Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes

Double-stranded RNA interference (RNAi) is an effective method for disrupting expression of specific genes in Caenorhabditis elegans and other organisms. Applications of this reverse-genetics tool, however, are somewhat restricted in nematodes because introduced dsRNA is not stably inherited. Another difficulty is that RNAi disruption of late-acting genes has been generally less consistent than that of embryonically expressed genes, perhaps because the concentration of dsRNA becomes lower as cellular division proceeds or as developmental time advances. In particular, some neuronally expressed genes appear refractory to dsRNA-mediated interference. We sought to extend the applicability of RNAi by in vivo expression of heritable inverted-repeat (IR) genes. We assayed the efficacy of in vivo-driven RNAi in three situations for which heritable, inducible RNAi would be advantageous: (i) production of large numbers of animals deficient for gene activities required for viability or reproduction; (ii) generation of large populations of phenocopy mutants for biochemical analysis; and (iii) effective gene inactivation in the nervous system. We report that heritable IR genes confer potent and specific gene inactivation for each of these applications. We suggest that a similar strategy might be used to test for dsRNA interference effects in higher organisms in which it is feasible to construct transgenic animals, but impossible to directly or transiently introduce high concentrations of dsRNA.     

The coconut pathosystem: weed hosts of nymphs of the American palm Cixiid Haplaxius crudus (Hemiptera: Fulgoroidea)

Lethal yellowing (LY) of coconut palm (Cocos nucifera L., Arecaceae) is a disease of economic importance that is caused by the phytoplasma ‘Candidatus Phytoplasma palmae’ and is transmitted by the planthopper Haplaxius crudus (Van Duzee) (Hemiptera: Cixiidae). This study explores the weeds used by H. crudus nymphs and other Cixiidae in a coconut pathosystem in southern Mexico. Nymphs were collected directly from the root system of each weed by hand or with the help of a vacuum after carefully opening the culm. This study included 11 species of weeds: nine Poaceae [Brachiaria decumbens Stapf, B. humidicola (Rendle) Schweick, B. mutica (Forssk.) Stapf, Digitaria abyssinica (Hochst. Ex A. Rich.) Stapf, Eustachys petraea (Sw.) Desv., Leersia hexandra Sw., Panicum laxum Sw., P. maximum Jacq., Paspalum notatum Flüggé]; one Cyperaceae [Cyperus ligularis L.], and one Portulacaceae: [Portulaca pilosa L.]. Brachiaria mutica, E. petraea, B. humidicola, P. maximum were identified as the principal host species for H. crudus nymphs. Brachiaria decumbens, D. abyssinica, and C. ligularis are new host records for the nymphs of H. crudus. Additionally, it was found that H. crudus may coexist with its cogeners H. skarphion Kramer (Cixiidae) and H. caldwelli Kramer (Cixiidae), on B. mutica. On C. ligularis, H. crudus may coexist with Oecleus snowi Ball (Cixiidae) nymphs. These results suggest that in the coconut pathosystem there is a complex of multitrophic interactions that should be considered in integrated management of LY.     

The p53 tumour suppressor gene

The cell cycle is composed of a series of steps which can be negatively or positively regulated by various factors. Chief among the negative regulators is the p53 protein. Alteration or inactivation of p53 by mutation, or by its interactions with oncogene products of DNA tumour viruses, can lead to cancer. These mutations seem to be the most common genetic change in human cancers.     

