Haplotype variation and linkage disequilibrium in 313 human genes.

Variation within genes has important implications for all biological traits. We identified 3899 single nucleotide polymorphisms (SNPs) that were present within 313 genes from 82 unrelated individuals of diverse ancestry, and we organized the SNPs into 4304 different haplotypes. Each gene had several variable SNPs and haplotypes that were present in all populations, as well as a number that were population-specific. Pairs of SNPs exhibited variability in the degree of linkage disequilibrium that was a function of their location within a gene, distance from each other, population distribution, and population frequency. Haplotypes generally had more information content (heterozygosity) than did individual SNPs. Our analysis of the pattern of variation strongly supports the recent expansion of the human population.

Helicopter transport of patients to tertiary care centers after cardiac arrest.

Air transport is commonly used to transfer survivors of cardiac arrest from rural hospitals to large tertiary-care centers, presumably to improve outcome. To examine this issue, a retrospective review of patients stabilized after a cardiac arrest was conducted; 157 transports were reviewed. The mean age of patients was 37.9 +/- 27.8 yrs, with a male to female ratio of 2.2:1. Survivors were significantly older than nonsurvivors. Thirty-one of 69 patients (45%) with primary cardiac disease were discharged alive from the hospital, 75% without neurological sequelae. Only a minority of patients with noncardiac medical illness (7%), electrical injury (33%), suffocation (15%), near-drowning (15%), and inhalation (0%) were discharged alive from the hospital. Outcomes for cardiac arrest in adult patients older than 65 years (32.3% survival) were similar to those for adult patients younger than 65 years (36.2% survival) (P = .887). These results show that survivors of a primary cardiac event have a favorable outcome when transferred by air to tertiary centers when compared with historical controls that were transported by ground. On the other hand, cardiac arrests from noncardiac medical illness, suffocation, near-drowning, and inhalation have a grim prognosis. Prospective studies should clarify the role of air transport in these patients.

Three mechanistic steps detected by FRET after presynaptic filament formation in homologous recombination. ATP hydrolysis required for release of oligonucleotide heteroduplex product from RecA.

The Escherichia coli RecA protein promotes DNA strand exchange in homologous recombination and recombinational DNA repair. Stopped-flow kinetics and fluorescence resonance energy transfer (FRET) were used to study RecA-mediated strand exchange between a 30-bp duplex DNA and a homologous single-stranded 50mer. In our standard assay, one end of the dsDNA helix was labeled at apposing 5' and 3' ends with hexachlorofluorescein and fluorescein, respectively. Strand exchange was monitored by the increase in fluorescence emission resulting upon displacement of the fluorescein-labeled strand from the initial duplex. The potential advantages of FRET in study of strand exchange are that it noninvasively measures real-time kinetics in the previously inaccessible millisecond time regime and offers great sensitivity. The oligonucleotide substrates model short-range mechanistic effects that might occur within a localized region of the ternary complex formed between RecA and long DNA molecules during strand exchange. Reactions in the presence of ATP with 0.1 microM duplex and 0.1-1.0 microM ss50mer showed triphasic kinetics in 600 s time courses, implying the existence of three mechanistic steps subsequent to presynaptic filament formation. The observed rate constants for the intermediate phase were independent of the concentration of ss50mer and most likely characterize a unimolecular isomerization of the ternary complex. The observed rate constants for the first and third phases decreased with increasing ss50mer concentration. Kinetic experiments performed with the nonhydrolyzable analogue ATPgammaS showed overall changes in fluorescence emission identical to those observed in the presence of ATP. In addition, the observed rate constants for the two fastest reaction phases were identical in ATP or ATPgammaS. The observed rate constant for the slowest phase showed a 4-fold reduction in the presence of ATPgammaS. Results in ATPgammaS using an alternate fluorophore labeling pattern suggest a third ternary intermediate may form prior to ssDNA product release. The existence of two or three ternary intermediates in strand exchange with a 30 bp duplex suggests the possibility that the step size for base pair switching may be 10-15 bp. Products of reactions in the presence of ATP and ATPgammaS, with and without proteinase K treatment, were analyzed on native polyacrylamide gels. In reactions in which only short-range RecA-DNA interactions were important, ATP hydrolysis was not required for recycling of RecA from both oligonucleotide products. Hydrolysis or deproteinization was required for RecA to release the heteroduplex product, but not the outgoing single strand.

Preferential interactions of the Escherichia coli LexA repressor with anions and protons are coupled to binding the recA operator.

