A modification of Representational Difference Analysis, with application to the cloning of a candidate in the Reelin signalling pathway

Background  

cDNA-RDA is one of the subtractive cloning techniques used to isolate differentially expressed genes between two complex cDNA populations. In the present study we present a modification of the protocol described by Hubank and Schatz.     

Apelin signalling: a promising pathway from cloning to pharmacology

The discovery of new signalling pathways is always followed by the development of pharmacological agents as drugs that can be used in the treatment of diseases resulting from a dysfunction of the signalling pathway in question. Apelin signalling plays a role in the central and peripheral regulation of the cardiovascular system, in water and food intake, and possibly in immune function. Up-regulation of ligand and receptor is also associated with pathophysiological states such as cardiac dysfunction and neovascularisation. Finally, the apelin receptor is a coreceptor for the entry of several HIV-1 and SIV strains. In view of these features, the apelin receptor constitutes a very interesting target for the design of new drugs for treating the prime causes of human mortality.     

Identification of Virulence Genes in a Pathogenic Strain of Pseudomonas aeruginosa by Representational Difference Analysis

Pseudomonas aeruginosa is an opportunistic pathogen that may cause severe infections in humans and other vertebrates. In addition, a human clinical isolate of P. aeruginosa, strain PA14, also causes disease in a variety of nonvertebrate hosts, including plants, Caenorhabditis elegans, and the greater wax moth, Galleria mellonella. This has led to the development of a multihost pathogenesis system in which plants, nematodes, and insects have been used as adjuncts to animal models for the identification of P. aeruginosa virulence factors. Another approach to identifying virulence genes in bacteria is to take advantage of the natural differences in pathogenicity between isolates of the same species and to use a subtractive hybridization technique to recover relevant genomic differences. The sequenced strain of P. aeruginosa, strain PAO1, has substantial differences in virulence from strain PA14 in several of the multihost models of pathogenicity, and we have utilized the technique of representational difference analysis (RDA) to directly identify genomic differences between P. aeruginosa strains PA14 and PAO1. We have found that the pilC, pilA, and uvrD genes in strain PA14 differ substantially from their counterparts in strain PAO1. In addition, we have recovered a gene homologous to the ybtQ gene from Yersinia, which is specifically present in strain PA14 but absent in strain PAO1. Mutation of the ybtQ homolog in P. aeruginosa strain PA14 significantly attenuates the virulence of this strain in both G. mellonella and a burned mouse model of sepsis to levels comparable to those seen with PAO1. This suggests that the increased virulence of P. aeruginosa strain PA14 compared to PAO1 may relate to specific genomic differences identifiable by RDA.     

Patched represses the Hedgehog signalling pathway by promoting modification of the Smoothened protein

Hedgehog (Hh) signalling plays a central role in many developmental processes in both vertebrates and invertebrates [1]. The multipass membrane-spanning proteins Patched (Ptc) [2-4] and Smoothened (Smo) [5-7] have been proposed to act as subunits of a putative Hh receptor complex. According to this view, Smo functions as the transducing subunit, the activity of which is blocked by a direct interaction with the ligand-binding subunit, Ptc [8]. Activation of the intracellular signalling pathway occurs when Hh binds to Ptc [8-11], an event assumed to release Smo from Ptc-mediated inhibition. Evidence for a physical interaction between Smo and Ptc is so far limited to studies of the vertebrate versions of these proteins when overexpressed in tissue culture systems [8,12]. To test this model, we have overexpressed the Drosophila Smo protein in vivo and found that increasing the levels of Smo protein per se was not sufficient for activation of the pathway. Immunohistochemical staining of wild-type and transgenic embryos revealed distinct patterns of Smo distribution, depending on which region of the protein was detected by the antibody. Our findings suggest that Smo is modified to yield a non-functional form and this modification is promoted by Ptc in a non-stoichiometric manner.     

