The genome of the natural genetic engineer Agrobacterium tumefaciens C58.

The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

Effects of 5' regulatory-region polymorphisms on paraoxonase-gene (PON1) expression.

Human HDL-associated paraoxonase (PON1) hydrolyzes a number of toxic organophosphorous compounds and reduces oxidation of LDLs and HDLs. These properties of PON1 account for its ability to protect against pesticide poisonings and atherosclerosis. PON1 also hydrolyzes a number of lactone and cyclic-carbonate drugs. Among individuals in a population, PON1 levels vary widely. We previously identified three polymorphisms in the PON1 regulatory region that affect expression levels in cultured human hepatocytes. In this study, we determined the genotypes of three regulatory-region polymorphisms for 376 white individuals and examined their effect on plasma-PON1 levels, determined by rates of phenylacetate hydrolysis. The -108 polymorphism had a significant effect on PON1-activity level, whereas the -162 polymorphism had a lesser effect. The -909 polymorphism, which is in linkage disequilibrium with the other sites, appears to have little or no independent effect on PON1-activity level in vivo. Other studies have found that the L55M polymorphism in the PON1-coding region is associated with differences in both PON1-mRNA and PON1-activity levels. The results presented here indicate that the L55M effect of lowered activity is not due to the amino acid change but is, rather, largely due to linkage disequilibrium with the -108 regulatory-region polymorphism. The codon 55 polymorphism marginally appeared to account for 15.3% of the variance in PON1 activity, but this dropped to 5% after adjustments for the effects of the -108 and Q192R polymorphisms were made. The -108C/T polymorphism accounted for 22.8% of the observed variability in PON1-expression levels, which was much greater than that attributable to the other PON1 polymorphisms. We also identified four sequence differences in the 3' UTR of the PON1 mRNA.

Polymorphisms in the human paraoxonase (PON1) promoter.

Paraoxonase (PON1) is a protein component of high-density lipoprotein (HDL) particles that protects against oxidative damage to both low-density lipoprotein and HDL and detoxifies organophosphorus pesticides and nerve agents. A wide range of expression levels of PON1 among individuals has been observed. We examined the promoter region of PON1 for genetic factors that might affect PON1 activity levels. We conducted a deletion analysis of the PON1 promoter region in transient transfection assays and found that cell-type specific promoter elements for liver and kidney are present in the first 200bp upstream of the coding sequence. Sequence analysis of DNA from a BAC clone and a YAC clone identified five polymorphisms in the first 1000 bases upstream of the coding region at positions -108, -126, -162, -832 and -909. Additionally, the promoter sequences of two individuals expressing high levels of PON1 and two individuals expressing low levels of PON1 were analysed. The two polymorphisms at -126 and -832 had no apparent effect on expression level in the reporter gene assay. The polymorphisms at position -909, -162 (a potential NF-I transcription factor binding site) and -108 (a potential SP1 binding site) each have approximately a two-fold effect on expression level. The expression level effects of the three polymorphisms appear not to be strictly additive and may depend on context effects.

Construction of biosensors using a gold-binding polypeptide and a miniature integrated surface plasmon resonance sensor.

Surface plasmon resonance (SPR) biosensors were constructed on miniature integrated sensors. Recognition elements were attached to the sensor surface using a gold-binding repeating polypeptide. Biosensors with fluorescyl groups attached to their surfaces were functional for at least 1 month of daily use with little decrease in response to the binding of an anti-fluorescyl monoclonal antibody. The coupling of protein A to the gold-binding polypeptide on the sensor surface enabled the biosensor to detect the binding of antibodies to the protein A and provided a sensor with convertible specificity. The system described herein provides a simple and rapid approach for the fabrication of highly specific, durable, portable and low cost SPR-based biosensors.

Effect of bending strain on the torsion elastic constant of DNA.