The binding of Escherichia coli LexA repressor to the recA operator was examined as a function of the concentration of NaCl, KCl, NaF, and MgCl2 at pH 7.5, 21 degrees C. The effects of pH at 100 mM NaCl were also examined. Changes both in the qualitative appearance of the binding isotherms and in the magnitude of the apparent binding affinity with changes in solution conditions suggest that binding of anions and protons by LexA repressor is linked to oligomerization and/or operator binding. Binding of LexA repressor to the recA operator in the presence of NaCl ranging from 25 to 400 mM at picomolar DNA concentration showed a broad, apparently noncooperative, binding isotherm. Binding of LexA repressor in NaF at the same [DNA] yielded binding isotherms with a narrow transition, reflecting an apparently cooperative binding process. Also, the apparent binding affinity was weaker in NaF than in NaCl. Furthermore, the binding affinity and also the apparent binding mode, cooperative vs noncooperative, were pH dependent. The binding affinity of LexA repressor for operator was greatest near neutral pH. The apparent binding mode was noncooperative at pH 7-9 but was cooperative at pH 6 or 9.3. These observations suggest that the specific cation and anion composition and concentrations must be considered in understanding the details of regulation of the SOS system.

Equilibrium constants for nonspecific binding of proteins to DNA may be obtained by quantitative zonal DNA affinity chromatography.

Quantitative zonal DNA affinity chromatography may be used to determine accurate equilibrium constants for the binding of proteins nonspecifically to DNA. Zonal quantitative affinity chromatography has not previously been applied to the determination of binding constants of proteins to DNA, although its use is quite commonplace for determination of affinity constants for protein-protein or protein-small ligand interactions. Equilibrium constants were measured for the nonspecific binding of bovine pancreatic ribonuclease A and Escherichia coli lac repressor to double-stranded DNA immobilized on cellulose. The equilibrium constants determined agree with literature values evaluated using other techniques. The experimental advantages of the zonal technique, when it can be applied, are that collection of data is fast and data analysis is simple. Detection of the protein elution profile by absorbance at 220 nm with an in-line detector can provide adequate sensitivity when binding constants are in the range 10(2)-10(4) M-1.

Binding of Escherichia coli LexA repressor to the RecA operator.

Equilibrium binding of Escherichia coli LexA repressor to the recA operator was studied by the polyacrylamide gel mobility shift assay as a function of solution conditions. In the presence of NaCl at 20 degrees C, there was a significant salt dependence in binding to the recA operator, typical for protein-nucleic acid interactions with some electrostatic contribution to the binding free energy. In preliminary experiments in which the anion of the Na+ salt was changed from chloride to fluoride, little change was found with anion identity. This indicates that the salt effect on the binding interaction arises solely from the polyelectrolyte effect, not from anion binding or release by the protein upon complex formation. Increasing the temperature to 37 degrees C changed the binding affinity for complex formation at any given salt concentration and resulted in a change in the sensitivity of complex formation to NaCl concentration. Quantitative analysis of the data to obtain equilibrium binding constants is discussed.

Flight crew physical fitness: a baseline analysis.

INTRODUCTION: The purpose of this pilot study was to determine whether flight crew personnel are physically fit in comparison to published standards for the average American adult. SETTING: The study group consisted of pilots, paramedics and nurses in two similarly configured and geographically located rotor-wing air medical transport programs. METHODS: A physical fitness assessment of flight crew members was conducted. The results were compared with published standards for average adult males and females (AVG). Percentage of fat in body composition (FM%), aerobic fitness (VO2MAX), muscular endurance (ME), muscular strength (MS) and flexibility (FL) measurements were obtained using accepted testing methods. RESULTS: The study population consisted of 29 male and 21 female individuals. The following were their mean scores. Males averaged: pFAT = 19% (AVG = 20.0%); VO2MAX = 41.0 (AVG = 42.5); ME = 37.0 (AVG = 28.5); MS = 125.0 (AVG = 86.5); FL = 5.2 (AVG = 1.4); Females averaged: pFAT = 28.0% (AVG = 26.5%); VO2MAX (AVG = 34.0); ME = 27.0 (AVG = 21.0); MS = 83.0 (AVG = 76.5); FL = 4.5 (AVG = 3.4). CONCLUSION: These baseline data suggest the study population of air medical flight crew was physically fit compared to the average American adult.

The distribution of Escherichia coli recA protein bound to duplex DNA with single-stranded ends.

A short single-stranded tail on one end of an otherwise duplex DNA molecule enables recA protein, in the presence of ATP and MgCl2, to form a complex with the DNA which extends into the duplex portion of the molecule. Nuclease protection studies at a concentration of MgCl2 which permits homologous pairing showed that cleavage by restriction endonucleases at sites throughout the duplex region was inhibited, whereas digestion by DNase I was not affected. These results indicate that recA protein binds to the duplex portion of tailed DNA allowing access by DNase I to a random sample of the many sites at which it cleaves, but providing limited protection of the relatively rare restriction sites. Electron microscopy revealed that the recA nucleoprotein complex with duplex DNA is indeed a segmented or interrupted filament that, with time, extends further from the single-stranded tail into the duplex region. recA protein binding extended into the duplex region more rapidly for duplexes with 5' tails than for those with 3' tails. These observations show that recA protein translocates from a single-stranded region into duplex DNA in the form of a segmented filament by a mechanism that is not strongly polarized.

Translocation of Escherichia coli recA protein from a single-stranded tail to contiguous duplex DNA.

