Spectroscopic studies of the nature of the iron clusters in the soluble hydrogenase from Desulfovibrio desulfuricans (strain Norway 4)

57Fe-enriched samples of the soluble hydrogenase from Desulfovibrio desulfuricans (Norway) have been investigated in both the native (oxidized) and the dithionite-reduced states using M?ssbauer spectroscopy. The data clearly show that the iron in this enzyme is predominantly in the form of iron-sulphur clusters which are closely similar to the [4Fe-4S] clusters found in a large number of ferredoxins, such as that from Bacillus stearothermophilus. There appear to be two [4Fe-4S] clusters. The iron-sulphur clusters in the oxidized protein are virtually diamagnetic, as indicated by M?ssbauer, electron spin resonance and magnetic circular dichroic spectroscopy. On reduction by dithionite + methyl viologen, M?ssbauer spectroscopy showed that only 50% of the [4Fe-4S] clusters were reduced. Even reduction with hydrogen up to a pressure of 23 GPa did not reduce the iron-sulphur clusters completely. An ESR signal due to a rapidly relaxing species with g = 2.03, 1.89 was observed in the reduced protein, together with a weaker spectrum from a slower-relaxing species at g = 2.34, 2.12.     

Biotin-tubulin incorporates into kinetochore fiber microtubules during early but not late anaphase

The dynamic behavior of kinetochore fiber microtubules has been examined in PtK1 cells during anaphase of mitosis. Cells in anaphase were injected with biotin-tubulin and, at various intervals after injection, fixed for light or electron microscopic immunolocalization of biotin-tubulin-containing microtubules. When cells in early to mid anaphase were injected with biotin-tubulin and fixed 1-2 min later, fluorescence was observed throughout the spindle, including the region of the kinetochore fibers. Electron microscopy of early to mid anaphase cells, after processing with immunogold methods, revealed both labeled and unlabeled microtubules in the kinetochore fibers; some labeled microtubules contacted the kinetochores. When late anaphase cells were injected with biotin-tubulin, and fixed a few minutes later, little fluorescence was observed in the kinetochore fibers. Electron microscopy confirmed that kinetochore fibers in late anaphase cells were refractory to tubulin incorporation. The results of these experiments demonstrate that the kinetochore fiber incorporates new microtubules during early anaphase but that this incorporation ceases in mid to late anaphase. Thus, microtubule turnover within the kinetochore fiber does not abruptly cease at the onset of anaphase and anaphase kinetochore fiber microtubules are more dynamic than previously suspected.     

Meiosis in Drosophila males

The conjunctive mechanism of the XY bivalent is believed to differ from that of the autosomal bivalents in the achiasmate Drosophila melanogaster male. It has been proposed that hypothetical cohesive elements, termed collochores, hold the X and Y chromosomes together at or near their nucleolar organizing regions (NORs) and that collochores are not exhibited by autosomal bivalents. In electron micrographs, unique fibrillar material is observed between the X and Y chromosomes at the synaptic site. Recently, the 240 bp nontranscribed spacer associated with rRNA genes at the NOR has been implicated as the essential DNA sequence for XY pairing. To test whether this DNA sequence is always associated with XY pairing and to determine its relationship to the unique fibrillar material, we studied the XY bivalent in Drosophila simulans. The D. simulans Y chromosome has few, if any, rRNA genes, but does have a large block (3,000 kb or 12,500 copies) of the nontranscribed spacer repeat located at the distal end of its long arm. This is in contrast to the D. melanogaster Y, which has the repeat located among rRNA genes on its short arm. Using light and electron microscopy, we show that the X does indeed pair with the distal end of the long arm of the D. simulans Y. However, no fibrillar material is evident in serial thin sections of the D. simulans XY bivalent, suggesting that this material (in D. melanogaster) may be remnants of the NOR rather than a morphological manifestation of the hypothetical collochores. Indeed, in electron micrographs, the synaptic regions of the XY and autosomal bivalents appear similar with no obvious pairing structures, suggesting that the conjunctive mechanism holding homologous chromosomes together is the same for the XY and autosomal bivalents.     

Fine structure of crystalline inclusions in B-cells of the islets of Langerhans in the alligator

Summary The ultrastructure of crystalline beta granules of the islets of Langerhans in the alligator has been investigated. From optical diffraction analysis and serial sectioning, the existence of four distinct types of crystalline inclusions was established in ultrathin sections. The first type is the most frequent and is interpreted as a rhombohedroni with a base, the ortho-hexagonal unit-cell edges being a=18.9 nm, c=23.0 nm. The second type of crystal (not observed in serial sections) is found compatible with a rhomb-dodecahedron which indexes on a cubic cell with a=9.6 nm. The third type of crystal was assigned to dipyramids. Dipyramids are extremely rare, and only two diffraction patterns were obtained; their crystal system could not be determined. Prisms, which are second in abundance, represent the fourth type of crystal. Spacings as well as the symmetry differ from those of the above three crystal types and indicate a tetragonal cell with a=4.2 nm, c=14.2 nm. The data for the prismatic crystals are strikingly similar to those of proinsulin and may represent the first case of agreement between crystals (i) formed in vitro and studied by X-ray diffraction and (ii) those investigated in situ by electron microscopy.     

