Evolution of Single-Domain Globins in Hydrothermal Vent Scale-Worms.

Hypoxia at deep-sea hydrothermal vents represents one of the most basic challenges for metazoans, which then requires specific adaptations to acquire oxygen to meet their metabolic needs. Hydrothermal vent scale-worms (Polychaeta; Polynoidae) express large amounts of extracellular single- and multi-domain hemoglobins, in contrast with their shallow-water relatives that only possess intracellular globins in their nervous system (neuroglobins). We sequenced the gene encoding the single-domain (SD) globin from nine species of polynoids found in various vent and deep-sea reduced microhabitats (and associated constraints) to determine if the Polynoidae SD globins have been the targets of diversifying selection. Although extracellular, all the SD globins (and multi-domain ones) form a monophyletic clade that clusters within the intracellular globin group of other annelids, indicating that these hemoglobins have evolved from an intracellular myoglobin-like form. Positive selection could not be detected at the major ecological changes that the colonization of the deep-sea and hydrothermal vents represents. This suggests that no major structural modification was necessary to allow the globins to function under these conditions. The mere expression of these globins extracellularly may have been sufficiently advantageous for the polynoids living in hypoxic hydrothermal vents. Among hydrothermal vent species, positively selected amino acids were only detected in the phylogenetic lineage leading to the two mussel-commensal species (Branchipolynoe). In this lineage, the multiplicity of hemoglobins could have lessened the selective pressure on the SD hemoglobin, allowing the acquisition of novel functions by positive Darwinian selection. Conversely, the colonization of hotter environments (species of Branchinotogluma) does not seem to have required additional modifications.

[Specialist Training in Psychiatry and Psychotherapy : Evaluation of the East Westphalia-Lippe Academy curriculum]. / Facharztweiterbildung Psychiatrie und Psychotherapie : Evaluation des Curriculums eines Weiterbildungsverbunds in Ostwestfalen-Lippe.

BACKGROUND: Despite of the undisputed impact of education quality on becoming a specialist in psychiatry and psychotherapist, systematic studies in this field do not exist in Germany, apart from the evaluation of practical education by the medical councils. MATERIALS AND METHODS: The Akademie-OWL, an education union of psychiatric clinics in the East Westphalia region of Germany, has provided 12-item questionnaire-based routine evaluations of theoretical education seminars since 2009. Seminars are held by consultants of psychiatry and psychotherapy within six separate weeks over a period of four years. All lecturers receive feedback from the evaluation. The results of this ongoing evaluation are presented using a typical example and in an aggregated format as a time series on the basis of one global item. RESULTS: The data show that (1) the full range of a five-point Likert scale is used by the candidates, (2) psychiatric competences of the lecturers are more positively evaluated than their didactic competences, (3) neither within the four-years period of psychiatric education nor between different generations of candidates are systematic changes of evaluation results observed, and (4) minor to moderate differences of evaluation were observed between the different thematic fields of psychiatry and psychotherapy. DISCUSSION: These results indicate the need of more systematic education research on establishing valid and reliable specific instruments as well as the need of systematic training of didactic competences of teaching consultants.

Restless legs syndrome associated with exercise intolerance: Data from a retrospective observational clinical neuromuscular center study.

INTRODUCTION: Exercise intolerance (EI) is a frequent motive for seeking neuromuscular consultation and may be a sign of metabolic disease or, rarely, muscular dystrophy. The diagnosis is not established in many patients with a typical clinical presentation. Nevertheless, some of them complain of sleep disorders and more especially of restless legs syndrome (RLS). OBJECTIVE: The objective of our study was to estimate the frequency of RLS in patients presenting with EI. METHODS: Our retrospective observational study included all patients seen in the center from 2005 to 2011, who were subsequently investigated for EI in the neuromuscular department of the Caen University hospital. Data were collected on clinical RLS and muscular investigations (creatine kinase [CK], EMG, maximal exercise tests magnetic resonance imaging [MRI] and muscle biopsy obtained along with muscle exploration). RESULTS: Of the 318 patient records analyzed, 84 showed patients accurately complaining of EI. RLS was diagnosed in 25 of these patients (29.7%). This percentage was significantly higher (P<0.001) than found in the general population. Improvement was seen in 91.3% of the patients receiving specific treatment. CONCLUSION: RLS can sometimes present with pain, potentially worsening with exercise, inappropriately leading to a hypothesis of EI. Clinicians should thus explore the possible diagnosis of RLS when a muscular disease is not found in patients presenting with such symptoms.

