Isolation and characterization of a protein with high affinity for DNA: the glutamine synthetase of Thermus thermophilus 111

In a search of proteins from the thermophilic bacterium Thermus thermophilus 111 with a high affinity for DNA, the selected protein from this screening appears to be the glutamine synthetase (GS). The purified product gives one band in SDS-polyacrylamide gel electrophoresis (53,700 Da). The N-terminal 32 residues have been identified and present an homology of 80% with the glutamine synthetase of Bacillus subtilis and 76% with that of Thermotoga maritima. The protein displays the characteristic dodecameric structure of the eubacteria glutamine synthetase. From a detailed study of the interaction of this protein with DNA by dark-field electron microscopy and agarose gel electrophoresis, it is concluded that double-stranded DNA wraps the protein by a full turn of 150 bp length. An even number of GS molecules bound to a closed relaxed plasmid DNA does not alter its null topology. By using an inverted dimer DNA fragment, which contains twice a curved kinetoplast DNA insert in its central part, it is shown that DNA curvature rules the order in which GS binds to the DNA. DNA ends are also sites of high affinity for the GS. Supercoiling does not favor the binding of GS to the DNA with the exception of the apices that are by essence bent regions. By saturating a DNA molecule with GS one obtains a novel characteristic scalloped configuration in which the DNA undulates from one GS to the next. The DNA is condensed at least three times in these structures. By increasing the ratio of GS to DNA in solution the resulting material migrates as discrete bands relative to the free DNA in an agarose gel. By gel retardation and EM statistical distribution analysis of GS within the complexes, an average affinity constant of 10(7) M-1 was obtained. The potential implications of this novel interaction of the glutamine synthetase with DNA for the regulation of its own gene are briefly discussed.     

Interaction of homopyrimidazole derivatives with biopolymers. II. Binding to chemically modified albumins; attempt to correlate chemical structure and binding affinity

The degree of human serum albumin (HSA) binding for 16 different homopyrimidazole derivatives has been studied. The positions of UV absorption maximum, the corresponding molar extinction coefficients and the binding parameters obtained by equilibrium dialysis experiments at 4 degrees C, in pH 7.35 phosphate buffer, are surveyed. In most cases compounds with high toxicity show low binding tendency. Saturation of the ring system decreases binding, while an increase of the number of double bonds enhances the binding affinity. Important requirements for HSA binding of homopyrimidazole derivatives are the presence of a carbethoxy (or carboxyl) group in position 3 and a C6-methyl group. The highest binding affinity could be demonstrated in the case of MZ 211, an acid-type metabolite of 1,6-dimethyl-3-carbethoxy-4-oxo-6,7,8,9-tetrahydro-4H-pyrido-(1,2-alpha)-pyrimidin-1-ium-methyl-sulphate (MZ 144, Probon). Binding of MZ 144 to chemically modified albumins was studied under standard conditions. Binding studies with acetylated and methylated HSA indicated that the presence of free carboxyl groups is a requirement for binding, while the amino groups play a less important role. More selective chemical modifications of HSA were realised with acetylsalicylic acid, diethylpyrocarbonate (DEP) and o-nitrophenylsulfenyl-chloride (NPS-C1). Modifications with DEP and acetylsalicylic acid result in an increase of affinity in the binding area, probably due to blocking of a particular amino group in the vicinity of the binding site. Histidine residues are believed to be of minor importance in the binding process. When the indole ring is modified with NPS-C1, affinity at the strong binding site is significantly reduced, indicating that the Trp residue must be involved in the binding of MZ 144.     