The torsion constants of both circular and linear forms of the same 181 bp DNA were investigated by time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium. The ratio of intrinsic ethidium binding constants of the circular and linear species was determined from the relative fluorescence intensities of intercalated and non-intercalated dye in each case. Possible changes in secondary structure were also probed by circular dichroism (CD) spectroscopy. Upon circularization, the torsion constant increased by a factor of 1.42, the intrinsic binding constant for ethidium increased by about fourfold, and the CD spectrum underwent a significant change. These effects are attributed to an altered secondary structure induced by the bending strain. Quantitative agreement between torsion constants obtained from the present FPA studies and previous topoisomer distribution measurements on circular DNAs containing 205 to 217 bp removes a long-standing apparent discrepancy between those two methods. After storage at 4 degrees C for eight months, the torsion constant of the circular DNA increased by about 1.25-fold, whereas that of the linear DNA remained unchanged. For these aged circles, both the torsion constant and intrinsic binding constant ratio lie close to the corresponding values obtained previously for a 247 bp DNA by analyzing topoisomer distributions created in the presence of various amounts of ethidium. The available evidence strongly implies that torsion constants measured for small circular DNAs with less than 250 bp are specific to the altered secondary structure(s) therein, and are not applicable to linear and much larger circular DNAs with lower mean bending strains.

Structural organization of the human PON1 gene.

Serum paraoxonase/arylesterase (PON) is an "A" esterase found in the HDL2 fraction of mammalian sera closely associated with apolipoproteins A1 and J. This enzyme hydrolyzes the active metabolites (oxons) of several organophosphate (OP) insecticides as well as the P-F bond of the nerve agents soman and sarin. PON also destroys biologically active, multioxygenated phospholipids. Two factors result in large individual variations in PON serum levels, a substrate-dependent activity polymorphism and large individual differences in PON protein levels that are stable over time. Animal model studies indicate that PON activity levels are likely to play a major role in determining sensitivity to OPs. The arg192 PON isoform appears to be a risk factor in coronary artery disease. We report here the characterization of a 28-kb contig encompassing 300 bp of 5' sequence, the entire coding region, and 2 kb of 3'-flanking sequence of the PON gene. The structural portion of the paraoxonase protein is encoded by nine exons that form the primary transcript through the use of typical splice donor and acceptor sites. DNA sequences of the regions surrounding all the coding exons have been determined. A polymorphic CA repeat is located in intron 4.

Monte Carlo simulations of supercoiling free energies for unknotted and trefoil knotted DNAs.

A new Monte Carlo (MC) algorithm is proposed for simulating inextensible circular chains with finite twisting and bending rigidity. This new algorithm samples the relevant Riemann volume elements in a uniform manner, when the constraining potential vanishes. Simulations are performed for filaments comprising 170 subunits, each containing approximately 28 bp, which corresponds to a DNA length of 4770 bp. The bending rigidity is chosen to yield a persistence length, P = 500 A, and the intersubunit potential is taken to be a hard-cylinder potential with diameter d = 50 A. This value of d yields the same second virial coefficient as the electrostatic potential obtained by numerical solution of the Poisson-Boltzmann equation for 150 mM salt. Simulations are performed for unknotted circles and also for trefoil knotted circles using two different values of the torsional rigidity, C = (2.0 and 3.0) x 10(-19) dyne cm2. In the case of unknotted circles, the simulated supercoiling free energy varies practically quadratically with linking difference delta l. The simulated twist energy parameter ET, its slope dET/dT, and the mean reduced writhe /delta l for C = 3 x 10(-19) dyne cm2 all agree well with recent simulations for unknotted circles using the polygon-folding algorithm with identical P, d, and C. The simulated ET vs. delta l data for C = 2.0 x 10(-19) dyne cm2 agree rather well with recent experimental data for p30 delta DNA (4752 bp), for which the torsional rigidity, C = 2.07 x 10(-19) dyne cm2, was independently measured. The experimental data for p30 delta are enormously more likely to have arisen from C = 2.0 x 10(-19) than from C = 3.0 x 10(-19) dyne cm2. Serious problems with the reported experimental assessments of ET for pBR322 and their comparison with simulated data are noted. In the case of a trefoil knotted DNA, the simulated value, (ET)tre, exceeds that of the unknotted DNA, (ET)unk, by approximately equal to 1.40-fold at magnitude of delta l = 1.0, but declines to a plateau about 1.09-fold larger than (ET)unk when magnitude of delta l > or = 15. Although the predicted ratio, (ET)tre/(ET)unk approximately equal to 1.40, agrees fairly well with recent experimental measurements on a 5600-bp DNA, the individual measured ET values, like some of those reported for pBR322, are so large that they cannot be simulated using P = 500 A, d = 50 A, and any previous experimental estimate of C.

