A principal challenge currently facing biologists is how to connect the complete DNA sequence of an organism to its development and behaviour. Large-scale targeted-deletions have been successful in defining gene functions in the single-celled yeast Saccharomyces cerevisiae, but comparable analyses have yet to be performed in an animal. Here we describe the use of RNA interference to inhibit the function of ∼86% of the 19,427 predicted genes of C. elegans. We identified mutant phenotypes for 1,722 genes, about two-thirds of which were not previously associated with a phenotype. We find that genes of similar functions are clustered in distinct, multi-megabase regions of individual chromosomes; genes in these regions tend to share transcriptional profiles. Our resulting data set and reusable RNAi library of 16,757 bacterial clones will facilitate systematic analyses of the connections among gene sequence, chromosomal location and gene function in C. elegans.

http://llama.mshri.on.ca/courses/Biophysics205/Papers/Kamath_2003.pdf

Systematic functional analysis of the Caenorhabditis elegans genome using RNAi

5-Noncoding sequences have been tabulated for 211 messenger RNAs from higher eukaryotic cells. The 5'-proximal AUG triplet serves as the initiator codon in 95% of the mRNAs examined. The most conspicuous conserved feature is the presence of a purine (most often A) three nucleotides upstream from the AUG initiator codon; only 6 of the mRNAs in the survey have a pyrimidine in that position. There is a predominance of C in positions -1, -2, -4 and -5, just upstream from the initiator codon. The sequence CCAGCCAUG (G) thus emerges as a consensus sequence for eukaryotic initiation sites. The extent to which the ribosome binding site in a given mRNA matches the -1 to -5 consensus sequence varies: more than half of the mRNAs in the tabulation have 3 or 4 nucleotides in common with the CCACC consensus, but only ten mRNAs conform perfectly.

Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs

Our previous study demonstrated increased expression of Heat shock protein (Hsp) 90 in the skin of patients with systemic sclerosis (SSc). We aimed to evaluate plasma Hsp90 in SSc and characterize its association with SSc-related features. Ninety-two SSc patients and 92 age-/sex-matched healthy controls were recruited for the cross-sectional analysis. The longitudinal analysis comprised 30 patients with SSc associated interstitial lung disease (ILD) routinely treated with cyclophosphamide. Hsp90 was increased in SSc compared to healthy controls. Hsp90 correlated positively with C-reactive protein and negatively with pulmonary function tests: forced vital capacity and diffusing capacity for carbon monoxide (DLCO). In patients with diffuse cutaneous (dc) SSc, Hsp90 positively correlated with the modified Rodnan skin score. In SSc-ILD patients treated with cyclophosphamide, no differences in Hsp90 were found between baseline and after 1, 6, or 12 months of therapy. However, baseline Hsp90 predicts the 12-month change in DLCO. This study shows that Hsp90 plasma levels are increased in SSc patients compared to age-/sex-matched healthy controls. Elevated Hsp90 in SSc is associated with increased inflammatory activity, worse lung functions, and in dcSSc, with the extent of skin involvement. Baseline plasma Hsp90 predicts the 12-month change in DLCO in SSc-ILD patients treated with cyclophosphamide.

/pdf/plasma-hsp90-levels-in-patients-with-systemic-sclerosis-and-1ftthcf8xe.pdf

Plasma Hsp90 levels in patients with systemic sclerosis and relation to lung and skin involvement: a cross-sectional and longitudinal study.

The neurohypophysial peptide oxytocin (OT) and OT-like hormones facilitate reproduction in all vertebrates at several levels. The major site of OT gene expression is the magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei. In response to a variety of stimuli such as suckling, parturition, or certain kinds of stress, the processed OT peptide is released from the posterior pituitary into the systemic circulation. Such stimuli also lead to an intranuclear release of OT. Moreover, oxytocinergic neurons display widespread projections throughout the central nervous system. However, OT is also synthesized in peripheral tissues, e.g., uterus, placenta, amnion, corpus luteum, testis, and heart. The OT receptor is a typical class I G protein-coupled receptor that is primarily coupled via Gq proteins to phospholipase C-β. The high-affinity receptor state requires both Mg2+ and cholesterol, which probably function as allosteric modulators. The agonist-binding region of the receptor has bee...

