A Common Molecular Mechanism Underlies Two Phenotypically Distinct 17p13.1 Microdeletion Syndromes

DNA copy-number variations (CNVs) underlie many neuropsychiatric conditions, but they have been less studied in cancer. We report the association of a 17p13.1 CNV, childhood-onset developmental delay (DD), and cancer. Through a screen of over 4000 patients with diverse diagnoses, we identified eight probands harboring microdeletions at TP53 (17p13.1). We used a purpose-built high-resolution array with 93.75% breakpoint accuracy to fine map these microdeletions. Four patients were found to have a common phenotype including DD, hypotonia, and hand and foot abnormalities, constituting a unique syndrome. Notably, these patients were not affected with cancer. Moreover, none of the TP53-deletion patients affected with cancer (n = 4) had neurocognitive impairments. DD patients have larger deletions, which encompass but do not disrupt TP53, whereas cancer-affected patients harbor CNVs with at least one breakpoint within TP53. Most 17p13.1 deletions arise by Alu-mediated nonallelic homologous recombination. Furthermore, we identify a critical genomic region associated with DD and containing six underexpressed genes. We conclude that, although they overlap, 17p13.1 CNVs are associated with distinct phenotypes depending on the position of the breakpoint with respect to TP53. Further, detailed characterization of breakpoints revealed a common formation signature. Future studies should consider whether other loci in the genome also give rise to phenotypically distinct disorders by means of a common mechanism, resulting in a similar formation signature.     

Polarity of microtubules nucleated by centrosomes and chromosomes of Chinese hamster ovary cells in vitro

The structural and growth polarities of centrosomal and chromosomal microtubules were studied by analyzing the kinetics of growth of these microtubules and those initiated by flagellar seeds. By comparing rates of elongation of centrosomal and flagellar-seeded microtubules, we determined whether the centrosomal microtubules were free to grow at their plus ends only, minus ends ony, or at both ends. Our results show that centrosomal microtubules elongate at a rate corresponding to the addition of subunits at the plus end only. The depolymerization rate was also equivalent to that for the plus end only. Chromosomal microtubule elongation was similar to the centrosome-initiated growth. Since the data do not support the hypothesis that both ends of these spindle microtubules are able to interact with monomer in solution, then growth must occur only distal or only proximal to the organizing centers, implying tha the opposite ends in unavailable for exchange of subunits. Experiments with flagellar-seeded microtubules serving as internal controls indicated that the inactivity of the minus end could not be accounted for by a diffusible inhibitor, suggesting a structural explanation. Since there is no apparent way in which the distal ends may be capped, whereas the proximal ends are embedded in the pericentriolar cloud, we conclude that centrosomal microtubules are oriented with their plus ends distal to the site of nucleation. A similar analysis for chromosomal microtubules suggests that they too must be oriented with their plus ends distal to the site of initiation.     

Synthesis of salicyl-peptides and their effect on human platelet aggregationin vitro

Summary A series of leucine dipeptide amides containing at their N-terminal amino group the salicyl-residue [(o)-RO-C₆H₄-CO-, where R=H or CH₃CO] have been synthesized by conventional solution techniques and tested for their inhibitory activity on human platelet aggregationin vitro induced by collagen, ADP or adrenaline. The salicyl-peptide (o)-HO-C₆H₄-CO-Leu-Asp(OBzl)-NH₂ was found to exert strong inhibitory activity on platelet aggregation induced by collagen with an IC₅₀ value 4.5 mM. The corresponding dipetide H₂N-Leu-Asp(OVzl)-NH₂ was also examined and was found to be less active, indicating that the presence of the lipophilic-benzyl ester group in combination with the salicyl group enhance the inhibitory activity. All the other salicyl-peptides examined either didn't show any inhibitory or aggregatory activity or a slight inhibition at the concentration of 9–10 mm.The abbreviations used are in accordance with the rules of IUPAC-IUB Joint Commission on Biochemical Nomenclature: Eur J Biochem (1984) 138: 9–37; J Biol Chem (1989) 264: 663–673. Additional abbreviations are: ADP adenosine diphosphate - Boc tert-butyloxycarbonyl - Bzl benzyl - DIEA N,N-diisopropylethylamine - DMSO dimethyl sulfoxide - DMF N,N-dimethylformamide - EtOAc ethyl acetate - FCC flash column chromatography - IC₅₀ molar concentration of salicyl-peptide for 50% inhibition of platelet aggregation - iso-BCF isobutyl chloroformate - NMM N-methylmorpholine - PyBOP (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate - TLC thin layer chromatography - TMS tetramethylsilane     

Recolonization history and large-scale dispersal in the open sea: the case study of the North Atlantic cod, Gadus morhua L.

