马铃薯甲虫结节性硬化复合物基因LdTSC1和LdTSC2基因的克隆及功能分析

【目的】通过RNAi技术明确马铃薯甲虫TOR上游的关键信号集成节点及类胰岛素信号通道下游基因结节性硬化复合物TSC1和TSC2的功能。旨在为探明马铃薯甲虫类胰岛素信号转导提供更多理论支持。【方法】在NCBI(美国国家生物技术信息中心)获取马铃薯甲虫LdTSC1/2序列,分别利用多重序列比对和系统发育分析确定该基因的完整性和系统发育关系;采用喂食幼虫dsRNA的方法,观察该基因的调低对马铃薯甲虫幼虫生长发育、糖脂代谢的影响。【结果】克隆得到马铃薯甲虫TSC1编码蛋白的氨基酸序列与鞘翅目白蜡窄吉丁直系同源蛋白的氨基酸序列的自展一致度为100%,聚为一支;TSC2编码蛋白的氨基酸序列与鞘翅目白蜡窄吉丁和赤拟谷盗的同源蛋白氨基酸序列的自展一致度为100%,聚为一支。通过分别喂食2龄幼虫LdTSC1/2的dsRNA能有效降低靶标基因的表达量,幼虫出现体重减轻,化蛹率和羽化率显著下降,葡萄糖的吸收转化效率降低,海藻糖含量升高和甘油三酯均减少。【结论】下调2龄幼虫LdTSC1/2的表达量,导致试虫出现抑制了糖脂代谢、脂肪体减少、体重减轻以及发育延迟;结果表明LdTSC1/2调控了马铃薯甲虫幼虫的糖脂代谢过程,显著影响幼虫化蛹和蜕皮过程。     

RTA2, a novel gene involved in azole resistance in Candida albicans

Widespread and repeated use of azoles, particularly fluconazole, has led to the rapid development of azole resistance in Candida albicans. Overexpression of CDR1, CDR2, and CaMDR1 has been reported contributing to azole resistance in C. albicans. In this study, hyper-resistant C. albicans mutant, with the above three genes deleted, was obtained by exposure to fluconazole and fluphenezine for 28 passages. Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; among the 13 up-regulated genes, we successfully constructed the rta2 and ipf14030 null mutants in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1. Using spot dilution assay, we demonstrated that the disruption of RTA2 increased the susceptibility of C. albicans to azoles while the disruption of IPF14030 did not influence the sensitivity of C. albicans to azoles. Meanwhile, we found that ectopic overexpression of RTA2 in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1 conferred resistance to azoles. RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans. In conclusion, our findings suggest that RTA2 is involved in the development of azole resistance in C. albicans.     

The chitin synthase genes chs-1 and chs-2 are essential for C. elegans development and responsible for chitin deposition in the eggshell and pharynx, respectively

It is widely accepted that chitin is present in nematodes. However, its precise role in embryogenesis is unclear and it is unknown if chitin is necessary in other nematode tissues. Here, we determined the roles of chitin and the two predicted chitin synthase genes in Caenorhabditis elegans by chitin localization and gene disruption. Using a novel probe, we detected chitin in the eggshell and discovered elaborate chitin localization patterns in the pharyngeal lumen walls. Chitin deposition in these two sites is likely regulated by the activities of chs-1 (T25G3.2) and chs-2 (F48A11.1), respectively. Reducing chs-1 gene activity by RNAi led to eggs that were fragile and permeable to small molecules, and in the most severe case, absence of embryonic cell division. Complete loss of function in a chs-1 deletion resulted in embryos that lacked chitin in their eggshells and failed to divide. These results showed that eggshell chitin provides both mechanical support and chemical impermeability essential to developing embryos. Knocking down chs-2 by RNAi caused a defect in the pharynx and led to L1 larval arrest, indicating that chitin is involved in the development and function of the pharynx.     

Downregulation of MDM2 expression by RNAi inhibits LoVo human colorectal adenocarcinoma cells growth and the treatment of LoVo cells with mdm2siRNA3 enhances the sensitivity to cisplatin

To investigate the biological effect of mdm2 in human colorectal adenocarcinoma LoVo cells, three mdm2siRNA constructions were recombined and transient transfected into human colorectal adenocarcinoma LoVo cells with low differentiation character in vitro. The results showed that mdm2siRNA3 reduced mRNA level of mdm2 and protein level of mdm2, leading to proliferation inhibition on LoVo cells, and reduced tumor growth in nude mice. It was found that depletion of MDM2 in this pattern promoted apoptosis of LoVo cells and Cisplatin (DDP) treated in the mdm2siRNA3 transfected cell population would result in a substantial decrease by MTT colorimetry. Decreasing the MDM2 protein level in LoVo cells by RNAi could significantly inhibit tumor growth both in vitro and in vivo, which indicated that mdm2 gene played a definite role in the development and aggressiveness of human colon carcinoma. It also could be a therapeutic target in colorectal carcinoma. The synergistic activation of RNAi and cell toxicity agents indicated that the combination of chemotherapy and gene therapy will be a promising approach in the future.     

