DNA damaged by ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene

A DNA-binding protein was partially purified from extracts of HeLa cells by high-speed centrifugation and chromatography on DEAE-cellulose, phosphocellulose and ultraviolet light-irradiated DNA-cellulose columns. It eluted from the phosphocellulose column with 0.375 M potassium phosphate and from the ultraviolet light-irradiated DNA-cellulose column between 0.5 M and 1 M NaCl. The protein binds preferentially to supercoiled PM2 DNA treated with ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene, as compared to native supercoiled PM2 DNA. The binding is non-cooperative. Nicked or linear forms of PM2 DNA (damaged or untreated) are not efficient substrates, indicating a requirement of DNA supercoiling for DNA binding. The sedimentation coefficient of the protein estimated by glycerol gradient centrifugation is 2.0–2.5 S, corresponding to a molecular weight of about 20 000–25 000 if the protein is spherical. The binding to DNA irradiated with ultraviolet light or treated with acetoxyacetylaminofluorene is optimal at around 100–200 mM NaCl and is relatively independent of temperature and pH. MgCl₂ and MnCl₂ at concentrations between 1 and 5 mM do not markedly affect the binding, but it is inhibited by sucrose, ATP and caffeine. The biological significance of the DNA-binding protein remains to be determined. It does not possess significant glycosylase, endonuclease or exonuclease activities. The dissociation equilibrium constant for the binding reaction of the protein to the ultraviolet light or acetoxyacetylaminofluorene-induced binding sites on DNA is estimated to be 4·10^?11 M. There are at least 1·10⁵ DNA-binding protein molecules/HeLa cell.     

Studies on the interaction of the food colorant tartrazine with double stranded deoxyribonucleic acid

Interaction of the food additive tartrazine with double-stranded DNA was studied by spectroscopic and calorimetric techniques. Absorbance studies revealed that tartrazine exhibited hypochromism in the presence of DNA without any bathochromic effects. Minor groove displacement assay of DAPI and Hoechst 33258 suggested that tartrazine binds in the minor groove of DNA. The complexation was predominantly entropy driven with a smaller but favorable enthalpic contribution to the standard molar Gibbs energy. The equilibrium constant was evaluated to be (3.68?±?.08)?×?10⁴?M^?1 at 298.15?K. The negative standard molar heat capacity value along with an enthalpy–entropy compensation phenomenon proposed the involvement of dominant hydrophobic forces in the binding process. Tartrazine enhanced the thermal stability of DNA by 7.53?K under saturation conditions.     

Pf1 bacteriophage replication-assembly complex: X-ray fibre diffraction and scanning transmission electron microscopy

X-ray fibre diffraction and scanning transmission electron microscopy have been used to investigate the structure of an intracellular complex between circular single-stranded viral DNA and a viral DNA-binding protein. This complex is an intermediate between replication and assembly of the filamentous bacteriophage Pf1. By scanning transmission electron microscopy, the complex has a length of 1.00 μm and M_r = 29.6 × 10⁶. It consists of 1770 protein subunits, each of 15,400 M_r, and one viral DNA molecule of 2.3 × 10⁶M_r: there are 4.2 ± 0.5 nucleotides per subunit. The structure is flexible in solution, but in oriented dry fibres it forms a regular helix of 45 Å pitch having 6.0 dimeric protein subunits per turn, with an axial spacing of 7.5 Å between dimers and 1.9 Å between adjacent nucleotides. Model calculations suggest that the protein dimers may be oriented in a direction approximately perpendicular to the 45 Å helix, so that each dimer spans the two anti-parallel DNA chains. The results imply that conformational changes are required of the DNA as it is transferred from the double-stranded form to the replication-assembly complex, and subsequently to the virion.     

Characterization of a hormone receptor defect in the androgen-insensitivity mutant

Mouse kidney cytosol contains specific receptors that reversibly bind dihydrotestosterone at a concentration of 43 f moles/mg protein. [Nonstandard abbreviation: DHT, dihydrotestosterone, 17 β-hydroxy-5 α-androstan-3-one.] The equilibrium dissociation constant of the receptor-dihydrotestosterone complex is 1.3 × 10^?9M for females and 1.7 × 10^?9M for castrated males. The complex sediments at 8–9S in glycerol gradients. In males bearing the androgen-insensitivity mutation (analogous to human testicular feminization), the specific dihydrotestosterone receptor activity is decreased about 8 fold. The residual binding activity has wild type affinity (K_D = 1.5 × 10^?9M) for dihydrotestosterone and also sediments at 8–9S. Kidney cytosol from castrated mutant mice displays a new binding component with low affinity and high capacity for dihydrotestosterone.     