应用代表性差异分析法筛选人癌候选抑癌基因

运用cDNA代表性差异分析法(cDNA representational difference analysis,cDNA RDA),以正常人鼻咽上皮细胞及鼻咽癌HNE1细胞作为比较的样品来源,分离了四个在鼻咽癌中缺失的cDNA片段.以此四个片段作探针,分别进行DNA杂交、RNA杂交,结果显示,这些差异性的cDNA序列确实来自正常人鼻咽上皮且只在其中表达和/或在鼻咽癌HNE1中表达降低,并在鼻咽癌病人中存在不同程度的缺失.序列分析结果表明这些差异性表达的基因为具有相当抑癌基因功能的已知基因和可能与鼻咽癌相关的抑癌基因的新基因.从而说明cDNA RDA是一种高效、敏感、假阳性低的克隆抑癌基因的有效方法.     

Isolation and cloning of a Drosophila homolog to the mammalian RACK1 gene,implicated in PKC-mediated signalling

The mammalian RACK1 protein binds activated protein kinase C, acting as an intracellular receptor to anchor the activated PKC to the cytoskeleton. Genes encoding RACK1-like proteins have been isolated from a wide range of eucaryotic organisms; we report the isolation of a Drosophila member of this family. This Drosophila RACK1-like protein shows 76% identity to the mammalian RACK1 proteins, but only about 60% identity to related proteins from plants and fungi. The Drosophila rack1 gene has a dynamic pattern of expression during early embryogenesis with the highest expression in the mesodermal and endodermal lineages.     

Notch signalling in Drosophila: three ways to use a pathway

Cell-cell interactions mediated by Notch are critical at multiple stages of development. Our current understanding of the Notch signalling pathway suggests a comparatively simple transduction mechanism. However, this core pathway can be deployed in three different types of developmental process: lateral inhibition, lineage decisions and boundary formation. These illustrate how the activity of the pathway can be modulated both at the cell surface, through availability and effectiveness of ligand interactions, and inside the cell, through effects on the transduction pathway and the responsiveness of target genes.     

Positional cloning of a candidate gene for resistance to the sunflower downy mildew, Plasmopara halstedii race 300

The resistance of sunflower to Plasmopara halstedii is conferred by major resistance genes denoted Pl. Previous genetic studies indicated that the majority of these genes are clustered on linkage groups 8 and 13. The Pl6 locus is one of the main clusters to have been identified, and confers resistance to several P. halstedii races. In this study, a map-based cloning strategy was implemented using a large segregating F2 population to establish a fine physical map of this cluster. A marker derived from a bacterial artificial chromosome (BAC) clone was found to be very tightly linked to the gene conferring resistance to race 300, and the corresponding BAC clone was sequenced and annotated. It contains several putative genes including three toll-interleukin receptor-nucleotide binding site-leucine rich repeats (TIR-NBS-LRR) genes. However, only one TIR-NBS-LRR appeared to be expressed, and thus constitutes a candidate gene for resistance to P. halstedii race 300.     

Ethylene signalling and response pathway: a unique signalling cascade with a multitude of inputs and outputs

Plants as immobile organisms need to constantly monitor the changes in the environment to modify and adjust developmental and metabolic pathways accordingly. The responses to these environmental cues require an integrative mechanism where external and internal signals are detected and processed to trigger an appropriate 'reaction' in the plant. Hormones play a key role in mediating some of these integrative processes and in generating the response reactions. The identification and characterization of the basic hormone signalling components and their interactions represent the first step towards comprehensive understanding of plant responses to intrinsic and extrinsic cues. A relatively well-characterized ethylene signalling and response pathway, together with numerous evidences of its interactions with other signalling/response pathways, provide an excellent example to illustrate our current knowledge and perspective on how signal integration occurs in plants.     

A novel protein modification pathway related to the ubiquitin system.

Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N- and C-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitin-conjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitin-proteasome system and may play a regulatory role.     

