Decades of population genetic research reveal the need for harmonization of molecular markers: the grey wolf Canis lupus as a case study

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The last bastion? X chromosome genotyping of Anopheles gambiae species pair males from a hybrid zone reveals complex recombination within the major candidate ‘genomic island of speciation’

Speciation with gene flow may be aided by reduced recombination helping to build linkage between genes involved in the early stages of reproductive isolation. Reduced recombination on chromosome X has been implicated in speciation within the Anopheles gambiae complex, species of which represent the major Afrotropical malaria vectors. The most recently diverged, morphologically indistinguishable, species pair, A. gambiae and Anopheles coluzzii, ubiquitously displays a ‘genomic island of divergence’ spanning over 4 Mb from chromosome X centromere, which represents a particularly promising candidate region for reproductive isolation genes, in addition to containing the diagnostic markers used to distinguish the species. Very low recombination makes the island intractable for experimental recombination studies, but an extreme hybrid zone in Guinea Bissau offers the opportunity for natural investigation of X‐island recombination. SNP analysis of chromosome X hemizygous males revealed: (i) strong divergence in the X‐island despite a lack of autosomal divergence; (ii) individuals with multiple‐recombinant genotypes, including likely double crossovers and localized gene conversion; (iii) recombination‐driven discontinuity both within and between the molecular species markers, suggesting that the utility of the diagnostics is undermined under high hybridization. The largely, but incompletely protected nature of the X centromeric genomic island is consistent with a primary candidate area for accumulation of adaptive variants driving speciation with gene flow, while permitting some selective shuffling and removal of genetic variation.     

The Protozoan Parasite Toxoplasma gondii Targets Proteins to Dense Granules and the Vacuolar Space Using Both Conserved and Unusual Mechanisms

All known proteins that accumulate in the vacuolar space surrounding the obligate intracellular protozoan parasite Toxoplasma gondii are derived from parasite dense granules. To determine if constitutive secretory vesicles could also mediate delivery to the vacuolar space, T. gondii was stably transfected with soluble Escherichia coli alkaline phosphatase and E. coli β-lactamase. Surprisingly, both foreign secretory reporters were delivered quantitatively into parasite dense granules and efficiently secreted into the vacuolar space. Addition of a glycosylphosphatidylinositol membrane anchor rerouted alkaline phosphatase to the parasite surface. Alkaline phosphatase fused to the transmembrane domain and cytoplasmic tail from the endogenous dense granule protein GRA4 localized to dense granules. The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent upon the cytoplasmic tail, but not upon a tyrosine-based motif within the tail. Alkaline phosphatase fused to the vesicular stomatitis virus G protein transmembrane domain and cytoplasmic tail localized primarily to the Golgi, although staining of dense granules and the intravacuolar network was also detected; truncating the cytoplasmic tail decreased Golgi staining and increased delivery to dense granules but blocked delivery to the intravacuolar network. Targeting of secreted proteins to T. gondii dense granules and the plasma membrane uses general mechanisms identified in higher eukaryotic cells but is simplified and exaggerated in scope, while targeting of secreted proteins beyond the boundaries of the parasite involves unusual sorting events.     

Substrate Specificity of Barley Cysteine Endoproteases EP-A and EP-B

The cysteine endoproteases (EP)-A and EP-B were purified from green barley (Hordeum vulgare L.) malt, and their identity was confirmed by N-terminal amino acid sequencing. EP-B cleavage sites in recombinant type-C hordein were determined by N-terminal amino acid sequencing of the cleavage products, and were used to design internally quenched, fluorogenic peptide substrates. Tetrapeptide substrates of the general formula 2-aminobenzoyl-P₂-P₁-P₁′-P₂′-tyrosine(NO₂)-aspartic acid, in which cleavage occurs between P₁ and P₁′, showed that the cysteine EPs preferred phenylalanine, leucine, or valine at P₂. Arginine was preferred to glutamine at P₁, whereas proline at P₂, P₁, or P₁′ greatly reduced substrate kinetic specificity. Enzyme cleavage of C hordein was mainly determined by the primary sequence at the cleavage site, because elongation of substrates, based on the C hordein sequence, did not make them more suitable substrates. Site-directed mutagenesis of C hordein, in which serine or proline replaced leucine, destroyed primary cleavage sites. EP-A and EP-B were both more active than papain, mostly because of their much lower K_m values.     

