Mutation analysis of the coding sequence of the MECP2 gene in infantile autism

Mutations in the coding region of the methyl-CpG-binding protein 2 ( MECP2) gene cause Rett syndrome and have also been reported in a number of X-linked mental retardation syndromes. Furthermore, such mutations have recently been described in a few autistic patients. In this study, a large sample of individuals with autism was screened in order to elucidate systematically whether specific mutations in MECP2 play a role in autism. The mutation analysis of the coding sequence of the gene was performed by denaturing high-pressure liquid chromatography and direct sequencing. Taken together, 14 sequence variants were identified in 152 autistic patients from 134 German families and 50 unrelated patients from the International Molecular Genetic Study of Autism Consortium affected relative-pair sample. Eleven of these variants were excluded for having an aetiological role as they were either silent mutations, did not cosegregate with autism in the pedigrees of the patients or represented known polymorphisms. The relevance of the three remaining mutations towards the aetiology of autism could not be ruled out, although they were not localised within functional domains of MeCP2 and may be rare polymorphisms. Taking into account the large size of our sample, we conclude that mutations in the coding region of MECP2 do not play a major role in autism susceptibility. Therefore, infantile autism and Rett syndrome probably represent two distinct entities at the molecular genetic level.     

Genetic dissection of a TIR‐NB‐LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine

The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR‐NB‐LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated r esistance to P lasmopara v iticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south‐eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1‐mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR‐NB‐LRR genes at this locus share a common ancestor.     

Binding affinity of Escherichia coli RNA polymerase*sigma54 holoenzyme for the glnAp2, nifH and nifL promoters

Escherichia coli RNA polymerase associated with the sigma54 factor (RNAP*sigma54) is a holoenzyme form that transcribes a special class of promoters not recognized by the standard RNA polymerase*sigma70 com plex. Promoters for RNAP*sigma54 vary in their overall 'strength' and show differences in their response to the presence of DNA curvature between enhancer and promoter. In order to examine whether these effects are related to the promoter affinity, we have determined the equilibrium dissociation constant K(d) for the binding of RNAP*sigma54 to the three promoters glnAp2, nifH and nifL. Binding studies were conducted by monitoring the changes in fluorescence anisotropy upon titrating RNAP*sigma54 to carboxyrhodamine-labeled DNA duplexes. For the glnAp2 and nifH promoters similar values of K(d) = 0.94 +/- 0.55 nM and K(d) = 0.85 +/- 0.30 nM were determined at physiological ionic strength, while the nifL promoter displayed a significantly weaker affinity with K(d) = 8.5 +/- 1.9 nM. The logarithmic dependence of K(d) on the ionic strength I was -Deltalog(K(d))/Deltalog(I) = 6.1 +/- 0.5 for the glnAp2, 5.2 +/- 1.2 for the nifH and 2.1 +/- 0.1 for the nifL promoter. This suggests that the polymerase can form fewer ion pairs with the nifL promoter, which would account for its weaker binding affinity.     

From “Haus Tambaran” to Church: Continuity and Change in Contemporary Papua New Guinean Architecture

ABSTRACT

This article examines contemporary Papua New Guinean architecture created in the oscillation between Melanesian culture and Western impact. Some of the country's indigenous buildings are truly remarkable, especially the prestigious structures known as “Haus Tambaran” in the Sepik River area which have lent inspiration to many outstanding samples of contemporary Papua New Guinean design. We have studied both the indigenous and contemporary architecture of the Sepik region in the field and in additional research in collaboration with the late Professor Wallace Ruff and are comparing and contrasting contemporary architectural design with indigenous buildings. Here, we focus on a church at Ambunti, East Sepik Province. There is no electricity available on the site to facilitate climate control. Moreover, this contemporary building serves Western political, social, religious and educational functions. At the same time, the church incorporates aspects of the country's architectural heritage, including aesthetics as well as site-design and design with climate. Our comparison contrasts the modern building's functions, aesthetics, and design solutions with the area's indigenous local equivalent, the Haus Tambaran, and reveals elements borrowed, left aside, or altered in the process of adaptation. Issues discussed here include historic precedent, aesthetics, ownership, gender, and other social issues, as well as design and construction. Formal aspects, their meanings and functions, as well as environmental considerations and design solutions are borrowed, translated, or transformed, recalling but also differing from “Green Architecture,” to serve contemporary Western needs.     

A new yeast poly(A) polymerase complex involved in RNA quality control

Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNA^Met (tRNA_i^Met). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNA_i^Met with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNA_i^Met by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A) tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover.     

Interaction network of the ribosome assembly machinery from a eukaryotic thermophile

Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre‐ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre‐ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (～180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in‐depth analysis of their protein–protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2‐hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein–protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre‐ribosome factors forming the ctUTP‐A and ctUTP‐B modules, and the Brix‐domain containing assembly factors of the pre‐60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.     

Cre-mediated germline mosaicism: a new transgenic mouse for the selective removal of residual markers from tri-lox conditional alleles

The binary Cre-lox conditional knockout system requires an essential part of the target gene to be flanked by loxP sites, enabling excision in vivo upon Cre expression. LoxP sites are introduced by homologous recombination, together with a selectable marker. However, this marker can disturb gene expression and should be removed. The marker is therefore often prepared with a third, flanking loxP site (tri-lox construct), facilitating its selective removal by partial Cre-lox recombination. We have shown that this excision can be achieved in vivo in the germline using EIIaCre transgenic mice, and have described the advantages of in vivo over in vitro removal. We show here that MeuCre40, a new transgenic mouse, more reliably and reproducibly generates an optimal partial mosaic Cre-lox recombination pattern in the early embryo. This mosaicism was transmitted to the germline and to many other tissues. Alleles with partial deletions, in particular floxed alleles from which the selectable marker was removed, were readily recovered in the next generation, after segregation from the transgene. Segregation via paternal or maternal transmission led to successful recovery of the alleles of interest. We also obtained total deletion of the floxed regions in the same experiment, making this transgene a polyvalent Cre-lox tool. We rigorously tested the ability of MeuCre40 to solve tri-lox problems, by using it for the in vivo removal of neo^R- and hprt-expression cassettes from three different tri-lox mutants.     

From Single Variants to Protein Cascades: MULTISCALE MODELING OF SINGLE NUCLEOTIDE VARIANT SETS IN GENETIC DISORDERS*

Understanding the role of genetics in disease has become a central part of medical research. Non-synonymous single nucleotide variants (nsSNVs) in coding regions of human genes frequently lead to pathological phenotypes. Beyond single variations, the individual combination of nsSNVs may add to pathogenic processes. We developed a multiscale pipeline to systematically analyze the existence of quantitative effects of multiple nsSNVs and gene combinations in single individuals on pathogenicity. Based on this pipeline, we detected in a data set of 842 nsSNVs discovered in 76 genes related to cardiomyopathies, associated nsSNV combinations in seven genes present in at least 70% of all 639 patient samples, but not in a control cohort of healthy humans. Structural analyses of these revealed primarily an influence on the protein stability. For amino acid substitutions located at the protein surface, we generally observed a proximity to putative binding pockets. To computationally analyze cumulative effects and their impact, pathogenicity methods are currently being developed. Our approach supports this process, as shown on the example of a cardiac phenotype but can be likewise applied to other diseases such as cancer.