Assessment of cryogenic pretreatment for simulating environmental weathering in the formation of surrogate micro- and nanoplastics from agricultural mulch film.

Microplastics (MPs) and nanoplastics (NPs) from mulch films and other plastic materials employed in vegetable and small fruit production pose a major threat to agricultural ecosystems. For conducting controlled studies on MPs' and NPs' (MNPs') ecotoxicity to soil organisms and plants and fate and transport in soil, surrogate MNPs are required that mimic MNPs that form in agricultural fields. We have developed a procedure to prepare MPs from plastic films or pellets using mechanical milling and sieving, and conversion of the resultant MPs into NPs through wet grinding, both steps of which mimic the degradation and fragmentation of plastics in nature. The major goal of this study was to determine if cryogenic exposure of two biodegradable mulch films effectively mimics the embrittlement caused by environmental weathering in terms of the dimensional, thermal, chemical, and biodegradability properties of the formed MNPs. We found differences in size, surface charge, thermal and chemical properties, and biodegradability in soil between MNPs prepared from cryogenically treated vs. environmentally weathered films, related to the photochemical reactions occurring in the environment that were not mimicked by cryogenic treatment, such as depolymerization and cross-link formation. We also investigated the size reduction process for NPs and found that the size distribution was bimodal, with populations centered at 50 nm and 150-300 nm, and as the size reduction process progressed, the former subpopulation's proportion increased. The biodegradability of MPs in soil was greater than for NPs, a counter-intuitive trend since greater surface area exposure for NPs would increase biodegradability. The result isassociated with differences in surface and chemical properties and to minor components that are readily leached out during the formation of NPs. In summary, the use of weathered plastics as feedstock would likely produce MNPs that are more realistic than cryogenically-treated unweathered films for use in experimental studies.

Origin and evolution of the unique tetra-domain hemoglobin from the hydrothermal vent scale worm Branchipolynoe.

Hemoglobin is the most common respiratory pigment in annelids. It can be intra or extracellular, and this latter type can form large multimeric complexes. The hydrothermal vent scale worms Branchipolynoe symmytilida and Branchipolynoe seepensis express an extracellular tetra-domain hemoglobin (Hb) that is unique in annelids. We sequenced the gene for the single-domain and tetra-domain globins in these two species. The single-domain gene codes for a mature protein of 137 amino acids, and the tetra-domain gene codes for a mature protein of 552 amino acids. The single-domain gene has a typical three exon/two intron structure, with introns located at their typical positions (B12.2 and G7.0). This structure is repeated four times in the tetra-domain gene, with no bridge introns or linker sequences between domains. The phylogenetic position of Branchipolynoe globins among known annelid globins revealed that, although extracellular, they cluster within the annelid intracellular globins clade, suggesting that the extracellular state of these Hbs is the result of convergent evolution. The tetra-domain structure likely resulted from two tandem duplications, domain 1 giving rise to domain 2 and after this the two-domain gene duplicated to produce domains 3 and 4. The high O(2) affinity of Branchipolynoe extracellular globins may be explained by the two key residues (B10Y and E7Q) in the heme pocket in each of the domains of the single and tetra-domain globins, which have been shown to be essential in the oxygen-avid Hb from the nematode Ascaris suum. This peculiar globin evolutionary path seems to be very different from other annelid extracellular globins and is most likely the product of evolutionary tinkering associated with the strong selective pressure to adapt to chronic hypoxia that characterizes hydrothermal vents.

[Mitochondrial diseases in adults: An update]. / Les maladies mitochondriales de l'adulte : mise au point.