Field Studies on Growth and Regenerative Capacity in the Foliose Macrolichen Xanthoria parietina (Teloschistales,Ascomycotina)

Growth and development of the foliose macrolichen Xanthoria parietina and adjacent lichen and bryophyte spp. was photographically monitored over a period of five years. This association developed on a plane quadrangle on the top surface of a sandstone block in the botanical garden of the University of Zürich. Line tracings of the thalline outlines were used for a quantitative estimation of laminal growth by means of gravimetric morphometry. Average radial growth of approximately 6 mm yr^?1 was recorded, with maxima around 7 mm yr^?1. The oldest, apothecia-covered central parts of the thalli lost contact with the substratum and broke off along stress cracks but the blank substratum was rapidly re-colonized by new thallus lobes which regenerated along the wound margins, to our present knowledge a peculiarity of X. parietina and few related species among the Teloschistales. Approximately 50% of the surface area of the experimental plot was covered by X. parietina at the beginning and again at the end of this five years period, with significant fluctuations in the time between. New growth in the range of 87% of the total surface area almost compensated the losses of approximately 90% per surface area in five years. New growth and losses within these five years accounted for more than 170% of the initial coverage. X. parietina gains a significant ecological advantage from its very remarkable regenerative capacity.     

Development of a New Selective Plating Medium for Rhodococcus equi

A new selective plating medium for Rhodococcus equi containing ceftazidime (20 mg/l) and novobiocin (25 mg/l) on a Mueller-Hinton agar basis is described. It proved to be less inhibitory for R. equi than selective plating media devised earlier and grew only very few other nocardioform bacteria.     

The concentration of Ca,Sr, Ba and Mn in successive needle age classes of Norway spruce [Picea abies (L.) Karst.]

The concentrations of Ca, Sr, Ba and Mn were determined in up to five successive needle age classes from 54 individual Norway spruce trees [Picea abies (L.) Karst] from nine different sites. The accumulation behaviour was found to be very nonuniform, going from an increase with needle age to a decrease; irregular patterns were also found. The type of accumulation is largely site specific. The increasing behaviour can in most cases be approximated by a simple arithmetic function. All four elements usually show the same accumulation pattern, the similarities being closest between Ca and Mn and least between Ca and Ba. It is postulated that the similarity between the four elements is due to their precipitation and storage as oxalates. The similarity between Ca, Sr and Ba is observed at all concentrations, that with Mn only at concentrations larger than 300 g/g. Mn at small concentrations (< 50 g/g) shows a decreasing pattern and no similarity at all with Ca, Sr and Ba, but behaves similar to mobile elements.     

The MECP2-TRD domain interacts with the DNMT3A-ADD domain at the H3-tail binding site

The DNMT3A DNA methyltransferase and MECP2 methylation reader are highly expressed in neurons. Both proteins interact via their DNMT3A-ADD and MECP2-TRD domains, and the MECP2 interaction regulates the activity and subnuclear localization of DNMT3A. Here, we mapped the interface of both domains using peptide SPOT array binding, protein pull-down, equilibrium peptide binding assays, and structural analyses. The region D529-D531 on the surface of the ADD domain was identified as interaction point with the TRD domain. This includes important residues of the histone H3 N-terminal tail binding site to the ADD domain, explaining why TRD and H3 binding to the ADD domain is competitive. On the TRD domain, residues 214–228 containing K219 and K223 were found to be essential for the ADD interaction. This part represents a folded patch within the otherwise largely disordered TRD domain. A crystal structure analysis of ADD revealed that the identified H3/TDR lysine binding pocket is occupied by an arginine residue from a crystallographic neighbor in the ADD apoprotein structure. Finally, we show that mutations in the interface of ADD and TRD domains disrupt the cellular interaction of both proteins in NIH3T3 cells. In summary, our data show that the H3 peptide binding cleft of the ADD domain also mediates the interaction with the MECP2-TRD domain suggesting that this binding site may have a broader role also in the interaction of DNMT3A with other proteins leading to complex regulation options by competitive and PTM specific binding.     

Lack of correspondence between mRNA expression for a putative cell death molecule (SGP-2) and neuronal cell death in the central nervous system

Neuronal death during nervous system development, a widely observed phenomenon, occurs through unknown mechanisms. Recent evidence suggests an active, destructive process requiring new gene expression. Sulfated glycoprotein-2 (SGP-2), a secretory product of testicular Sertoli cells has been shown to up-regulate in several nonneural tissues undergoing programmed cell death and in several types of neuronal degeneration. In order to determine if this message up-regulates in neurons undergoing developmentally determined cell death, we have studied the expression of SGP-2 mRNA in the developing and adult rat central nervous system (CNS) with in situ hybridization. We also report on the expression of this message in nonneural tissues from several regions of the developing embryo. The developing and adult rat central nervous system as well as widely varied tissues in the rat embryo express SGP-2 mRNA in a pattern that does not correlate with regions undergoing developmental cell death. In the nervous system, SGP-2 mRNA is expressed in neuronal populations including motor neurons, cortical neurons, and hypothalamic neurons at ages when the period of developmental cell death has passed. In a nonneural tissue (palatal shelve epithelium) for which a developmental cell death period has been described, SGP-2 mRNA was not present in the region where cell death occurs. We conclude that SGP-2 mRNA expression cannot be correlated with programmed cell death in neural or nonneural tissues. The results of this study as well as recently reported SGP-2 homologies indicate a possible role for this protein in secretion and lipid transport.