Ligands with affinity to specific sequences of DNA base pairs. X. Synthesis and binding of netropsin analogs, containing a chelating copper ion peptide, with DNA

An analogue of netropsin has been synthesized consisting of two N-propylpyrrolcarboxamide units linked covalently to a copper-chelating tripeptide Gly-Gly-L-His by means of two and three glycine residues. Binding to DNA and synthetic polynucleotides of netropsin analogue containing three glycine residues between Gly-Gly-L-His tripeptide and the N-end of netropsin analogue (His-Nt) has been studied. It is shown that this netropsin analogue chelates a copper ion with 1:1 stoichiometry, similar to a free Gly-Gly-L-His peptide. It is found that this netropsin analogue occupies 3 to 4 base pairs upon binding to poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] polymers, irrespective of whether it binds in Cu(2+)-ligated or unligated forms. Binding constants and binding site sizes have been calculated for netropsin analogue complexes with DNA, poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] polymers at the [Cu2+]/[His-Nt] ratio equal to 0 and 1.0. In the three-component system including His-Nt and Cu(2+)-His-Nt, cooperative effects are recognized which can be explained by heterodimer generation on interaction of His-Nt and Cu(2+)-His-Nt at adjacent binding sites.     

In vitro cytotoxicity, cellular pharmacology, and DNA lesions induced by annamycin, an anthracycline derivative with high affinity for lipid membranes

Annamycin (AN) is an anthracycline antibiotic with high affinity for lipid membranes which is being developed for clinical studies formulated in liposomes. We studied the in vitro cytotoxicity, cellular pharmacology, and DNA damage induced by AN in P388 cells sensitive and resistant to doxorubicin (DOX). AN was as cytotoxic as DOX against P388-sensitive cells and about 50 times more cytotoxic than DOX against P388-resistant cells (resistance index 5 for AN versus 250 for DOX). Cellular uptake of AN by sensitive cells was 2-3-fold higher than that of DOX. In resistant cells, cellular uptake of AN and DOX was approximately 65% and 30%, respectively, of the cellular uptake in sensitive cells. As a result, cellular uptake of AN by resistant cells was higher than uptake of DOX by sensitive cells. DOX was fully retained in sensitive cells while it was effluxed rapidly from resistant cells. In contrast, efflux of AN was similar in sensitive and resistant cells, thus suggesting that it is not mediated by P-glycoprotein. AN was more effective than DOX in inducing single DNA breaks, double DNA breaks, and DNA-protein cross-links, both in sensitive and resistant cells, although DNA damage was lower in resistant cells than in sensitive cells. DNA lesions induced by AN in resistant cells were similar to or greater than those induced by DOX in sensitive cells. These studies indicate that the lack of cross-resistance between DOX and AN appears to be related, at least in part, to the relatively higher cellular uptake of AN compared with DOX and is associated with the ability of AN to induce significant DNA damage in resistant cells.     

The androgenic regulation of prostate proteins with a high affinity for deoxyribonucleic acid. Evidence for a prostate deoxyribonucleic acid-unwinding protein

1. When testosterone is injected into castrated rats in vivo, a significant increase in the incorporation of [35S]methionine into prostate proteins may be detected under conditions in vitro. 2. Studies based on DNA-cellulose chromatography show that the synthesis of prostate proteins with a high affinity for DNA is particularly enhanced by androgenic stimulation. 3. These changes in protein synthesis are negated when the anti-androgen, cyproterone acetate, is administered concomitantly with testosterone in vivo. 4. Two assays were developed for measuring the strand separation of prostate DNA; first, the retention of 3H-labelled native DNA on nitrocellulose membranes, and second, the activation of native DNA as a template for 9S prostate DNA polymerase. On the basis of these criteria, DNA-unwinding activity is present in the prostate gland and it is regulated by androgens in a steroid-and tissue-specific manner. 5. The results are discussed in the context of the mechanism of action of androgens, particularly since the changes provoked in DNA-unwinding activity by androgens precede the onset of DNA replication and mitosis.     

New Approach to Designing Multilayer Feedforward Neural Network Architecture for Modeling Nonlinear Restoring Forces. II: Applications

Based on the basic formulation developed in a companion paper, the writers now present the application of an artificial neural network approach to designing streamlined network models to simulate and identify the nonlinear dynamic response of single-degree-of-freedom oscillators using the restoring force-state mapping interpretation. The neural networks which use sigmoidal activation functions are shown to be highly robust in modeling a wide variety of commonly observed nonlinear structural dynamic response behaviors. By streamlining the networks, individual network model parameters take on physically or geometrically interpretable meaning, and hence, the network initialization can be achieved through an engineered approach rather than through less physically meaningful numerical initialization schemes. Although not proven in general, examples show that by starting with a more meaningful initial design, identification convergence is improved, and the final identified model parameters are seen to have a more physical meaning. A set of model architecture prototypes is developed to capture commonly observed nonlinear response behaviors.     