Mitochondrial diseases, characterized by a respiratory chain deficiency, are considered as rare genetic diseases but are the most frequent among inherited metabolic disorders. The complexity of their diagnosis is due to the dual control by the mitochondrial (mtDNA) and the nuclear DNA (nDNA), and to the heterogeneous clinical presentations; illegitimate association of symptoms should prompt the clinician to evoke a mitochondrial disorder. The goals of this review are to provide clinicians a better understanding of mitochondrial diseases in adults. After a brief overview on the mitochondrial origin and functions, especially their role in the energy metabolism, we will describe the genetic bases for mitochondrial diseases, then we will describe the various clinical presentations with the different affected tissues as well as the main symptoms encountered. Even if the new sequencing approaches have profoundly changed the diagnostic process, the brain imaging, the biological, the biochemical, and the histological explorations are still important highlighting the need for a multidisciplinary approach. While for most of the patients with a mitochondrial disease, only supportive and symptomatic therapies are available, recent advances in the understanding of the pathophysiological mechanisms have been made and new therapies are being developed and are evaluated in human clinical trials.

Gene Copy Number Quantification of SHOX, VAMP7, and SRY for the Detection of Sex Chromosome Aneuploidies in Neonates.

Aims: To explore the feasibility of detecting sex chromosome aneuploidies (SCAs) by means of gene copy number quantification of short stature homeobox (SHOX), vesicle-associated membrane protein 7 (VAMP7), and SRY in newborns. Materials and Methods: Gene doses of SHOX, VAMP7, and SRY were determined by quantitative polymerase chain reaction (qPCR) using DNA obtained from dried blood samples from newborns. Relative quantification values were obtained. An aneuploidy profile was established according to cutoff values. Samples with ≥2 gene doses (out of range) were reanalyzed, and those with aneuploidy profiles were confirmed by karyotyping. Sensitivity, specificity, and positive and negative predictive values were obtained. Results: A total of 10,033 samples were collected (4945 females and 5088 males). Of 244 (2.43%) samples with ≥2 gene doses that were retested, 20 cases were confirmed. The overall incidence of SCAs was 1 in 500 live newborns. There were six cases of Turner syndrome (1/824), 3 cases of XXX (1/1648), 7 cases of Klinefelter syndrome (1/726), and 4 cases of of XYY (1/1272). The sensitivity was 0.952 (95.42%); the specificity was 0.975 (97.56%); the positive predictive value was 0.909 (90.91%) and the negative predictive value was 0.987 (98.77%). Conclusions: Gene copy number analyses of the VAMP7, SHOX, and SRY genes by qPCR from blood samples spotted onto filter paper is a highly reliable method for the early detection of male and female SCAs.

Membrane recycling in the cone cell endings of the turtle retina.

The ultrastructural effects of dark, light, and low temperature were investigated in the cone cell endings of the red-eared turtle (Pseudemys scripta elegans). Thin sections revealed that in dark-adapted retinas maintained at 22 degrees C, the neural processes which contact the cone cells at the invaginating synapses penetrated deeply into the photoreceptor endings. When dark-adapted retinas were illuminated for 1 h at 22 degrees C, the invaginating processes were apparently extruded from the synaptic endings. On the other hand, 1-h exposure to a temperature of 4 degrees C in the dark caused the invaginating processes to become much more strikingly inserted than at room temperature. A morphometric analysis showed that the ratio between the synaptic surface density of the endings and their total surface density decreased in the light and increased in the dark and cold. Freeze-fracturing documented fusion of synaptic vesicles with the presynaptic membrane in all conditions tested. These observations suggest that the changes in configuration of the pedicles in the light, dark, and cold reflect a different balance between addition and retrieval of synaptic vesicle membrane from the plasmalemma; in the dark, the rate of vesicle fusion is increased, whereas in the cold, membrane retrieval is blocked. When the eyecups were warmed up and illuminated for 30-45 min after cold exposure, a striking number of vacuoles and cisterns appeared in the cytoplasm and coated vesicles were commonly seen budding from the plasmalemma. 60-90 min after returning to room temperature, the endings had reverted to their normal configuration, and the vast majority of vacuoles, cisterns, and coated vesicles had disappeared. When horseradish peroxidase was included in the incubation medium, very few synaptic vesicles were labeled at the end of the period of cold exposure. 30-45 min after returning to 22 degrees C, vacuoles and cisterns contained peroxidase, whereas most synaptic vesicles were devoid of reaction product. 2 h after returning to 22 degrees C, coated vesicles, vacuoles, and cisterns had disappeared and a number of synaptic vesicles were labeled. These experiments suggest that vacuoles, cisterns, and coated vesicles mediate the retrieval of the synaptic vesicle membrane that has been added to the plasmalemma during cold exposure.

Membrane interactions between adjacent mucols secretion granules.

