Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans

A key challenge of functional genomics today is to generate well-annotated data sets that can be interpreted across different platforms and technologies. Large-scale functional genomics data often fail to connect to standard experimental approaches of gene characterization in individual laboratories. Furthermore, a lack of universal annotation standards for phenotypic data sets makes it difficult to compare different screening approaches. Here we address this problem in a screen designed to identify all genes required for the first two rounds of cell division in the Caenorhabditis elegans embryo. We used RNA-mediated interference to target 98% of all genes predicted in the C. elegans genome in combination with differential interference contrast time-lapse microscopy. Through systematic annotation of the resulting movies, we developed a phenotypic profiling system, which shows high correlation with cellular processes and biochemical pathways, thus enabling us to predict new functions for previously uncharacterized genes.     

The phylogenetic position of the 'giant deer' Megaloceros giganteus

The giant deer, or 'Irish elk', has featured extensively in debates on adaptation, sexual selection, and extinction. Its huge antlers--the largest of any deer species, living or extinct--formed a focus of much past work. Yet the phylogenetic position of the giant deer has remained an enigma. On the basis of its flattened antlers, the species was previously regarded as closely related to the living fallow deer. Recent morphological studies, however, have challenged that view and placed the giant deer closer to the living red deer or wapiti. Here we present a new phylogenetic analysis encompassing morphological and DNA sequence evidence, and find that both sets of data independently support a sister-group relationship of giant and fallow deer. Our results include the successful extraction and sequencing of DNA from this extinct species, and highlight the value of a joint molecular and morphological approach.     

T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand

The binding of a T-cell antigen receptor (TCR) to peptide antigen presented by major histocompatibility antigens (pMHC) on antigen-presenting cells (APCs) is a central event in adaptive immune responses. The mechanism by which TCR-pMHC ligation initiates signalling, a process termed TCR triggering, remains controversial. It has been proposed that TCR triggering is promoted by segregation at the T cell-APC interface of cell-surface molecules with small ectodomains (such as TCR-pMHC and accessory receptors) from molecules with large ectodomains (such as the receptor protein tyrosine phosphatases CD45 and CD148). Here we show that increasing the dimensions of the TCR-pMHC interaction by elongating the pMHC ectodomain greatly reduces TCR triggering without affecting TCR-pMHC ligation. A similar dependence on receptor-ligand complex dimensions was observed with artificial TCR-ligand systems that span the same dimensions as the TCR-pMHC complex. Interfaces between T cells and APCs expressing elongated pMHC showed an increased intermembrane separation distance and less depletion of CD45. These results show the importance of the small size of the TCR-pMHC complex and support a role for size-based segregation of cell-surface molecules in TCR triggering.     

Localization and analysis of nonpolar regions in onconase

A detailed analysis of the composition and properties of hydrophobic nuclei and microclusters has been carried out for onconase. Two main hydrophobic nuclei in the onconase structure were detected. Their composition and shape were found to be very similar to those of RNase A, in accordance with the predictions made. The nuclei in onconase are more compact, the side-chain atoms of residues included in the nuclei in onconase form more contacts with the environment than in RNase A. The hydrophobic nuclei should be considered as individual structural units along with elements of the secondary structure. Differences in composition and conformation of exposed loops between onconase and RNase A were found. The additional hydrophobic clusters attached to the nuclei in onconase might be involved in the fixation of an appropriate conformation of site(s) for manifestation of the biological activity of onconase. A comparison of amphibian representatives of the RNase A superfamily was also made. The results obtained suggest that the availability of nonpolar residues in established key positions of amino acid sequences determines the characteristic fold of homologous proteins and the structure of the active site cleft.     

A new gene involved in X-linked mental retardation identified by analysis of an X;2 balanced translocation

X-linked forms of mental retardation (MR) affect approximately 1 in 600 males and are likely to be highly heterogeneous. They can be categorized into syndromic (MRXS) and nonspecific (MRX) forms. In MRX forms, affected patients have no distinctive clinical or biochemical features. At least five MRX genes have been identified by positional cloning, but each accounts for only 0.5%-1.0% of MRX cases. Here we show that the gene TM4SF2 at Xp11.4 is inactivated by the X breakpoint of an X;2 balanced translocation in a patient with MR. Further investigation led to identification of TM4SF2 mutations in 2 of 33 other MRX families. RNA in situ hybridization showed that TM4SF2 is highly expressed in the central nervous system, including the cerebral cortex and hippocampus. TM4SF2 encodes a member of the tetraspanin family of proteins, which are known to contribute in molecular complexes including beta-1 integrins. We speculate that through this interaction, TM4SF2 might have a role in the control of neurite outgrowth.     

Cardiac defects and renal failure in mice with targeted mutations in Pkd2

PKD2, mutations in which cause autosomal dominant polycystic kidney disease (ADPKD), encodes an integral membrane glycoprotein with similarity to calcium channel subunits. We induced two mutations in the mouse homologue Pkd2 (ref.4): an unstable allele (WS25; hereafter denoted Pkd2WS25) that can undergo homologous-recombination-based somatic rearrangement to form a null allele; and a true null mutation (WS183; hereafter denoted Pkd2-). We examined these mutations to understand the function of polycystin-2, the protein product of Pkd2, and to provide evidence that kidney and liver cyst formation associated with Pkd2 deficiency occurs by a two-hit mechanism. Pkd2-/- mice die in utero between embryonic day (E) 13.5 and parturition. They have structural defects in cardiac septation and cyst formation in maturing nephrons and pancreatic ducts. Pancreatic ductal cysts also occur in adult Pkd2WS25/- mice, suggesting that this clinical manifestation of ADPKD also occurs by a two-hit mechanism. As in human ADPKD, formation of kidney cysts in adult Pkd2WS25/- mice is associated with renal failure and early death (median survival, 65 weeks versus 94 weeks for controls). Adult Pkd2+/- mice have intermediate survival in the absence of cystic disease or renal failure, providing the first indication of a deleterious effect of haploinsufficiency at Pkd2on long-term survival. Our studies advance our understanding of the function of polycystin-2 in development and our mouse models recapitulate the complex human ADPKD phenotype.     

Senescence bypass screen identifies TBX2, which represses Cdkn2a (p19(ARF)) and is amplified in a subset of human breast cancers

To identify new immortalizing genes with potential roles in tumorigenesis, we performed a genetic screen aimed to bypass the rapid and tight senescence arrest of primary fibroblasts deficient for the oncogene Bmi1. We identified the T-box member TBX2 as a potent immortalizing gene that acts by downregulating Cdkn2a (p19(ARF)). TBX2 represses the Cdkn2a (p19(ARF)) promoter and attenuates E2F1, Myc or HRAS-mediated induction of Cdkn2a (p19(ARF)). We found TBX2 to be amplified in a subset of primary human breast cancers, indicating that it might contribute to breast cancer development.