Reactions of the eco RV restriction endonuclease with fluorescent oligodeoxynucleotides: identical equilibrium constants for binding to specific and non-specific DNA

The EcoRV restriction endonuclease cleaves DNA specifically at its recognition sequence in the presence of magnesium ions, but several studies have indicated that it binds to DNA in the absence of Mg2+ without any preference for its recognition site. However, specific binding to the recognition site has also been reported. To distinguish between these reports, oligodeoxynucleotides were tagged with either dansyl or eosin fluorophores at their 5' termini and annealed to form duplexes of 12 to 16 base-pairs. For each length of duplex, one derivative had the EcoRV recognition sequence while another lacked this sequence. For the duplexes with the recognition site, the fluorophores had no effect on DNA cleavage rates by EcoRV in the presence of Mg2+. The binding of the specific and non-specific duplexes to EcoRV in the absence of Mg2+ was measured by fluorescence resonance energy transfer and by fluorescence depolarization. In both procedures, the signal from the specific complex differed from the complex with non-specific DNA, with the depolarization data indicating that non-specific DNA bound to EcoRV retains a higher rotational freedom than specific DNA. Even so, the equilibrium constant for the binding of specific DNA was identical, within error limits, to that for non-specific DNA.     

Subcycling-PCR for multiplex long-distance amplification of regions with high and low GC content: application to the inversion hotspot in the factor VIII gene

OBJECTIVE: Treatment of long or multiple anterior urethral stricture(s) when Monseur technique is not applicable. Our technique entails augmentation of the dorsally slit open stenosed urethra using pedicled non-hair bearing penile skin. PATIENTS AND METHODS: Between June 1991 and May 1996, 26 men (median age 34 years) with anterior urethral strictures underwent roofing urethroplasty. Nine patients had long stricture (average 3.2 cm) and 17 had multiple short segment strictures (average 7 cm). All patients were circumcised, and dorsal urethral augmentation was performed using transversely oriented non-hair bearing penile skin pedicled flap. RESULTS: Median follow-up was 38 months (range 3-50). A successful outcome with no recurrent stricture as evidenced by normal retrograde urethrography and voiding history was achieved in 23 of 26 men (88%). Two patients had fistula in early postoperative period; one of them needed surgical closure. CONCLUSION: Roofing urethroplasty is a practical alternative for repair of long anterior urethral stricture(s) when Monseur technique cannot be applied.     

A partially functional DNA helicase II mutant defective in forming stable binary complexes with ATP and DNA. A role for helicase motif III

To address the functional significance of motif III in Escherichia coli DNA helicase II, the conserved aspartic acid at position 248 was changed to asparagine. UvrDD248N failed to form stable binary complexes with either DNA or ATP. However, UvrDD248N was capable of forming an active ternary complex when both ATP and single-stranded DNA were present. The DNA-stimulated ATPase activity of UvrDD248N was reduced relative to that of wild-type UvrD with no significant change in the apparent Km for ATP. The mutant protein also demonstrated a reduced DNA unwinding activity. The requirement for high concentrations of UvrDD248N to achieve unwinding of long duplex substrates likely reflects the reduced stability of various binary and ternary complexes that must exist in the catalytic cycle of a helicase. The data suggest that motif III may act as an interface between the ATP binding and DNA binding domains of a helicase. The uvrDD248N allele was also characterized in genetic assays. The D248N protein complemented the UV-sensitive phenotype of a uvrD deletion strain to levels nearly equivalent to wild-type helicase II. In contrast, the mutant protein only partially complemented the mutator phenotype. A correlation between the level of genetic complementation and the helicase activity of UvrDD248N is discussed.     

