Transcriptome sequencing and high-resolution melt analysis advance single nucleotide polymorphism discovery in duplicated salmonids.

Until recently, single nucleotide polymorphism (SNP) discovery in nonmodel organisms faced many challenges, often depending upon a targeted-gene approach and Sanger sequencing of many individuals. The advent of next-generation sequencing technologies has dramatically improved discovery, but validating and testing SNPs for use in population studies remain labour intensive. Here, we detail a SNP discovery and validation pipeline that incorporates 454 pyrosequencing, high-resolution melt analysis (HRMA) and 5' nuclease genotyping. We generated 4.59×10(8) bp of redundant sequence from transcriptomes of two individual chum salmon, a highly valued species across the Pacific Rim. Nearly 26000 putative SNPs were identified--some as heterozygotes and some as homozygous for different nucleotides in the two individuals. For validation, we selected 202 templates containing single putative SNPs and conducted HRMA on 10 individuals from each of 19 populations from across the species range. Finally, 5' nuclease genotyping validated 37 SNPs that conformed to Hardy-Weinberg equilibrium expectations. Putative SNPs expressed as heterozygotes in an ascertainment individual had more than twice the validation rate of those homozygous for different alleles in the two fish, suggesting that many of the latter may have been paralogous sequence variants. Overall, this validation rate of 37/202 suggests that we have found more than 4500 templates containing SNPs for use in this population set. We anticipate using this pipeline to significantly expand the number of SNPs available for the studies of population structure and mixture analyses as well as for the studies of adaptive genetic variation in nonmodel organisms.

Impact of hypericum (St.-John's-wort) given prenatally on cognition of mice offspring.

This study investigated the cognitive impact of prenatal exposure to the herbal antidepressant hypericum in CD-1 mice. Hypericum (182 mg/kg/day) or a placebo was consumed in food bars for 2 weeks before mating and throughout gestation. The hypericin content in our hypericum formulation was in the middle range of standardized hypericum products. One offspring per gender from each litter (hypericum 13, placebo 12) was tested on each of the following tasks: juvenile runway with adult memory, adult Morris maze, adult passive avoidance, or adult straight water runway followed by a dry Cincinnati maze. Learning occurred in both genders in all tasks (P<.003) with no significant differences between treatments at the final trial. Female offspring exposed to hypericum, rather than to a placebo, required more time to learn the Morris maze task (P<.05). Postlearning sessions did not show any significant differences. In conclusion, prenatal exposure to a therapeutic dose of hypericum did not have a major impact on certain cognitive tasks in mice offspring.

Topoisomerase II drives DNA transport by hydrolyzing one ATP.

DNA topoisomerase II is a homodimeric molecular machine that couples ATP usage to the transport of one DNA segment through a transient break in another segment. In the presence of a nonhydrolyzable ATP analog, the enzyme is known to promote a single turnover of DNA transport. Current models for the enzyme's mechanism based on this result have hydrolysis of two ATPs as the last step, used only to reset the enzyme for another round of reaction. Using rapid-quench techniques, topoisomerase II recently was shown to hydrolyze its two bound ATPs in a strictly sequential manner. This result is incongruous with the models based on the nonhydrolyzable ATP analog data. Here we present evidence that hydrolysis of one ATP by topoisomerase II precedes, and accelerates, DNA transport. These results indicate that important features of this enzyme's mechanism previously have been overlooked because of the reliance on nonhydrolyzable analogs for studying a single reaction turnover. A model for the mechanism of topoisomerase II is presented to show how hydrolysis of one ATP could drive DNA transport.

Kinetic and thermodynamic analysis of mutant type II DNA topoisomerases that cannot covalently cleave DNA.

DNA topoisomerase II catalyzes two different chemical reactions as part of its DNA transport cycle: ATP hydrolysis and DNA breakage/religation. The coordination between these reactions was studied using mutants of yeast topoisomerase II that are unable to covalently cleave DNA. In the absence of DNA, the ATPase activities of these mutant enzymes are identical to the wild type activity. DNA binding stimulates the ATPase activity of the mutant enzymes, but with steady-state parameters different from those of the wild type enzyme. These differences were examined through DNA binding experiments and pre-steady-state ATPase assays. One mutant protein, Y782F, binds DNA with the same affinity as wild type protein. This mutant topologically traps one DNA circle in the presence of a nonhydrolyzable ATP analog under the same conditions that the wild type protein catenates two circles. Rapid chemical quench and pulse-chase ATPase experiments reveal that the mutant proteins bound to DNA have the same sequential hydrolysis reaction cycle as the wild type enzyme. Binding of ATP to the mutants is not notably impaired, but hydrolysis of the first ATP is slower than for the wild type enzyme. Models to explain these results in the context of the entire DNA topoisomerase II reaction cycle are discussed.

Pre-steady-state analysis of ATP hydrolysis by Saccharomyces cerevisiae DNA topoisomerase II. 2. Kinetic mechanism for the sequential hydrolysis of two ATP.

