Identification of genes that function in the TNF-alpha-mediated apoptotic pathway using randomized hybrid ribozyme libraries

Now that the sequences of many genomes are available, methods are required for the rapid identification of functional genes. We describe here a simple system for the isolation of genes that function in the tumor necrosis factor-alpha (TNF-alpha)-mediated pathway of apoptosis, using RNA helicase-associated ribozyme libraries with randomized substrate-binding arms. Because target-site accessibility considerably limits the effective use of intracellular ribozymes, the effectiveness of a conventional ribozyme library has been low. To overcome this obstacle, we attached to ribozymes an RNA motif (poly(A)-tail) able to interact with endogenous RNA helicase(s) so that the resulting helicase-attached, hybrid ribozymes can more easily attack target sites regardless of their secondary or tertiary structures. When the phenotype of cells changes upon introduction of a ribozyme library, genes responsible for these changes may be identified by sequencing the active ribozyme clones. In the case of TNF-alpha-mediated apoptosis, when a ribozyme library was introduced into MCF-7 cells, surviving clones were completely or partially resistant to TNF-alpha-induced apoptosis. We identified many pro-apoptotic genes and partial sequences of previously uncharacterized genes using this method. Our gene discovery system should be generally applicable to the identification of functional genes in various systems.     

Role of the Rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway and its connection to calcineurin function

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.     

Class VI myosin moves processively along actin filaments backward with large steps.

Among a superfamily of myosin, class VI myosin moves actin filaments backwards. Here we show that myosin VI moves processively on actin filaments backwards with large ( approximately 36 nm) steps, nevertheless it has an extremely short neck domain. Myosin V also moves processively with large ( approximately 36 nm) steps and it is believed that myosin V strides along the actin helical repeat with its elongated neck domain that is critical for its processive movement with large steps. Myosin VI having a short neck cannot take this scenario. We found by electron microscopy that myosin VI cooperatively binds to an actin filament at approximately 36 nm intervals in the presence of ATP, raising a hypothesis that the binding of myosin VI evokes "hot spots" on actin filaments that attract myosin heads. Myosin VI may step on these "hot spots" on actin filaments in every helical pitch, thus producing processive movement with 36 nm steps.     

Polyphyletic origin of cultivated rice: based on the interspersion pattern of SINEs

The wild rice species Oryza rufipogon with wide intraspecific variation is thought to be the progenitor of the cultivated rice species Oryza sativa with two ecotypes, japonica and indica. To determine the origin of cultivated rice, subfamily members of the rice retroposon p-SINE1, which show insertion polymorphism in the O. sativa -O. rufipogon population, were identified and used to "bar code" each of 101 cultivated and wild rice strains based on the presence or absence of the p-SINE1 members at the respective loci. A phylogenetic tree constructed based on the bar codes given to the rice strains showed that O. sativa strains were classified into two groups corresponding to japonica and indica, whereas O. rufipogon strains were in four groups, in which annual O. rufipogon strains formed a single group, differing from the perennial O. rufipogon strains of the other three groups. Japonica strains were closely related to the O. rufipogon perennial strains of one group, and the indica strains were closely related to the O. rufipogon annual strains, indicating that O. sativa has been derived polyphyletically from O. rufipogon. The subfamily members of p-SINE1 constitute a powerful tool for studying the classification and relationship of rice strains, even when one has limited knowledge of morphology, taxonomy, physiology, and biochemistry of rice strains.     

Existence of ghrelin-immunopositive and -expressing cells in the proventriculus of the hatching and adult chicken

Ghrelin was isolated from the rat stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) and has been found in the gastrointestinal tract of many vertebrates. Although the sequence and structure of chicken ghrelin has recently been determined, morphological characteristics of ghrelin cells in the chicken gastrointestinal tract are still obscure. In this study, we investigated ghrelin expression and distribution of ghrelin-producing cells in the hatching and adult chicken gastrointestinal tract by RT-PCR, immunohistochemistry and in situ hybridization. Ghrelin mRNA expression was observed mainly in the proventriculus in the hatching chicken and in the proventriculus, pylorus and duodenum of the adult chicken by RT-PCR. Ghrelin-immunopositive (ghrelin-ip) cells in the proventriculus were located at the mucosal layer but not in the myenteric plexus or smooth muscle layer. The number of ghrelin-ip cells in the adult chicken was greater than that in the hatching chicken. Interestingly, in the adult chicken, the number of ghrelin-ip cells were almost the same as that of ghrelin mRNA-expressing (ghrelin-ex) cells; however, in the hatching chicken, the number of ghrelin-ex cells was greater than that of ghrelin-ip cells. These results clearly demonstrate that ghrelin-producing cells exist in the chicken gastrointestinal tract, especially in the proventriculus, from hatching to adult stages of development, as well as in mammals.     