Effect of ethidium binding and superhelix density on the supercoiling free energy and torsion and bending constants of p30 delta DNA.

Topoisomer distributions created by the action of topoisomerase I on p30 delta DNA in the presence of various concentrations of ethidium are measured and analyzed using recently developed theory to obtain the twist energy parameter (ET) that governs the free energy of supercoiling in each case. Competitive dialysis experiments to investigate the relative affinity of ethidium for linear and supercoiled DNAs at different binding ratios are assayed fluorometrically and the results are analyzed using related theory. The topoisomer distributions and fluorescence intensity ratios agree well with the theory, which is based on the assumption that the supercoiling free energy varies quadratically with the effective linking difference, regardless of ethidium binding or superhelix density. The topoisomer distribution experiments alone yield an average best-fit value, ET = 950 +/- 80, independent of ethidium binding ratio from r = 0 to 0.082, while the combined topoisomer distribution and ethidium binding experiments yield an average best-fit value, ET = 1030 +/- 90, which is essentially independent of ethidium binding ratio from r = 0 to 0.082 and superhelix density from sigma = 0 to (-)0.053. One may conclude that the supercoiling free-energy-varies quadratically with effective linking difference over the entire range of observed ethidium binding ratios and superhelix densities. The independently measured torsion constant (alpha) of p30 delta DNA is likewise essentially independent of superhelix density and ethidium binding ratio. The observed invariance of ET and alpha implies that the bending constant kappa beta is similarly invariant to superhelix density and ethidium binding ratio. The apparently ideal behavior displayed by p30 delta DNA is not exhibited by pBR322 DNA, which is discussed in the following companion paper.

Effect of ethidium binding and superhelix density on the apparent supercoiling free energy and torsion constant of pBR322 DNA.

The value of the twist energy parameter (ET) of pBR322 is determined near zero superhelix density from topoisomer distributions created under various conditions. The resulting value, ET = 1155 +/- 65, at 37 degrees C is essentially unaffected by adding 10 mM Mg2+, or by changing the kind of Topo I from chicken-red-cell to calf-thymus. This value significantly exceeds that (ET = 950 +/- 80) measured for p30 delta DNA under identical conditions by the same method in the preceding paper. Decreasing the temperature from 37 to 21 degrees C yields a slightly larger value, ET = 1340 +/- 130, but the statistical significance of the increase is marginal. Attempts to determine reliable ET values for pBR322 at higher superhelix densities by ethidium binding were frustrated by the fact that good fits of the equilibrium dialysis results could not be achieved using a single value of ET. Moreover, the curves of apparent ET versus binding ratio r vary considerably from one preparation to another, and for a given preparation vary with time after cell lysis up to about seven weeks, after which they settle in to nearly reproducible behavior. The apparent ET values obtained from competitive dialysis experiments are typically rather low (ET approximately 700) for small r and nearly native superhelix density, and rise up to 1300 to 1500 with increasing binding ratio (up to r = 0.055) and decreasing negative superhelix density.(ABSTRACT TRUNCATED AT 250 WORDS)

A model for the binding of E. coli single-strand binding protein to supercoiled DNA.

A model is proposed for the binding of E. coli single strand binding protein (SSB) to supercoiled DNA. The basic tetrameric binding units of SSB are assumed to bind in pairs to the complementary single strands of a locally melted region. The cooperativity of the binding includes contributions from both protein-protein and base-pair stacking interactions. Each bound SSB tetramer is assumed to unwind l = 34 bp, which implies an unwinding angle of 3.27 turns. The resulting loss of superhelical strain is the essential driving force for binding SSB to supercoiled DNAs. All molecular parameters entering into the theory are estimated from available data, except for the composite binding constant (Ka), which is adjusted to best-fit the theory to the fluorescence quenching (FQ) and diffusion coefficient (D0) data of Langowski et al. Very good fits are obtained with optimum values of Ka that are consistent with estimates from other data. This binding model predicts several noteworthy features. (1) SSB binds essentially always in a single contiguous stack on a supercoiled plasmid, and relative fluctuations in stack length are quite small, in agreement with results of electron microscopy studies. (2) The progressive loss of superhelical strain with increasing bound ligand decreases the affinity of the DNA for SSB. This anti-cooperativity offsets the cooperativity of the binding and causes apparent saturation of the binding at rather low binding ratios. Consequently, over the limited span of the measurements, the FQ data can also be satisfactorily fitted by a non-cooperative model comprising a small number of independent sites. (3) When SSB binds to a population of different topoisomers, the distribution of linking differences of the resulting complexes is extremely narrow. Thus, SSB acts to level any differences in superhelical strain in a population of topoisomers. Finally, the effects of restricting binding to a region comprising only part of the plasmid are assessed.