Allopatric and sympatric diversification within roach (Rutilus rutilus) of large pre‐alpine lakes

Intraspecific differentiation in response to divergent natural selection between environments is a common phenomenon in some northern freshwater fishes, especially salmonids and stickleback. Understanding why these taxa diversify and undergo adaptive radiations while most other fish species in the same environments do not, remains an open question. The possibility for intraspecific diversification has rarely been evaluated for most northern freshwater fish species. Here, we assess the potential for intraspecific differentiation between and within lake populations of roach (Rutilus rutilus)—a widespread and abundant cyprinid species—in lakes in which salmonids have evolved endemic adaptive radiations. Based on more than 3,000 polymorphic RADseq markers, we detected low but significant genetic differentiation between roach populations of two ultraoligotrophic lakes and between these and populations from other lakes. This, together with differentiation in head morphology and stable isotope signatures, suggests evolutionary and ecological differentiation among some of our studied populations. Next, we tested for intralacustrine diversification of roach within Lake Brienz, the most pristine lake surveyed in this study. We found significant phenotypic evidence for ecological intralacustrine differentiation between roach caught over a muddy substrate and those caught over a rocky substrate. However, evidence for intralacustrine genetic differentiation is at best subtle and phenotypic changes may therefore be mostly plastic. Overall, our findings suggest roach can differ between ecologically distinct lakes, but the extent of intralacustrine ecological differentiation is weak, which contrasts with the strong differentiation among endemic species of whitefish in the same lakes.     

Exome Sequencing of Phenotypic Extremes Identifies CAV2 and TMC6 as Interacting Modifiers of Chronic Pseudomonas aeruginosa Infection in Cystic Fibrosis

Discovery of rare or low frequency variants in exome or genome data that are associated with complex traits often will require use of very large sample sizes to achieve adequate statistical power. For a fixed sample size, sequencing of individuals sampled from the tails of a phenotype distribution (i.e., extreme phenotypes design) maximizes power and this approach was recently validated empirically with the discovery of variants in DCTN4 that influence the natural history of P. aeruginosa airway infection in persons with cystic fibrosis (CF; MIM219700). The increasing availability of large exome/genome sequence datasets that serve as proxies for population-based controls affords the opportunity to test an alternative, potentially more powerful and generalizable strategy, in which the frequency of rare variants in a single extreme phenotypic group is compared to a control group (i.e., extreme phenotype vs. control population design). As proof-of-principle, we applied this approach to search for variants associated with risk for age-of-onset of chronic P. aeruginosa airway infection among individuals with CF and identified variants in CAV2 and TMC6 that were significantly associated with group status. These results were validated using a large, prospective, longitudinal CF cohort and confirmed a significant association of a variant in CAV2 with increased age-of-onset of P. aeruginosa airway infection (hazard ratio = 0.48, 95% CI=[0.32, 0.88]) and variants in TMC6 with diminished age-of-onset of P. aeruginosa airway infection (HR = 5.4, 95% CI=[2.2, 13.5]) A strong interaction between CAV2 and TMC6 variants was observed (HR=12.1, 95% CI=[3.8, 39]) for children with the deleterious TMC6 variant and without the CAV2 protective variant. Neither gene showed a significant association using an extreme phenotypes design, and conditions for which the power of an extreme phenotype vs. control population design was greater than that for the extreme phenotypes design were explored.     

Efficient selection of tagging single-nucleotide polymorphisms in multiple populations

Common genetic polymorphism may explain a portion of the heritable risk for common diseases, so considerable effort has been devoted to finding and typing common single-nucleotide polymorphisms (SNPs) in the human genome. Many SNPs show correlated genotypes, or linkage disequilibrium (LD), suggesting that only a subset of all SNPs (known as tagging SNPs, or tagSNPs) need to be genotyped for disease association studies. Based on the genetic differences that exist among human populations, most tagSNP sets are defined in a single population and applied only in populations that are closely related. To improve the efficiency of multi-population analyses, we have developed an algorithm called MultiPop-TagSelect that finds a near-minimal union of population-specific tagSNP sets across an arbitrary number of populations. We present this approach as an extension of LD-select, a tagSNP selection method that uses a greedy algorithm to group SNPs into bins based on their pairwise association patterns, although the MultiPop-TagSelect algorithm could be used with any SNP tagging approach that allows choices between nearly equivalent SNPs. We evaluate the algorithm by considering tagSNP selection in candidate-gene resequencing data and lower density whole-chromosome data. Our analysis reveals that an exhaustive search is often intractable, while the developed algorithm can quickly and reliably find near-optimal solutions even for difficult tagSNP selection problems. Using populations of African, Asian, and European ancestry, we also show that an optimal multi-population set of tagSNPs can be substantially smaller (up to 44%) than a typical set obtained through independent or sequential selection.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.