A novel pathway of intercellular signalling in Bacillus subtilis involves a protein with similarity to a component of type III secretion channels

During spore formation in Bacillus subtilis, sigma(E)-directed gene expression in the mother-cell compartment of the sporangium triggers the activation of sigma(G) in the forespore by a pathway of intercellular signalling that is composed of multiple proteins of unknown function. Here, we confirm that the vegetative protein SpoIIIJ, the forespore protein SpoIIQ and eight membrane proteins (SpoIIIAA through SpoIIIAH) produced in the mother cell under the control of sigma(E) are ordinarily required for intercellular signalling. In contrast, an anti-sigma(G) factor previously implicated in the pathway is shown to be dispensable. We also present evidence suggesting that SpoIIIJ is a membrane protein translocase that facilitates the insertion of SpoIIIAE into the membrane. In addition, we report the isolation of a mutation that partially bypasses the requirement for SpoIIIJ and for SpoIIIAA through SpoIIIAG, but not for SpoIIIAH or SpoIIQ, in the activation of sigma(G). We therefore propose that under certain genetic conditions, SpoIIIAH and SpoIIQ can constitute a minimal pathway for the activation of sigma(G). Finally, based on the similarity of SpoIIIAH to a component of type III secretion systems, we speculate that signalling is mediated by a channel that links the mother cell to the forespore.     

Posttranslational modification of ras proteins: detection of a modification prior to fatty acid acylation and cloning of a gene responsible for the modification

Products of ras genes are synthesized as precursors in the cytosol and transported to the plasma membrane by a process which involves posttraslational modification by fatty acid. In this paper, we present evidence for the occurrence in the cytosol of an intermediate modification of ras proteins prior to the fatty acid acylation. The modification is detected by a slight shift in the mobility of the protein on SDS polyacrylamide gel. The fatty acid acylation does not contribute to this mobility shift. This modification is affected by the dprl mutation which has recently been shown to affect the processing of yeast RAS proteins. To further characterize the nature of the modification event, we have cloned DPR1 gene from the DNA of Saccharomyces cerevisiae. The gene is actively transcribed in yeast cells producing mRNA of approximately 1.6 kb. Genes related to the DRP1 appear to be present in a distantly related yeast, Schizosaccharomyces pombe as well as in guinea pig and human cells.     

A model invalidation-based approach for elucidating biological signalling pathways,applied to the chemotaxis pathway in R. sphaeroides

Background  

Developing methods for understanding the connectivity of signalling pathways is a major challenge in biological research. For this purpose, mathematical models are routinely developed based on experimental observations, which also allow the prediction of the system behaviour under different experimental conditions. Often, however, the same experimental data can be represented by several competing network models.     

Roles of Axin in the Wnt signalling pathway

The Wnt signalling pathway is conserved in various species from worms to mammals, and plays important roles in development, cellular proliferation, and differentiation. The molecular mechanisms by which the Wnt signal regulates cellular functions are becoming increasingly well understood. Wnt stabilizes cytoplasmic beta-catenin, which stimulates the expression of genes including c-myc, c-jun, fra-1, and cyclin D1. Axin, newly recognized as a component of the Wnt signalling pathway, negatively regulates this pathway. Other components of the Wnt signalling pathway, including Dvl, glycogen synthase kinase-3beta, beta-catenin, and adenomatous polyposis coli, interact with Axin, and the phosphorylation and stability of beta-catenin are regulated in the Axin complex. Thus, Axin acts as a scaffold protein in the Wnt signalling pathway, thereby regulating cellular functions.     

A supramolecular complex in the environmental stress signalling pathway of Bacillus subtilis

SigmaB, an alternative sigma-factor of Bacillus subtilis, mediates the response of the cell to a variety of physical insults. Within the environmental stress signalling pathway RsbU, a protein phosphatase, is stimulated by its interaction with the protein kinase RsbT. In the absence of stress RsbT is expected to be trapped by an alternative binding partner, RsbS. Here, we have demonstrated that RsbS alone cannot act as an alternative partner for RsbT, but instead requires the presence of RsbR to create a high molecular mass RsbR:RsbS complex (approximately 1 MDa) able to capture RsbT. In this complex the phosphorylation state of RsbS, and not that of RsbR, controlled the binding to RsbT, whose kinase activity towards RsbS could be counterbalanced by the activity of RsbX, the phosphatase for RsbS-P. The RsbR:RsbS complex recruited RsbT from a mixture of RsbT and RsbU. The phosphorylated form of RsbR in the complex enhanced the kinase activity of RsbT towards RsbS. This supramolecular complex thus has the functional properties of an alternative partner for RsbT. Electron micrographs of this complex are presented, and the purification of the RsbR:RsbS complex from cellular extracts provides evidence for the existence of such a complex in vivo.