The Oxytocin Receptor System: Structure, Function, and Regulation

Maintenance of genome stability is essential for avoiding the passage to neoplasia. The DNA-damage response--a cornerstone of genome stability--occurs by a swift transduction of the DNA-damage signal to many cellular pathways. A prime example is the cellular response to DNA double-strand breaks, which activate the ATM protein kinase that, in turn, modulates numerous signalling pathways. ATM mutations lead to the cancer-predisposing genetic disorder ataxia-telangiectasia (A-T). Understanding ATM's mode of action provides new insights into the association between defective responses to DNA damage and cancer, and brings us closer to resolving the issue of cancer predisposition in some A-T carriers.

ATM and related protein kinases: safeguarding genome integrity

The estrogen receptor (ER) can be activated as a transcription factor either by binding of cognate estrogenic ligand or, indirectly, by a variety of other extracellular signals. As a first step towards elucidating the mechanism of 'steroid-independent activation' of the ER by the epidermal growth factor (EGF), we have mapped the ER target domain and determined the signaling pathway. We show that the N-terminal transcriptional activation function AF-1, but not the C-terminal AF-2, is necessary for the EGF response. Both the EGF-induced hyperphosphorylation and the transcriptional activation of the unliganded ER depend on a phosphorylatable serine residue at position 118. However, its phosphorylation is not sufficient and, hence, there must be other target domains or proteins which fulfill an additional requirement for EGF signaling through the ER. Using dominant-negative Ras and MAP kinase kinase (MAPK kinase) and constitutively active MAPK kinase mutants, we show that EGF activates the ER by signaling through the MAPK pathway suggesting that MAPK directly phosphorylates the critical serine 118. Our results also imply that the steroid-independent activation of a variety of ER mutants, which arise during the malignant progression of breast tumors, may contribute to tamoxifen resistance.

https://www.embopress.org/doi/epdf/10.1002/j.1460-2075.1996.tb00571.x

Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation.

Heat-shock protein 90 (Hsp90) is an abundant and highly conserved molecular chaperone that is essential for viability in eukaryotes. Hsp90 fulfills a housekeeping function in contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. A remarkable proportion of its substrates are proteins involved in cell cycle control and signal transduction. Hsp90 acts with a cohort of Hsp90 co-chaperones that modulate its substrate recognition, ATPase cycle and chaperone function. The large conformational flexibility of Hsp90 and a multitude of dynamic co-chaperone complexes contribute to generating functional diversity, and allow Hsp90 to assist a wide range of substrates.

Heat-shock protein 90, a chaperone for folding and regulation.

The G protein-coupled receptor GPR30 mediates c-fos up-regulation by 17beta-estradiol and phytoestrogens in breast cancer cells.

The glucocorticoid responses are shaped by molecular chaperones.

Steroid hormones such as estrogens are known to signal through ligand-regulated transcription factors of the nuclear receptor superfamily. In addition, they elicit rapid nongenomic responses from membrane-associated receptors. One of these receptors belongs to an entirely different family of proteins. The G protein-coupled and seven-transmembrane receptor, GPR30, is now widely recognized as an estrogen receptor (ER), hence its official new acronym GPER. It appears to mediate a wide range of responses to estrogen in a large variety of cell types. Its functions are clearly distinct from those of the classical nuclear ERs, although these pathways may overlap and interact in some cases. Here, we review the history of the discovery of this new ER, the evidence for the claim that it is an ER, its signal transduction, and its potential functions in physiology and disease.

/pdf/the-unfolding-stories-of-gpr30-a-new-membrane-bound-estrogen-4hqttxuix0.pdf

Didier Picard

Papers

Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation.

Heat-shock protein 90, a chaperone for folding and regulation.

The G protein-coupled receptor GPR30 mediates c-fos up-regulation by 17beta-estradiol and phytoestrogens in breast cancer cells.

The glucocorticoid responses are shaped by molecular chaperones.

The unfolding stories of GPR30, a new membrane-bound estrogen receptor