Copy-number variations measured by single-nucleotide-polymorphism oligonucleotide arrays in patients with mental retardation

Whole-genome analysis using high-density single-nucleotide-polymorphism oligonucleotide arrays allows identification of microdeletions, microduplications, and uniparental disomies. We studied 67 children with unexplained mental retardation with normal karyotypes, as assessed by G-banded chromosome analyses. Their DNAs were analyzed with Affymetrix 100K arrays. We detected 11 copy-number variations that most likely are causative of mental retardation, because they either arose de novo (9 cases) and/or overlapped with known microdeletions (2 cases). The eight deletions and three duplications varied in size from 200 kb to 7.5 Mb. Of the 11 copy-number variations, 5 were flanked by low-copy repeats. Two of those, on chromosomes 15q25.2 and Xp22.31, have not been described before and have a high probability of being causative of new deletion and duplication syndromes, respectively. In one patient, we found a deletion affecting only a single gene, MBD5, which codes for the methyl-CpG-binding domain protein 5. In addition to the 67 children, we investigated 4 mentally retarded children with apparent balanced translocations and detected four deletions at breakpoint regions ranging in size from 1.1 to 14 Mb.     

Non-random jumping translocations as a result of SV40 large T-antigen expression in benign human tumor cells.

In an attempt to analyse the cytogenetic effects caused by SV40 large T-antigen expression in cells of human benign tumors we transfected cells of an uterine leiomyoma characterized by a primary reciprocal translocation t(12;14)(q15;q24) and a pleomorphic adenoma of the salivary gland with an inversion inv(12) (q15q24.1) using a construct coding for SV40 large and small T-antigen. The most interesting finding was not a generally destabilized karyotype, but the strictly non-random involvement of two chromosomal breakpoints, i.e. 5p13 and 10q11 in jumping translocations, never described before as a result of SV40 transformation. In addition we were able to show by non-radioactive in situ hybridization that there was no direct relationship between the integration site of the construct and the pre-disposition of 5p13 and 10q11 to somatic recombination. The jumping translocations with consistent breakpoints observed closely resemble the cytogenetic situation seen in a variety of human tumors with specific translocations. Based on the findings described here it is tempting to assume that the expression of SV40 large T-antigen can induce specific karyotypic alterations following an unknown trans-acting mechanism.     

The regulation of osmotic and ionic balance in fish reproduction and in the early stages of ontogeny

On the basis of the modern literature data we analyzed the influence of the level of salt concentration and ion composition in a fish habitat during the processes of reproduction. The results of studies of the mechanisms of the reaction of fish to gamete hypo- and hyperosmotic stimulus in the external aqueous environment, as well as the role of mineral and organic osmolytes in the adaptation of mature eggs of fishes in the external environment, depend on the hydrochemical and hydrological conditions of the spawning grounds. The paper provides information about the features of the endocrine regulation of oocyte maturation in fish spawning in different hydrological conditions and the importance of humoral factors in the pathological process of the maturation of fish oocytes. The main scientific and practical aspects of the formation of the phys mechanisms that regulate the water-salt balance is the early ontogeny of fishes are discussed.     

The CONJUDOR pipeline for multiplexed knockdown of gene pairs identifies RBBP-5 as a germ cell reprogramming barrier in C. elegans

Multiple gene activities control complex biological processes such as cell fate specification during development and cellular reprogramming. Investigating the manifold gene functions in biological systems requires also simultaneous depletion of two or more gene activities. RNA interference-mediated knockdown (RNAi) is commonly used in Caenorhabditis elegans to assess essential genes, which otherwise lead to lethality or developmental arrest upon full knockout. RNAi application is straightforward by feeding worms with RNAi plasmid-containing bacteria. However, the general approach of mixing bacterial RNAi clones to deplete two genes simultaneously often yields poor results. To address this issue, we developed a bacterial conjugation-mediated double RNAi technique ‘CONJUDOR’. It allows combining RNAi bacteria for robust double RNAi with high-throughput. To demonstrate the power of CONJUDOR for large scale double RNAi screens we conjugated RNAi against the histone chaperone gene lin-53 with more than 700 other chromatin factor genes. Thereby, we identified the Set1/MLL methyltransferase complex member RBBP-5 as a novel germ cell reprogramming barrier. Our findings demonstrate that CONJUDOR increases efficiency and versatility of RNAi screens to examine interconnected biological processes in C. elegans with high-throughput.     