Identification of chromosomal locations associated with tail biting and being a victim of tail-biting behaviour in the domestic pig (Sus scrofa domesticus)

The objective of this study was to identify loci associated with tail biting or being a victim of tail biting in Norwegian crossbred pigs using a genome-wide association study with PLINK case?Ccontrol analysis. DNA was extracted from hair or blood samples collected from 98 trios of crossbred pigs located across Norway. Each trio came from the same pen and consisted of one pig observed to initiate tail biting, one pig which was the victim of tail biting and a control pig which was not involved in either behaviour. DNA was genotyped using the Illumina PorcineSNP60 BeadChip whole-genome single-nucleotide polymorphism (SNP) assay. After quality assurance filtering, 53,952 SNPs remained comprising 74 animals (37 pairs) for the tail biter versus control comparison and 53,419 SNPs remained comprising 80 animals (40 pairs) for the victim of tail biting versus control comparison. An association with being a tail biter was observed on Sus scrofa chromosome 16 (SSC16; p?=?1.6?×?10^?5) and an unassigned chromosome (p?=?3.9?×?10^?5). An association with being the victim of tail biting was observed on Sus scrofa chromosomes 1 (SSC1; p?=?4.7?×?10^?5), 9 (SSC9; p?=?3.9?×?10^?5), 18 (SSC18; p?=?7?×?10^?5 for 9,602,511?bp, p?=?3.4?×?10^?5 for 9,653,881?bp and p?=?5.3?×?10^?5 for 29,577,783 bp) and an unassigned chromosome (p?=?6.1?×?10^?5). An r ²?=?0.96 and a D???=?1 between the two SNPs at 9?Mb on SSC18 indicated extremely high linkage disequilibrium, suggesting that these two markers represent a single locus. These results provide evidence of a moderate genetic association between the propensity to participate in tail-biting behaviour and the likelihood of becoming a victim of this behaviour.     

Studies of nicotinic acetylcholine receptor protein from rat brain

Specific binding of ¹²⁵I-labeled α-bungarotoxin to a 34 800 × g pellet of a whole rat brain homogenate has been obtained at levels 2 pmol toxin per g of whole brain with a K_d of 8·10^?9 M. Binding is reduced 90% by 10^?5 M (+)- tubocurarine chloride and 10^?4 M nicotine, whereas concentrations of 10^?4 M choline chloride, atropine sulfate and eserine sulfate have essentially no effect on toxin binding. These results compare closely with those obtained from binding studies with ¹²⁵I-labeled α-bungarotoxin and soluble acetylcholine receptor protein preparations form Torpedo nobiliana; suggesting that this mammalian receptor protein is nicotinic in character.Extraction of the 34 800 × g pellet with 1% Emulphogene yields a soluble fraction with specifically binds ¹²⁵I-labeled α-bungarotoxin with a K_d of 5·10^?9 M. Nicotine and α-bungarotoxin at concentrations of 10^?5 M abolish toxin- receptor complex formation and carbachol and (+)-tubocurarine chloride reduce complex formation 35–40% at similar concentrations. Eserine sulfate, atropine sulfate, decamethonium, and pilocarpine had no effect on complex formation at concentrations of 10^?5 M.     

Effects of ecdysone analogues on development and metabolic activity of wing disks of the fleshfly, Sarcophaga peregrina,in vitro

Effects of ecdysone analogues on development and metabolic activities of Sarcophaga wing disks were studied in cultures. Development of disks was induced by ecdysterone, ponasterone A, and cyasterone in vitro, whereas rubrosterone was quite inactive in inducing development.As well as morphogenetic effects, a proper concentration (3 × 10^?5 M to 3 × 10^?7 M) was required to induce the incorporation of tritiated uridine, thymidine, and leucine into RNA, DNA, and protein, respectively. Higher concentration of the hormone was more favourable to development of disks and enhancement of RNA synthesis. However, the hormone at concentration higher than 2 × 10^?9 M seemed to be rather toxic to both development and metabolic activity.     

Absence of high-affinity adenosine 3',5'-monophosphate binding sites from the cytosol of three hepatic-derived cell lines

Binding of [³H]adenosine 3′,5′-monophosphate (cAMP) to specific sites was compared in liver and three hepatic-derived, cultured cell lines, HTC, RLC, and H4-II-E. At physiological pH, there are small though significant differences in total binding, with liver >H4-II-E = RLC > HTC. No specific inhibitor of binding was demonstrated in the cultured cells. Scatchard analysis of binding suggested the presence of two sites in liver with k_dl = 3.4 × 10^?8m and K_dll = ~ 10^?7m. The cultured cell cytosols all lack the higher-affinity site but have a site similar to K_d11 of liver, with K_d = 3.5 × 10^?7m. In all cell cytosols, basal protein kinase activity is higher than in liver, proportionally more of the kinase is cAMP independent, and the degree of cAMP stimulation of the kinase is much less than in liver. Ion exchange and exclusion chromatography revealed that the predominant protein kinase complex found in liver is virtually completely absent from HTC and RLC cells.