DNA "melting" proteins. IV. Fluorescence measurements of binding parameters for bacteriophage T4 gene 32-protein to mono-, oligo-, and polynucleotides

The quenching of the intrinsic tryptophan fluorescence of T4-coded gene 32-protein on binding to nucleotide ligands, which was described in the preceding paper, is here exploited to measure thermodynamic parameters of the single-stranded nucleic acid-gene 32-protein interaction. It is shown that binding of small ligands follows a single site binding isotherm, with association constants increasing from approximately 20 M-1 for phosphate, to approximately 10(3) M for ribose or deoxyribose 5'-phosphate, to approximately 10(4) M-1 for mononucleotides, and to approximately 10(5) M-1 for dinucleoside monophosphates (all in 0.1 M Na+). The measured binding constants appear to be about the same for homologous ribose- and deoxyribose-containing ligands and to be independent of oligonucleotide base sequence and composition. Furthermore, beyond the dinucleotide level and up to octanucleotides, the increase in binding constant with increasing chain length is only about that expected from the statistical factor resulting from the increased number of ways a longer oligonucleotide can form a protein complex. This suggests that the basic binding unit involved in gene 32-protein associations with single-stranded nucleic acids can be approximated by a dinucleoside monophosphate. Oligonucleotides long enough to accomodate two or more protein monomers are characterized by much larger association constants, indicating that binding is cooperative in protein concentration. A cooperativity parameter (omegac) of approximately 10(3) is estimated from these data, in good agreement with that deduced from the application of ligand-perturbed helix in equilibrium coil transition calculations. Values of association constants (Kcomegac) of approximately 10(8) M-1 (in 0.1 M Na+) and site size (nc) of approximately 5 (+/-1) nucleotide residues/protein monomer are determined by the fluorescence titration technique for the cooperative binding of gene 32-protein to both poly(dA) and poly(rA); these values are also in agreement with those measured by Jensen et al. (Jensen, D.E. Kelly, R.C., and von Hippel, P.H. (1976) J. Biol. Chem. 251, 7215-7228). Possible in vivo consequences and correlations of these findings with proposed roles for gene 32-protein in replication and recombination are discussed.     

Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. II. Structure of the active site with ADP or ATP bound to wild type and mutant ATPase fragment

The ATPase fragment of the bovine 70-kDa heat shock cognate protein is an attractive construct in which to study its mechanism of ATP hydrolysis. The three-dimensional structure suggests several residues that might participate in the ATPase reaction. Four acidic residues (Asp-10, Glu-175, Asp-199, and Asp-206) have been individually mutated to both the cognate amine (asparagine/glutamine) and to serine, and the effects of the mutations on the kinetics of the ATPase activity (Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 269, 12893-12898) and the structure of the mutant ATPase fragments have been determined, typically to approximately 2.4 A resolution. Additionally, the structures of the wild type protein complexed with MgADP and Pi, MgAMPPNP (5'-adenylyl-beta, gamma-imidodiphosphate) and CaAMPPNP have been refined to 2.1, 2.4, and 2.4 A, respectively. Combined, these structures provide models for the prehydrolysis, MgATP-bound state and the post-hydrolysis, MgADP-bound state of the ATPase fragment. These models suggest a pathway for the hydrolytic reaction in which 1) the gamma phosphate of bound ATP reorients to form a beta, gamma-bidentate phosphate complex with the Mg2+ ion, allowing 2) in-line nucleophilic attack on the gamma phosphate by a H2O molecule or OH- ion, with 3) subsequent release of inorganic phosphate.     

Stimulation of DNA synthesis in isolated chondrocytes by somatomedin. II. Validation of the assay for clinical use and comparison with stimulation of protein synthesis