Effect of a virus on accumulation of a tissue-specific cell-surface protein of the fungus Cryphonectria (Endothia) parasitica.

Hypovirulence and decreased sporulation of the plant pathogenic fungus Cryphonectria (Endothia) parasitica is caused by double-stranded (ds)RNAs. These symptoms of dsRNA infection are correlated with down-regulation of at least nine major fungal polypeptides. One of the regulated polypeptides was purified to homogeneity and antibody to it was prepared. This polypeptide (cryparin) has a -glycine-serine-repeating sequence near the amino-terminal end that is typical of structural proteins and has properties of a lectin. Antibody-staining showed that this 18.6-kDa polypeptide is specific to aerial hyphae and fruiting bodies and that it accumulates in large amounts on hyphal cell surfaces. The dsRNA affects accumulation of this protein, both in the fugal hyphae and in the growth medium. Cryparin is similar in physical properties to those of the putative phytotoxin cerato-ulmin produced by the Dutch elm disease fungus. Toxicity of cryparin is not detectable, but the striking similarities between the physical properties and locations of accumulation of cryparin and cerato-ulmin in fungal fruiting structures suggest either conservation of structure or convergent evolution in function of these two proteins.     

Antibodies to Borrelia sp. in wild foxes and coyotes from Wisconsin and Minnesota

Serum samples from 93 red foxes (Vulpes vulpes) and nine gray foxes (Urocyon cinereoargenteus) trapped in Wisconsin and 23 coyotes (Canis latrans) trapped in Wisconsin and Minnesota were tested for antibodies to Borrelia sp. with an indirect fluorescent antibody test which used Borrelia burgdorferi as the whole-cell antigen. Seven red foxes (8%) and two coyotes (9%) had antibody titers greater than or equal to 1:64. All the positive samples were from areas known to be endemic for human Lyme disease. Implications for the epizootiology of Lyme borreliosis in wild canids are not well understood, but even if these species are not actual reservoirs of B. burgdorferi they could serve to increase the range of the vector and establish new endemic foci of the spirochete.     

Cardiomyopathic mutations in essential light chain reveal mechanisms regulating the super relaxed state of myosin

In this study, we assessed the super relaxed (SRX) state of myosin and sarcomeric protein phosphorylation in two pathological models of cardiomyopathy and in a near-physiological model of cardiac hypertrophy. The cardiomyopathy models differ in disease progression and severity and express the hypertrophic (HCM-A57G) or restrictive (RCM-E143K) mutations in the human ventricular myosin essential light chain (ELC), which is encoded by the MYL3 gene. Their effects were compared with near-physiological heart remodeling, represented by the N-terminally truncated ELC (Δ43 ELC mice), and with nonmutated human ventricular WT-ELC mice. The HCM-A57G and RCM-E143K mutations had antagonistic effects on the ATP-dependent myosin energetic states, with HCM-A57G cross-bridges fostering the disordered relaxed (DRX) state and the RCM-E143K model favoring the energy-conserving SRX state. The HCM-A57G model promoted the switch from the SRX to DRX state and showed an ∼40% increase in myosin regulatory light chain (RLC) phosphorylation compared with the RLC of normal WT-ELC myocardium. On the contrary, the RCM-E143K–associated stabilization of the SRX state was accompanied by an approximately twofold lower level of myosin RLC phosphorylation compared with the RLC of WT-ELC. Upregulation of RLC phosphorylation was also observed in Δ43 versus WT-ELC hearts, and the Δ43 myosin favored the energy-saving SRX conformation. The two disease variants also differently affected the duration of force transients, with shorter (HCM-A57G) or longer (RCM-E143K) transients measured in electrically stimulated papillary muscles from these pathological models, while no changes were displayed by Δ43 fibers. We propose that the N terminus of ELC (N-ELC), which is missing in the hearts of Δ43 mice, works as an energetic switch promoting the SRX-to-DRX transition and contributing to the regulation of myosin RLC phosphorylation in full-length ELC mice by facilitating or sterically blocking RLC phosphorylation in HCM-A57G and RCM-E143K hearts, respectively.