Adrenomedullin in the eye

Adrenomedullin (AM) is a multifunctional regulatory peptide that is produced and secreted by various types of cells. We showed the presence of high concentrations of adrenomedullin-immunoreactivity in the vitreous fluid, and the levels were elevated in patients with proliferative vitreoretinopathy. Furthermore, adrenomedullin mRNA expression levels were elevated in the tissues of intraocular tumors and orbital tumors. Adrenomedullin is produced and secreted by cultured human retinal pigment epithelial (RPE) cells. Inflammatory cytokines and hypoxia are strong stimulators for the adrenomedullin expression in retinal pigment epithelial cells. Adrenomedullin stimulated the proliferation of retinal pigment epithelial cells both under normoxia and hypoxia. Dexamethasone (DEX) increased the adrenomedullin expression in two cultured cell lines of human retinal pigment epithelial cells; ARPE-19 cells and D407 cells, while it had no noticeable effects on the cytokine-induced adrenomedullin expression. These findings suggest that adrenomedullin is involved in the pathophysiology of inflammatory and neoplastic eye diseases as an autocrine or paracrine growth stimulator. The findings on glucocorticoid-induced AM expression raise the possibility that it may be related to the pathogenesis of some eye diseases, such as central serous chorioretinopathy and multifocal posterior pigment epitheliopathy, which are frequently seen in patients treated with high doses of glucocorticoids.     

WAVE2 deficiency reveals distinct roles in embryogenesis and Rac-mediated actin-based motility

The Wiskott-Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin-cytoskeletal reorganization downstream of the small Rho GTPase, Rac. We inactivated the WAVE2 gene by gene-targeted mutation to examine its role in murine development and in actin assembly. WAVE2-deficient embryos survived until approximately embryonic day 12.5 and displayed growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2-deficient embryonic stem cells displayed normal proliferation, whereas WAVE2-deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization. These results imply a non-redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin-based processes downstream of Rac that are essential for cell movement.     

A Physical Map of Arabidopsis thaliana Chromosome 3 Represented by Two Contigs of CIC YAC, P1, TAC and BAC Clones

We have constructed a physical map of Arabidopsis thaliana chromosome3 by ordering the clones from CIC YAC, P1, TAC and BAC librariesusing the sequences of a variety of genetic and EST markersand terminal sequences of clones. The markers used were 112DNA markers, 145 YAC end sequences, and 156 end sequences ofP1, TAC and BAC clones. The entire genome of chromosome 3, exceptfor the centromeric and telomeric regions, was covered by twolarge contigs, 13.6 Mb and 9.2 Mb long. This physical map willfacilitate map-based cloning experiments as well as genome sequencingof chromosome 3. The map and end sequence information are availableon the KAOS (Kazusa Arabidopsis data Opening Site) web siteat http://www.kazusa.or.jp/arabi/.     

Thermodynamic parameters of the interaction of Urtica dioica agglutinin with N-acetylglucosamine and its oligomers

The interaction between Urtica dioica agglutinin (UDA) and N-acetylglucosamine (GlcNAc) and its (1-4)-linked oligomers was studied by fluorescence titration and isothermal titration microcalorimetry. UDA possesses one significant binding site that can be measured calorimetrically. This site is composed of three subsites, each subsite accommodating one GlcNAc residue. The interaction is enthalpically driven, and the binding area of UDA is characterized by a H of interaction for a given oligosaccharide considerably smaller than that of wheat germ agglutinin (WGA), despite the fact that they both belong to a family of proteins composed entirely of hevein domains. Relatively high Cp values of the UDA-carbohydrate interactions and more favorable entropy term compared to WGA suggest that binding of the carbohydrate ligands by UDA has a higher hydrophobic component than that of WGA.