The p66shc adaptor protein controls oxidative stress response and life span in mammals

Gene mutations in invertebrates have been identified that extend life span and enhance resistance to environmental stresses such as ultraviolet light or reactive oxygen species. In mammals, the mechanisms that regulate stress response are poorly understood and no genes are known to increase individual life span. Here we report that targeted mutation of the mouse p66shc gene induces stress resistance and prolongs life span. p66shc is a splice variant of p52shc/p46shc (ref. 2), a cytoplasmic signal transducer involved in the transmission of mitogenic signals from activated receptors to Ras. We show that: (1) p66shc is serine phosphorylated upon treatment with hydrogen peroxide (H2O2) or irradiation with ultraviolet light; (2) ablation of p66shc enhances cellular resistance to apoptosis induced by H2O2 or ultraviolet light; (3) a serine-phosphorylation defective mutant of p66shc cannot restore the normal stress response in p66shc-/- cells; (4) the p53 and p21 stress response is impaired in p66shc-/- cells; (5) p66shc-/- mice have increased resistance to paraquat and a 30% increase in life span. We propose that p66shc is part of a signal transduction pathway that regulates stress apoptotic responses and life span in mammals.     

Podocalyxin enhances the adherence of cells to platelets

Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Δ) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin αVβ3/αVβ5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid. Received 14 August 2007; received after revision 12 September 2007; accepted 13 September 2007     

Population-based resequencing of ANGPTL4 uncovers variations that reduce triglycerides and increase HDL

Resequencing genes provides the opportunity to assess the full spectrum of variants that influence complex traits. Here we report the first application of resequencing to a large population (n = 3,551) to examine the role of the adipokine ANGPTL4 in lipid metabolism. Nonsynonymous variants in ANGPTL4 were more prevalent in individuals with triglyceride levels in the lowest quartile than in individuals with levels in the highest quartile (P = 0.016). One variant (E40K), present in approximately 3% of European Americans, was associated with significantly lower plasma levels of triglyceride and higher levels of high-density lipoprotein cholesterol in European Americans from the Atherosclerosis Risk in Communities Study and in Danes from the Copenhagen City Heart Study. The ratio of nonsynonymous to synonymous variants was higher in European Americans than in African Americans (4:1 versus 1.3:1), suggesting population-specific relaxation of purifying selection. Thus, resequencing of ANGPTL4 in a multiethnic population allowed analysis of the phenotypic effects of both rare and common variants while taking advantage of genetic variation arising from ethnic differences in population history.     

Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate

Numerous microRNAs (miRNAs) have been discovered in the genomes of higher eukaryotes, and functional studies indicate that they are important during development. However, little is known concerning the function of individual miRNAs. We approached this problem in zebrafish by combining identification of miRNA expression, functional analyses and experimental validation of potential targets. We show that miR-214 is expressed during early segmentation stages in somites and that varying its expression alters the expression of genes regulated by Hedgehog signaling. Inhibition of miR-214 results in a reduction or loss of slow-muscle cell types. We show that su(fu) mRNA, encoding a negative regulator of Hedgehog signaling, is targeted by miR-214. Through regulation of su(fu), miR-214 enables precise specification of muscle cell types by sharpening cellular responses to Hedgehog.     

Germline gain-of-function mutations in SOS1 cause Noonan syndrome

Noonan syndrome, the most common single-gene cause of congenital heart disease, is characterized by short stature, characteristic facies, learning problems and leukemia predisposition. Gain-of-function mutations in PTPN11, encoding the tyrosine phosphatase SHP2, cause approximately 50% of Noonan syndrome cases. SHP2 is required for RAS-ERK MAP kinase (MAPK) cascade activation, and Noonan syndrome mutants enhance ERK activation ex vivo and in mice. KRAS mutations account for <5% of cases of Noonan syndrome, but the gene(s) responsible for the remainder are unknown. We identified missense mutations in SOS1, which encodes an essential RAS guanine nucleotide-exchange factor (RAS-GEF), in approximately 20% of cases of Noonan syndrome without PTPN11 mutation. The prevalence of specific cardiac defects differs in SOS1 mutation-associated Noonan syndrome. Noonan syndrome-associated SOS1 mutations are hypermorphs encoding products that enhance RAS and ERK activation. Our results identify SOS1 mutants as a major cause of Noonan syndrome, representing the first example of activating GEF mutations associated with human disease and providing new insights into RAS-GEF regulation.