In the preceding paper, we showed that DNA topoisomerase II from Saccharomyces cerevisiae binds two ATP and rapidly hydrolyzes at least one of them before encountering a slow step in the reaction mechanism. These data are potentially consistent with two different types of reaction pathways: (1) sequential ATP hydrolysis or (2) simultaneous hydrolysis of both ATP. Here, we present results that are consistent only with topoisomerase II hydrolyzing its two bound ATP sequentially. Additionally, these results indicate that the products of the first hydrolysis are released from the enzyme before the second ATP is hydrolyzed. Release of products from both the first and second hydrolyses contributes to the rate-determining process. The proposed mechanism for ATP hydrolysis by topoisomerase II is complex, having nine rate constants. To calculate values for each of these rate constants, a technique of kinetic parameter estimation was developed. This technique involved using singular perturbation theory in order to estimate rate constants, and consequently identify kinetic steps following the rate-determining step.

Pre-steady-state analysis of ATP hydrolysis by Saccharomyces cerevisiae DNA topoisomerase II. 1. A DNA-dependent burst in ATP hydrolysis.

When bound to DNA, topoisomerase II from Saccharomyces cerevisiae exhibits burst kinetics with respect to ATP hydrolysis. Pre-steady-state analysis shows that the enzyme binds and hydrolyzes two ATP per reaction cycle. Our data indicate that at least one of the two ATP is rapidly hydrolyzed prior to the rate-determining step in the reaction mechanism. When DNA is not bound to topoisomerase II, the rate-determining step shifts to become either ATP binding or hydrolysis. Two possible mechanisms are proposed that agree with our observations.

Noninfectious retinopathy.

Noninfectious retinopathy is the most commonly observed condition of the retina in patients with acquired immunodeficiency syndrome (AIDS). The hallmark of this microvasculopathy is the cotton-wool spot, which is seen with greater frequency as the immune system becomes more suppressed. Noninfectious retinopathy does not threaten vision, but clinically it must be differentiated from infectious retinitis, which can cause blindness.

Magnetic field effects on B12 ethanolamine ammonia lyase: evidence for a radical mechanism.

A change in radical pair recombination rates is one of the few mechanisms by which a magnetic field can interact with a biological system. The kinetic parameter Vmax/Km (where Km is the Michaelis constant) for the coenzyme B12-dependent enzyme ethanolamine ammonia lyase was decreased 25 percent by a static magnetic field near 0.1 tesla (1000 gauss) with unlabeled ethanolamine and decreased 60 percent near 0.15 tesla with perdeuterated ethanolamine. This effect is likely caused by a magnetic field-induced change in intersystem crossing rates between the singlet and triplet spin states in the [cob(II)alamin:5'-deoxyadenosyl radical] spin-correlated radical pair.

Sexually transmitted diseases.

In the last decade, the incidence of sexually transmitted diseases in the United States has significantly increased. This paper discusses the systemic and ocular manifestations of several of them, specifically AIDS, syphilis, gonorrhea, and chlamydia. Its primary objective is to assist the clinician in establishing an accurate diagnosis and recommending appropriate therapy.

Medical management of acquired immune deficiency syndrome patients: a review.

Patients with acquired immunodeficiency syndrome (AIDS) may become infected by a number of different opportunistic pathogens. Treatment to control the Human Immune Virus (HIV) and its associated infections can be complex, and the medications required are not commonly used on patients with normal immunity.

Cytomegalovirus retinitis complicated by optic neuropathy: a longitudinal study.

Two cases of suspected cytomegalovirus (CMV) retinitis treated with ganciclovir and followed for 18 and 5 months are presented. Each patient developed optic neuropathy and experienced reduced vision despite effective control of their retinitis. In addition to objective ocular findings, subjective visual function changes (perimetry, contrast sensitivity and accommodation) in long term follow up of AIDS patients are reported for the first time. A discussion of CMV, its treatment and complications follows.

The BITA telescope: a first impression.

The Bilevel Telemicroscope Apparatus (BITA) is a new galilean telescope designed to offer improved cosmesis, weight, field of view and spatial orientation over more traditional spectacle-mounted telescopic systems. This paper describes the characteristics of the new telescope and presents two cases where the BITA was successfully prescribed.

Molecular basis for surface antigen size polymorphisms and conservation of a neutralization-sensitive epitope in Anaplasma marginale.

Anaplasmosis is one of several tick-borne diseases severely constraining cattle production and usage in many parts of the world. Cattle can be protected from anaplasmosis by immunization with major surface protein 1, a surface protein of Anaplasma marginale carrying a neutralization-sensitive epitope. Marked size polymorphisms exist among different isolates of A. marginale in the AmF105 subunit of major surface protein 1, yet all isolates still contain the neutralization-sensitive epitope. To clarify the basis for these observations, the mspl alpha gene encoding AmF105 was cloned from four isolates and sequenced. The encoded polypeptides share a high degree of overall homology between isolates but contain a domain with various numbers of tandemly repeated sequences and three regions of clustered amino acid substitutions outside the repeat domain. The polypeptide size differences are completely explained by the variations in the numbers of tandem repeat units. We have mapped the neutralization-sensitive epitope to a sequence that is present within each repeat unit. These results identify a basis for size polymorphisms of the surface polypeptide antigen concomitant with B-cell epitope conservation in rickettsiae.