In primate goblet cells, the membranes of adjacent mucous granules from contact areas which appear as extensive pentalaminar fusion sites in thin sections. In freeze-fracture replicas, the same membrane areas are smooth, except for a few 6-8-nm particles which adhere to the E face. These protein-poor membrane interaction sites are relatively long-lived, and it is proposed that further stimulus may be required to trigger membrane fission.

Perfusion CT Imaging Follows Clinical Severity in Left Hemispheric Strokes.

OBJECTIVE: The purpose of this study was to assess how imaging findings on admission perfusion CT (PCT) and follow-up noncontrast CT (NCT), and their changes over time, correlate with clinical scores of stroke severity measured on admission, at discharge and at 6-month follow-up. METHODS: Fifty-two patients with suspected hemispheric acute ischemic stroke underwent a PCT within the first 24 h of symptom onset and a follow-up NCT of the brain between 24 h and 3 months after the initial stroke CT study. NIH Stroke Scale (NIHSS) scores were recorded for each patient at admission, discharge and 6 months; modified Rankin scores were determined at discharge and 6 months. Baseline PCT and follow-up NCT were analyzed quantitatively (volume of ischemic/infarcted tissue) and semiquantitatively (anatomical grading score derived from the Alberta Stroke Program Early CT Score). The correlation between imaging volumes/scores and clinical scores was assessed. Analysis was performed for all patients considered together and separately for those with right and left hemispheric strokes. RESULTS: Significant correlations were found between clinical scores and both quantitative and semiquantitative imaging. The volume of the acute PCT mean transit time lesion showed best correlation with admission NIHSS scores (R2 = 0.61, p < 0.001). This association was significantly better for left hemispheric strokes (R(2) = 0.80, p < 0.001) than for right hemispheric strokes (R2 = 0.39, p = 0.131). Correlation between imaging and NIHSS scores was better than correlation between imaging and modified Rankin scores (p = 0.047). The correlation with discharge clinical scores was better than that with 6-month clinical scores (p = 0.012). CONCLUSIONS: Baseline PCT and follow-up NCT volumes predict stroke severity at baseline, discharge and, to a lesser extent, 6 months. The correlation is stronger for left-sided infarctions. This finding supports the use of PCT as a surrogate stroke outcome measure.

Perfusion-CT assessment of the cerebrovascular reserve: a revisit to the acetazolamide challenges.

BACKGROUND AND PURPOSE: Imaging techniques utilizing acetazolamide challenges classically measure cerebral blood flow (CBF). In addition to measuring CBF, Perfusion-CT (PCT) can also assess cerebral blood volume (CBV) and mean transit time (MTT), expanding but also complicating the results of acetazolamide challenges performed using PCT. The goal of this study is to clarify the interpretation of PCT studies obtained during acetazolamide challenges. METHODS: Four consecutive patients were referred for evaluation of their cerebrovascular reserve because of suspected or known large vessel stenosis or occlusion. In one patient, the potential stenosis was found to be artifactual, and this subject was considered as a normal control. The remaining three patients had clinical histories clearly suggestive of a worsening in cerebrovascular reserve (no.1 with a single transient ischemic attack (TIA), no.2 with several TIAs, no.3 with multiple, prolonged TIAs). All patients underwent a baseline PCT scan, followed by intravenous injection of 1g acetazolamide and, 20 min postinjection, by a second PCT scan at exactly the same locations as the first. PCT cerebral blood flow, volume, and mean transit time values were measured in regions of interest (ROIs) encompassing the brain tissue at-risk and the normal brain tissue, defined based on the site of occlusion and the anatomy of the Circle of Willis. Changes in PCT parameters were calculated in corresponding ROIs on pre- and postacetazolamide PCT maps. RESULTS: As compared to the normal control patient, baseline CBF values in the at-risk regions were similar in patients nos.1 and 2, and lower in patient no.3. After acetazolamide administration, CBF increased by 32% in the normal patient and decreased by 11, 11, and 9% in the at-risk regions in patients nos.1, 2, and 3, respectively; CBV was stable for all patients except no.3, who showed a 36% increase; MTT was the PCT parameter whose change best differentiated the four patients (-17% in the normal patient, +9% in patient no.1, +24% in patient no.2, +48% in patient no.3). Interestingly, the baseline MTT values, measured before acetazolamide injection, showed a similar, gradual increase in the four patients, ranging from 4.5 to 8.1s. CONCLUSION: The degree of impairment in cerebrovascular reserve, as assessed by clinical history, correlated most closely with the change in MTT in response to acetazolamide. Increased baseline MTT values may be a static, quantitative indicator of compromised cerebrovascular reserve in at-risk territories.