Duplex DNA with a contiguous single-stranded tail was nearly as effective as single-stranded DNA in acting as a cofactor for the ATPase activity of recA protein at neutral pH and concentrations of MgCl2 that support homologous pairing. The ATP hydrolysis reached a steady state rate that was proportional to the length of the duplex DNA attached to a short 5' single-stranded tail after a lag. Separation of the single-stranded tail from most of the duplex portion of the molecule by restriction enzyme cleavage led to a gradual decline in ATP hydrolysis. Measurement of the rate of hydrolysis as a function of DNA concentration for both tailed duplex DNA and single-stranded DNA cofactors indicated that the binding site size of recA protein on a duplex DNA lattice, about 4 base pairs, is similar to that on a single-stranded DNA lattice, about four nucleotides. The length of the lag phase preceding steady state hydrolysis depended on the DNA concentration, length of the duplex region, and the polarity of the single-stranded tail, but was comparatively independent of tail length for tails over 70 nucleotides in length. The lag was 5-10 times longer for 3' than for 5' single-stranded tailed duplex DNA molecules, whereas the steady state rates of hydrolysis were lower. These observations show that, after nucleation of a recA protein complex on the single-stranded tail, the protein samples the entire duplex region via an interaction that is labile and not strongly polarized.

Mechanism of the concerted action of recA protein and helix-destabilizing proteins in homologous recombination.

Secondary structure in single-stranded DNA impedes the presynaptic association of recA protein and consequently blocks the formation of joint molecules as evidenced by effects of temperature, nucleotide sequence, and ionic conditions. Escherichia coli single-strand-binding protein eliminates sequence-specific "cold spots" by removing folds even from sites of strong secondary structure. Thus, destabilization of secondary structure in single-stranded DNA is critical for the action of recA protein, whereas specific interactions directly between helix-destabilizing proteins and recA protein are unimportant.

Molecular weight and subunit stoichiometry of the chloroplast coupling factor 1 from Chlamydomonas reinhardi.

The molecular weight of the Chlamydomonas reinhardi coupling factor 1 (CF1) is 4.2 X 10(5) as determined by gel exclusion chromatography and sedimentation equilibrium. In addition, a measured sedimentation coefficient of 12.9 S results in a calculated molecular weight of 3.9 X 10(5). These molecular weight estimates are too high to support an alpha 2 beta 2-type subunit stoichiometry and are suggestive of an alpha 3 beta 3-type enzyme. The subunit stoichiometry of the C. reinhardi CF1 was determined from the distribution of label into the subunits of uniformly labeled CF1. An alpha: beta: gamma: epsilon ration of 2.9:2.9:1:1 was obtained.

Aggregation equilibria of Escherichia coli RNA polymerase: evidence for anion-linked conformational transitions in the protomers of core and holoenzyme.

The aggregation equilibria of Escherichia coli RNA polymerase core and holoenzyme have been studied by velocity sedimentation as a function of [NaCl] both in the presence and in the absence of MgCl2. Effects of other anions (F- and I-), pH, and temperature have also been examined. Diffusion coefficients obtained by quasi-elastic light scattering (QLS) at high and low salt concentrations were used in conjunction with sedimentation coefficients under these conditions to obtain molecular weights of the protomer and aggregates of the core enzyme. At low salt concentration, core aggregates to a tetramer in the absence of MgCl2 and to an octamer in the presence of MgCl2. Some ambiguity exists in the interpretation of the sedimentation and QLS data for holoenzyme. The sedimentation results are consistent with the formation of dimers at low salt, both in the presence and in the absence of MgCl2. In all cases, equilibrium constants were calculated assuming a simple monomer--j-mer stoichiometry. These equilibrium constants are extremely sensitive functions of the concentration and type of monovalent anion. In Cl-, aggregation of both core and holoenzyme begins abruptly when the salt concentration is reduced below approximately 0.2 M (at a protein concentration of approximately 0.30 mg/mL); for core, substitution of I- for Cl- suppresses aggregation while F- enhances aggregation at a fixed anion concentration. No specific effect of monovalent cations (Na+, NH4+) is observed; Mg2+ has no effect on holoenzyme dimerization and has little effect on the salt range of core aggregation, though the stoichiometries of the core aggregates in the presence and absence of Mg2+ differ. Anion effects on these equilibria were modeled by assuming that a class of anion-binding sites on the protomer is not present in the aggregate, so that anion release accompanies aggregation. Analytical expressions for several models of the effect of anions on the aggregation equilibria were derived by using the method of binding polynomials. The salt dependence of the aggregation equilibria in the absence of Mg2+ appears inconsistent with a model in which the anion-binding sites on the protomer are independent (noncooperative), but it is well described by a model in which anion binding to the protomers occurs in a completely cooperative manner. The molecular basis of this apparent cooperative effect of anions on the aggregation equilibria is proposed to be an allosteric effect of anions on conformational equilibria of the protomers of core polymerase and the holoenzyme. Implications of such a salt-dependent conformational transition for the DNA-binding interactions of the enzyme are considered.