Most studies of the genetic structure of Atlantic cod have focused on small geographical scales. In the present study, the genetic structure of cod sampled on spawning grounds in the North Atlantic was examined using eight microsatellite loci and the Pan I locus. A total of 954 cod was collected from nine different regions: the Baltic Sea, the North Sea, the Celtic Sea, the Irish Sea and Icelandic waters during spring 2002 and spring 2003, from Norwegian waters and the Faroe Islands (North and West spawning grounds) in spring 2003, and from Canadian waters in 1998. Temporal stability among spawning grounds was observed in Icelandic waters and the Celtic Sea, and no significant difference was observed between the samples from the Baltic Sea and between the samples from Faroese waters. F -statistics showed significant differences between most populations and a pattern of isolation-by-distance was described with microsatellite loci. The Pan I locus revealed the presence of two genetically distinguishable basins, the North-west Atlantic composed of the Icelandic and Canadian samples and the North-east Atlantic composed of all other samples. Permutation of allele sizes at each microsatellite locus among allelic states supported a mutational component to the genetic differentiation, indicating a historical origin of the observed variation. Estimation of the time of divergence was approximately 3000 generations, which places the origin of current genetic pattern of cod in the North Atlantic in the late Weichselian (Wisconsinian period), at last glacial maximum.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 315–329.     

Synergy between tetA and rpsL provides high-stringency positive and negative selection in bacterial artificial chromosome vectors

Bacterial artificial chromosome (bacmid) vectors are used to stably propagate large, complex fragments of cloned DNA and are a core technology for functional genomics. The simplest method of analyzing bacmid clones would involve a direct mutagenesis or allele exchange protocol utilizing positive and negative selectable markers. The utility of three different negative selectable markers to function in the context of a bacmid vector was therefore investigated: sacB from Bacillus subtilis, which confers sensitivity to sucrose; tetA from TN10, which confers resistance to tetracycline, osmotic sensitivity, and sensitivity to kanamycin and streptomycin; and rpsL from Escherichia coli, which confers sensitivity to streptomycin. When expressed individually in the context of a bacmid vector, each of these markers confers a similar stringency of negative selection, with plating efficiencies on selective media of 2.3 x 10(-5), 9.4 x 10(-4), and 5.7 x 10(-5), respectively. However coexpression of rpsL and tetA results in a synergistic enhancement of the osmotic, kanamycin, and streptomycin sensitivities, with a stringency of selection of approximately 50- to approximately 1000-fold over that obtained with rpsL or tetA alone and approximately 20-fold more than that obtained using sacB. The combination of rpsL and tetA thus serves as the most efficient positive and negative selectable marker system described to date.     

Architecture of a coat for the nuclear pore membrane

The symmetric core of the nuclear pore complex can be considered schematically as a series of concentric cylinders. A peripheral cylinder coating the pore membrane contains the previously characterized, elongated heptamer that harbors Sec13-Nup145C in its middle section. Strikingly, Sec13-Nup145C crystallizes as a hetero-octamer in two space groups. Oligomerization of Sec13-Nup145C was confirmed biochemically. Importantly, the numerous interacting surfaces in the hetero-octamer are evolutionarily highly conserved, further underlining the physiological relevance of the oligomerization. The hetero-octamer forms a slightly curved, yet rigid rod of sufficient length to span the entire height of the proposed membrane-adjacent cylinder. In concordance with the dimensions and symmetry of the nuclear pore complex core, we suggest that the cylinder is constructed of four antiparallel rings, each ring being composed of eight heptamers arranged in a head-to-tail fashion. Our model proposes that the hetero-octamer would vertically traverse and connect the four stacked rings.