The effects of GH on cartilage may be mediated by a variety of serum factors (somatomedins; SM). We have reported (Endocrinology 90: 1086, 1972) stimulation of thymidine incorporation in isolated chicken embryo chondrocytes by normal human serum. This was greater than that caused by serum from patients with hypopituitarism. We have now compared the stimulatory activity estimated by [3H]thymidine incorporation (SMT) with that estimated by [3H]leucine incorporation in 46 sera from children with GH deficiency; with short stature, but normal GH responsiveness; or with normal stature and normal GH responsiveness. These activities were also measured in sera from 9 normal adults and 12 acromegalics. Sera from GH deficient children had reduced SMT activity (.54 +/- .04; (mean +/- SE) P less than .01) compared to normal children (.83 +/- .08) whereas the sera from children with short stature and normal GH responsiveness had higher levels than normal (1.19 +/- .10: P less than .02). Acromegalic adults averaged higher SMT activity than normal adults (1.62 +/- .15 vs. 1.17 +/- .11; P less than .05). In sharp contrast, the leucine incorporation was essentially the same in the different groups of children. These studies have validated the use of the incorporation of thymidine into isolated chicken embryo chondrocytes as an adjunct in the evaluation of children with short stature (82.6% of the samples from children gave results that were consistent with their status as determined by provocative tests for GH). The disparity between the results with thymidine incorporation and those with leucine incorporation is as yet unexplained.     

Induction of allograft nonresponsiveness after intrathymic inoculation with donor class I allopeptides. II. Evidence for persistent chronic rejection despite high levels of donor microchimerism

We have recently demonstrated that three synthetic peptides corresponding to the donor class I RT1.Aa molecule induce long-term survival of cardiac allografts in the PVG.R8-to-PVG.1U rat strain combination (disparate for one isolated class I, RT1.A, molecule) when presented to the recipient immune system in the thymus. Long-term graft survivors had measurable levels of donor-reactive alloantibodies in their serum. In this study, we examined long-term allografts for the presence of chronic rejection and donor microchimerism to assess whether this regimen of immune modulation establishes true tolerance and whether this tolerance is dependent upon the presence of donor-recipient microchimerism. Histological examination of long-term heart grafts (>100 days) demonstrated chronic rejection, including a mild degree of myocardial infiltration by mononuclear cells, mild to moderate myocardial fibrosis, and various vascular changes ranging from focal intimal thickening to total vascular lumen blockade due to smooth muscle cell proliferation. In contrast, long-term syngeneic hearts transplanted under similar experimental conditions lacked these pathological manifestations. Donor microchimerism was analyzed using the polymerase chain reaction with a pair of oligonucleotides specific for the donor class I RT1.Aa gene and genomic DNA harvested from various tissues from graft recipients. We detected high levels of donor microchimerism in the heart, kidney, liver, skin, bone marrow, thymus, and lymph nodes of long-term graft recipients. Donor microchimerism was also detected in unmanipulated control graft recipients at rejection (7 days) and in intrathymically manipulated recipients that rejected allografts in a delayed fashion (12-82 days). These data clearly demonstrate that intrathymic inoculation of donor class I allopeptides induces long-term graft survival but does not prevent chronic rejection. Allograft rejection occurred despite high levels of donor microchimerism, providing direct evidence that donor-recipient microchimerism is not sufficient for the prevention of acute or chronic rejection in this model.     

Model for Predicting Floods due to Earthen Dam Breaching. II: Comparison with Other Methods and Predictive Use

A sensitivity analysis of the model presented in the companion paper is made and, on the basis of the results a criterion is proposed for the choice of values to assign to the side slopes of the breach, in order to use the model for prediction. Moreover, the sensitivity analysis shows that the outflow hydrograph and its peak value depend not only on the dam height and the stored volume, but also on the vertical distribution of the water mass in the reservoir. The model is compared with some previously published methods and the disadvantages, limitations, and errors that can be made using parametric models and predictive equations are pointed out. Finally, easy to use equations interpolating the numerical results of the model are provided that predict not only the peak discharge but the whole outflow hydrograph for overtopping failures.     

The physical mapping of human chromosomes. II. The production of unique chromosome-specific DNA fragments using the polymerase chain reaction with oligonucleotide primers to conservative regions of Alu repeats]

Unique DNA fragments localised between Alu-repeats have been produced by PCR. The reaction was carried out with oligonucleotide primers to conservative regions of Alu-repeats. The DNA fragments from different pulls, individual clones, chromosome-specific clonotecs derived from phage lambda, cosmids and individual human chromosomes served as matrixes. The possibilities are discussed of Alu-primer applying in production of exceptional physical features of DNA molecules, suitable for constructing clone couple groups and for direct physical mapping on the DNA of isolated chromosomes, missing the stage of cloning.