A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway

Members of the Hedgehog (Hh) family of signaling proteins are powerful regulators of developmental processes in many organisms and have been implicated in many human disease states. Here we report the results of a genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway. The screen identified hundreds of potential new regulators of Hh signaling, including many large protein complexes with pleiotropic effects, such as the coat protein complex I (COPI) complex, the ribosome and the proteasome. We identified the multimeric protein phosphatase 2A (PP2A) and two new kinases, the D. melanogaster orthologs of the vertebrate PITSLRE and cyclin-dependent kinase-9 (CDK9) kinases, as Hh regulators. We also identified a large group of constitutive and alternative splicing factors, two nucleoporins involved in mRNA export and several RNA-regulatory proteins as potent regulators of Hh signal transduction, indicating that splicing regulation and mRNA transport have a previously unrecognized role in Hh signaling. Finally, we showed that several of these genes have conserved roles in mammalian Hh signaling.     

A screen of the complete protein kinase gene family identifies diverse patterns of somatic mutations in human breast cancer

We examined the coding sequence of 518 protein kinases, approximately 1.3 Mb of DNA per sample, in 25 breast cancers. In many tumors, we detected no somatic mutations. But a few had numerous somatic mutations with distinctive patterns indicative of either a mutator phenotype or a past exposure.     

Evolutionary genomics: codon bias and selection on single genomes

The idea that natural selection on genes might be detected using only a single genome has been put forward by Plotkin and colleagues, who present a method that they claim can detect selection without the need for comparative data and which, if correct, would confer greater power of analysis with less information. Here we argue that their method depends on assumptions that confound their conclusions and that, even if these assumptions were valid, the authors' inferences about adaptive natural selection are unjustified.     

Genome-wide association study and mouse model identify interaction between RET and EDNRB pathways in Hirschsprung disease

Genetic studies of Hirschsprung disease, a common congenital malformation, have identified eight genes with mutations that can be associated with this condition. Mutations at individual loci are, however, neither necessary nor sufficient to cause clinical disease. We conducted a genome-wide association study in 43 Mennonite family trios using 2,083 microsatellites and single-nucleotide polymorphisms and a new multipoint linkage disequilibrium method that searches for association arising from common ancestry. We identified susceptibility loci at 10q11, 13q22 and 16q23; the gene at 13q22 is EDNRB, encoding a G protein-coupled receptor (GPCR) and the gene at 10q11 is RET, encoding a receptor tyrosine kinase (RTK). Statistically significant joint transmission of RET and EDNRB alleles in affected individuals and non-complementation of aganglionosis in mouse intercrosses between Ret null and the Ednrb hypomorphic piebald allele are suggestive of epistasis between EDNRB and RET. Thus, genetic interaction between mutations in RET and EDNRB is an underlying mechanism for this complex disorder.     

DNMT1 and DNMT3b cooperate to silence genes in human cancer cells

Inactivation of tumour suppressor genes is central to the development of all common forms of human cancer. This inactivation often results from epigenetic silencing associated with hypermethylation rather than intragenic mutations. In human cells, the mechanisms underlying locus-specific or global methylation patterns remain unclear. The prototypic DNA methyltransferase, Dnmt1, accounts for most methylation in mouse cells, but human cancer cells lacking DNMT1 retain significant genomic methylation and associated gene silencing. We disrupted the human DNMT3b gene in a colorectal cancer cell line. This deletion reduced global DNA methylation by less than 3%. Surprisingly, however, genetic disruption of both DNMT1 and DNMT3b nearly eliminated methyltransferase activity, and reduced genomic DNA methylation by greater than 95%. These marked changes resulted in demethylation of repeated sequences, loss of insulin-like growth factor II (IGF2) imprinting, abrogation of silencing of the tumour suppressor gene p16INK4a, and growth suppression. Here we demonstrate that two enzymes cooperatively maintain DNA methylation and gene silencing in human cancer cells, and provide compelling evidence that such methylation is essential for optimal neoplastic proliferation.     

Acrylamide is formed in the Maillard reaction

Reports of the presence of acrylamide in a range of fried and oven-cooked foods have caused worldwide concern because this compound has been classified as probably carcinogenic in humans. Here we show how acrylamide can be generated from food components during heat treatment as a result of the Maillard reaction between amino acids and reducing sugars. We find that asparagine, a major amino acid in potatoes and cereals, is a crucial participant in the production of acrylamide by this pathway.     

Observation and interpretation of a time-delayed mechanism in the hydrogen exchange reaction

Extensive theoretical and experimental studies have shown the hydrogen exchange reaction H+H2 --> H2+H to occur predominantly through a 'direct recoil' mechanism: the H--H bonds break and form concertedly while the system passes straight over a collinear transition state, with recoil from the collision causing the H2 product molecules to scatter backward. Theoretical predictions agree well with experimental observations of this scattering process. Indirect exchange mechanisms involving H3 intermediates have been suggested to occur as well, but these are difficult to test because bimolecular reactions cannot be studied by the femtosecond spectroscopies used to monitor unimolecular reactions. Moreover, full quantum simulations of the time evolution of bimolecular reactions have not been performed. For the isotopic variant of the hydrogen exchange reaction, H+D2 --> HD+D, forward scattering features observed in the product angular distribution have been attributed to possible scattering resonances associated with a quasibound collision complex. Here we extend these measurements to a wide range of collision energies and interpret the results using a full time-dependent quantum simulation of the reaction, thus showing that two different reaction mechanisms modulate the measured product angular distribution features. One of the mechanisms is direct and leads to backward scattering, the other is indirect and leads to forward scattering after a delay of about 25 femtoseconds.