Circularization of small DNAs in the presence of ethidium: a theoretical analysis.

A rigorous theory is developed for ethidium binding to linear and circular DNAs and for the ratios of topoisomers produced upon ligation of an equilibrium population of noncovalently closed circles in the presence of ethidium. Assuming an unwinding angle theta E = 26 degrees for intercalated ethidium, optimum values of the intrinsic binding constant, KE = 7.16 x 10(4) M-1, the intrinsic twist, l0 = 23.746 turns, and twist energy parameter, Et = 5250, are obtained by fitting the present theory to the data of Shore and Baldwin [(1993) Journal of Molecular Biology, Vol. 170, pp. 983-1007] for a 247 base pair DNA. A very good fit is achieved with these optimum values, but a poor fit results when the parameters estimated by Shore and Baldwin are employed in the same theory. Three assumptions employed in the analysis of Shore and Baldwin are found to be not strictly valid. Adoption of the present substantially larger Et value as representative of their short DNAs would allow the Et vs N data of Shore and Baldwin to conform to the shape predicted by Shimada and Yamakawa [(1985) Journal of Molecular Biology, Vol. 184, pp. 319-329] and Frank-Kamenetskii et al. [(1985) Journal of Biomolecular Structure and Dynamics, Vol. 2, pp. 1005-1012], and would imply that all of their DNAs exist in a substantially stiffer than normal state.

Interaction of chloroquine with linear and supercoiled DNAs. Effect on the torsional dynamics, rigidity, and twist energy parameter.

The magnitude and uniformity of the torsion elastic constant (alpha) of linear pBR322 DNA and supercoiled pBR322 DNAs with high-twist (sigma = -0.083) and normal-twist (sigma = -0.48) are measured in 0.1 M NaCl as a function of added chloroquine/base-pair ratio (chl/bp) by studying the fluorescence polarization anisotrophy (FPA) of intercalated ethidium dye. The time-resolved FPA is measured by using a picosecond dye laser for excitation and time-correlated single-photon counting detection. A general theory is developed for the binding of ligands that unwind superhelical DNAs, and the simultaneous binding of two different intercalators is treated in detail. The equilibrium constant (K) for binding chloroquine to linear pBR322 DNA and the number (r) of bound chloroquines per base pair are determined from the relative amplitude ratio of the slow (normally intercalated) and fast (free) components in the decay of the (probe) ethidium fluorescence intensity as a function of chl/bp. For chloroquine binding to supercoiled pBR322 DNAs, the intrinsic binding constant is assumed to be the same as for the linear DNA, but the twist energy parameter ET (N times the free energy to change the linking number from 0 to 1 in units of kBT) is regarded as adjustable. Using the best-fit ET, the binding ratios r are calculated for each chl/bp ratio. Twist energy parameters are also determined for ethidium binding to these supercoiled DNAs by competitive dialysis. For chloroquine binding, we obtain ET = 360 and 460 respectively for the normal-twist and high-twist supercoiled DNAs. For ethidium binding the corresponding values are ET = 280 +/- 70 and 347 +/- 50. Like other dye-binding values, these are substantially lower than those obtained by ligation methods. In the absence of chloroquine, the torsion constants of all three DNAs are virtually identical, alpha = (5.0 +/- 0.4) x 10(-12) dyn.cm. For linear pBR322 DNA, the magnitude and uniformity of alpha remain unaltered by intercalated chloroquine up to r = 0.19. This finding argues that the FPA is not significantly relaxed by diffusion of any kinks or solitons. If alpha d denotes the torsion constant between a dye and a base pair and alpha 0 that between two base pairs, then our data imply that alpha d/alpha 0 lies in the range 0.65-1.64, with a most probable value of 1.0.(ABSTRACT TRUNCATED AT 400 WORDS)