A new rhythm library for testing automatic external defibrillators: performance of three devices.

A library of arrhythmias obtained from patients with cardiac arrest was developed. Such a data base will permit both in vitro testing of the rhythm analysis system of automatic external defibrillators before clinical field trials are conducted and comparison of devices. Defibrillators equipped with voice/electrocardiographic tape recorders and used in the prehospital defibrillation programs in Iowa and King County, Washington provided the rhythm source. From these recordings, segments of ventricular fibrillation with minimal artifact and a duration of greater than or equal to 6 s were selected. Segments of ventricular fibrillation (n = 102) were categorized by average peak amplitude as fine (1 to less than 3 mm), medium (3 to less than 7 mm), coarse (7 to less than 12 mm) and extra coarse (greater than or equal to 12 mm), and transcribed onto high fidelity videocassette tapes. Nonventricular fibrillation cardiac arrest rhythms (n = 144), which included wide complex idioventricular rhythms, ventricular and supraventricular tachycardias, asystole and artifact, were also transcribed. Automatic external defibrillators developed by three manufacturers reached a treat (shock) decision on 88 to 93% of the ventricular fibrillation rhythms and on 5 to 10% of the nonventricular fibrillation rhythms. The latter decisions were defined as false positive, though for many rapid nonventricular fibrillation rhythms, countershock may be the appropriate treatment response. There were no statistically significant differences among the three devices in the shock/no shock decisions. A variety of ventricular fibrillation arrhythmias and terminology to express the preclinical performance of automatic external defibrillators are defined. Three commercially available automatic external defibrillators appear to successfully identify ventricular fibrillation and nonventricular fibrillation rhythms.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular population genetics of sex determination genes: the transformer gene of Drosophila melanogaster.

The transformer locus (tra) produces an RNA processing protein that alternatively splices the doublesex pre-mRNA in the sex determination hierarchy of Drosophila melanogaster. Comparisons of the tra coding region among Drosophila species have revealed an unusually high degree of divergence in synonymous and nonsynonymous sites. In this study, we tested the hypothesis that the tra gene will be polymorphic in synonymous and nonsynonymous sites within species by investigating nucleotide sequence variation in eleven tra alleles within D. melanogaster. Of the 1063 nucleotides examined, two synonymous sites were polymorphic and no amino acid variation was detected. Three statistical tests were used to detect departures from an equilibrium neutral model. Two tests failed to reject a neutral model of molecular evolution because of low statistical power associated with low levels of genetic variation (Tajima/Fu and Li). The Hudson, Kreitman, and Aguade test rejected a neutral model when the tra region was compared to the 5'-flanking region of alcohol dehydrogenase (Adh). The lack of variability in the tra gene is consistent with a recent selective sweep of a beneficial allele in or near the tra locus.

Nucleotide sequence of the Adh gene region of Drosophila pseudoobscura: evolutionary change and evidence for an ancient gene duplication.

The alcohol dehydrogenase (Adh) locus (ADH; alcohol: NAD+ oxidoreductase, EC 1.1.1.1) of Drosophila pseudoobscura was cloned and sequenced. Forty-five percent of the "effectively silent sites" have changed between Adh in D. pseudoobscura of the obscura species group and the homologous DNA sequence in D. mauritiana, the latter representing the melanogaster species group. The untranslated leader sequence of the adult transcript of D. pseudoobscura has two deletions relative to the D. mauritiana message. The ADH protein sequences of D. pseudoobscura is missing the third and fourth amino acids at the N-terminus relative to the D. mauritiana enzyme. Of the remaining 254 amino acid positions, 27 (10.64%) differ between the two species. Amino acid replacements are randomly distributed into hydrophilic and hydrophobic domains of ADH. However, replacement substitutions are distributed nonrandomly across the three exons among D. pseudoobscura and members of the melanogaster subgroup, suggesting that functional constraints across the exons are different. Surprisingly, silent substitutions are also nonrandomly distributed with the third exon being the most divergent. This pattern suggests possible selective constraints on supposedly neutral silent substitutions and/or variation in underlying mutation rates across the gene. The presence of transcriptional and translational signals at the beginning and end of conserved sequences 3' to Adh implies the existence of a previously undescribed gene. Codon usage and patterns of nucleotide divergence are consistent with a protein coding function for this gene. In addition, conservation of nucleotide and amino acid sequence and similarity in hydropathy plots suggests that the gene 3' to Adh represents an ancient duplication of the Adh gene.

Estimates of linkage disequilibrium and the recombination parameter determined from segregating nucleotide sites in the alcohol dehydrogenase region of Drosophila pseudoobscura.

The alcohol dehydrogenase (Adh) region of Drosophila pseudoobscura, which includes the two genes Adh and Adh-Dup, was used to examine the pattern and organization of linkage disequilibrium among pairs of segregating nucleotide sites. A collection of 99 strains from the geographic range of D. pseudoobscura were nucleotide-sequenced with polymerase chain reaction-mediated techniques. All pairs of the 359 polymorphic sites in the 3.5-kb Adh region were tested for significant linkage disequilibrium with Fisher's exact test. Of the 74,278 pairwise comparisons of segregating sites, 127 were in significant linkage disequilibrium at the 5% level. The distribution of five linkage disequilibrium estimators D(ij), D2, r(ij), r2 and D(ij) were compared to theoretical distributions. The observed distributions of D(ij), D2, r(ij) and r2 were consistent with the theoretical distribution given an infinite sites model. The observed distribution of D(ij) differed from the theoretical distribution because of an excess of values at -1 and 1. No spatial pattern was observed in the linkage disequilibrium pattern in the Adh region except for two clusters of sites nonrandomly associated in the adult intron and intron 2 of Adh. The magnitude of linkage disequilibrium decreases significantly as nucleotide distance increases, or a distance effect. Adh-Dup had a larger estimate of the recombination parameter, 4Nc, than Adh, where N is the effective population size and c is the recombination rate. A comparison of the mutation and recombination parameters shows that 7-17 recombination events occur for each mutation event. The heterogeneous estimates of the recombination parameter and the inverse relationship between linkage disequilibrium and nucleotide distance are no longer significant when the two clusters of Adh intron sites are excluded from analyses. The most likely explanation for the two clusters of linkage disequilibria is epistatic selection between sites in the cluster to maintain pre-mRNA secondary structure.

Molecular population genetics of an electrophoretically monomorphic protein in the alcohol dehydrogenase region of Drosophila pseudoobscura.

Nucleotide sequence data from the alcohol dehydrogenase (Adh) region of 18 isochromosomal strains of Drosophila pseudoobscura were used to determine whether the lack of amino acid polymorphism in ADH results from a low neutral mutation rate or a recent directional selection event. We estimated the neutral mutation parameter, 4Nmu, in synonymous sites for 17 subregions of Adh. The nucleotide diversity data were tested for departures from an equilibrium neutral model with two statistical tests. The Tajima test and the Hudson, Kreitman and Aguade test each failed to reject a neutral model. These results suggest that the ADH enzyme of D. pseudoobscura lacks amino acid polymorphisms because the neutral mutation rate of nonsynonymous sites is low. The neutral mutation parameter for synonymous sites is heterogeneous between domains of the Adh region. These data indicate that selective constrains on synonymous sites can vary between functional domains.

Estimates of gene flow in Drosophila pseudoobscura determined from nucleotide sequence analysis of the alcohol dehydrogenase region.

The genetic structure of Drosophila pseudoobscura populations was inferred from a nucleotide sequence analysis of a 3.4-kb segment of the alcohol dehydrogenase (Adh) region. A total of 99 isochromosomal strains collected from 13 populations in North and South America were used to determine if any population departed from a neutral model and to estimate levels of gene flow between populations. This study also included the nucleotide sequences from two sibling species, D. persimilis and D. miranda. We estimated the neutral mutation parameter, 4N mu, in synonymous and noncoding sites for 17 subregions of Adh in each of nine populations with sample sizes greater than three. The nucleotide diversity data in the nine populations was tested for departures from an equilibrium neutral model with two statistical tests. The Tajima and the Hudson, Kreitman, Aguade tests showed that each population fails to reject a neutral model. Tests for genetic differentiation between populations fail to show any population substructure among the North American populations of D. pseudoobscura. The nucleotide diversity data is consistent with direct and indirect measures of gene flow that show extensive dispersal between populations of D. pseudoobscura.

Polymorphism and divergence at a Drosophila pseudogene locus.

The larval cuticle protein (Lcp) cluster in Drosophila melanogaster contains four functional genes and a closely related pseudogene. A 630-bp fragment including the larval cuticle pseudogene locus (Lcp psi) was nucleotide sequenced in 10 strains of D. melanogaster and a 458-bp Lcp psi fragment from D. simulans was also sequenced. We used these data to test the hypotheses that the rates of synonymous and nonsynonymous substitution are equal, that the absolute levels of variation are higher than in functional genes, and that intraspecific polymorphism is correlated with interspecific divergence. As predicted, synonymous and nonsynonymous substitution rates were equivalent, and overall nucleotide divergence between D. melanogaster and D. simulans (Jukes-Cantor distance = 0.149 +/- 0.150) was extremely high. However, within-species DNA sequence comparisons at Lcp psi revealed lower levels of polymorphism (theta = 0.001 +/- 0.001) than at many functional loci in D. melanogaster. Using the HUDSON, KREITMAN, and AGUADE (HKA) test, we show that the level of polymorphism in Lcp psi within D. melanogaster is lower than expected given the amount of divergence between D. melanogaster and D. simulans when the pseudogene data are compared to the Adh 5' flanking region. Because the Lcp psi lies in a region of relatively infrequent recombination, we suggest that the low level of within-species polymorphism is the result of background selection.

Molecular population genetics of X-linked genes in Drosophila pseudoobscura.

This article presents a nucleotide sequence analysis of 500 bp determined in each of five X-linked genes, runt, sisterlessA, period, esterase 5, and Heat-shock protein 83, in 40 Drosophila pseudoobscura strains collected from two populations. Estimates of the neutral migration parameter for the five loci show that gene flow among D. pseudoobscura populations is sufficient to homogenize inversion frequencies across the range of the species. Nucleotide diversity at each locus fails to reject a neutral model of molecular evolution. The sample of 40 chromosomes included six Sex-ratio inversions, a series of three nonoverlapping inversions that are associated with a strong meiotic drive phenotype. The selection driven by the Sex-ratio meiotic drive element has not fixed variation across the X chromosome of D. pseudoobscura because, while significant linkage disequilibrium was observed within the sisterlessA, period, and esterase 5 genes, we did not find evidence for nonrandom association among loci. The Sex-ratio chromosome was estimated to be 25,000 years old based on the decomposition of linkage disequilibrium between esterase 5 and Heat-shock protein 83 or 1 million years old based on the net divergence of esterase 5 between Standard and Sex-ratio chromosomes. Genetic diversity was depressed within esterase 5 within Sex-ratio chromosomes, while the four other genes failed to show a reduction in heterozygosity in the Sex-ratio background. The reduced heterogeneity in esterase 5 is due either to its location near one of the Sex-ratio inversion breakpoints or that it is closely linked to a gene or genes responsible for the Sex-ratio meiotic drive system.

Natural selection and the frequency distributions of "silent" DNA polymorphism in Drosophila.

In Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster, codon bias may be maintained by a balance among mutation pressure, genetic drift, and natural selection favoring translationally superior codons. Under such an evolutionary model, silent mutations fall into two fitness categories: preferred mutations that increase codon bias and unpreferred changes in the opposite direction. This prediction can be tested by comparing the frequency spectra of synonymous changes segregating within populations; natural selection will elevate the frequencies of advantageous mutations relative to that of deleterious changes. The frequency distributions of preferred and unpreferred mutations differ in the predicted direction among 99 alleles of two D. pseudoobscura genes and five alleles of eight D. simulans genes. This result confirms the existence of fitness classes of silent mutations. Maximum likelihood estimates suggest that selection intensity at silent sites is, on average, very weak in both D. pseudoobscura and D. simulans (magnitude of NS approximately 1). Inference of evolutionary processes from within-species sequence variation is often hindered by the assumption of a stationary frequency distribution. This assumption can be avoided when identifying the action of selection and tested when estimating selection intensity.