The aurora kinase AIR-2 functions in the release of chromosome cohesion in Caenorhabditis elegans meiosis

Accurate chromosome segregation during cell division requires not only the establishment, but also the precise, regulated release of chromosome cohesion. Chromosome dynamics during meiosis are more complicated, because homologues separate at anaphase I whereas sister chromatids remain attached until anaphase II. How the selective release of chromosome cohesion is regulated during meiosis remains unclear. We show that the aurora-B kinase AIR-2 regulates the selective release of chromosome cohesion during Caenorhabditis elegans meiosis. AIR-2 localizes to subchromosomal regions corresponding to last points of contact between homologues in metaphase I and between sister chromatids in metaphase II. Depletion of AIR-2 by RNA interference (RNAi) prevents chromosome separation at both anaphases, with concomitant prevention of meiotic cohesin REC-8 release from meiotic chromosomes. We show that AIR-2 phosphorylates REC-8 at a major amino acid in vitro. Interestingly, depletion of two PP1 phosphatases, CeGLC-7alpha and CeGLC-7beta, abolishes the restricted localization pattern of AIR-2. In Ceglc-7alpha/beta(RNAi) embryos, AIR-2 is detected on the entire bivalent. Concurrently, chromosomal REC-8 is dramatically reduced and sister chromatids are separated precociously at anaphase I in Ceglc-7alpha/beta(RNAi) embryos. We propose that AIR-2 promotes the release of chromosome cohesion via phosphorylation of REC-8 at specific chromosomal locations and that CeGLC-7alpha/beta, directly or indirectly, antagonize AIR-2 activity.     

A locus controlling the activity of UDP-galactose:GM2(NeuGc) galactosyltransferase in mouse liver is linked to the H-2 complex

Genetic polymorphism in the expression of the G_M1(NeuGc) ganglioside has been shown in the liver of inbred strains of mice. Through analysis of the gangliosides of H-2 congenic and recombinant strains, this polymorphism was demonstrated to be controlled by a locus mapped left outside of the H-2 complex on chromosome 17, and the locus was assumed to control the level of the activity of G_M1(NeuGc) synthetase, UDP-galactose:G_M2(NeuGc) galactosyltransferase (E.C.2.4.1.62) [Hashimotoet al., J Biochem (1983) 94:2049-54].In the present study we analyzed the genetic linkage between the activity of the galactosyltransferase and the H-2 haplotype. For this purpose, we selected two inbred strains of mice, WHT/Ht and BALB/c, because they have different levels of the transferase activity and show different H-2 haplotypes; the specific activity of the transferase obtained with BALB/c was one-eighth of that with WHT/Ht, and BALB/c expressed the la.7 antigen as one of the products encoded in their H-2^d complex, whereas WHT/Ht did not. To analyze the linkage between these two phenotypes, WHT/Ht were mated with BALB/c to obtain the F₁ mice, and the female F₁ mice were then backcrossed to WHT/Ht. It was found that one half of the backcross generation expressed the la.7 antigen derived from BALB/c and had a significantly lower specific activity of the transferase than that of WHT/Ht, while the other half did not express the la.7 antigen but had the same specific activity of the transferase as that obtained with WHT/Ht.These results suggest that the locus controlling the level of the transferase activity in mouse liver is linked to the H-2 complex on chromosome 17.Abbreviations NeuGc N-glycolylneuraminic acid The ganglioside nomenclature is based on the system of Svennerholm, J Neurochem (1963) 10:613-23. The sialic acid species present is shown in parentheses after the ganglioside abbreviation.     

Drosophila CK2 phosphorylates Hairy and regulates its activity in vivo

Hairy is a repressor that regulates bristle patterning, and its loss elicits ectopic bristles (neural hyperplasia). However, it has remained unknown whether Hairy is regulated by phosphorylation. We describe here the interaction of protein kinase CK2 and Hairy. Hairy is robustly phosphorylated by the CK2-holoenzyme (CK2-HoloE) purified from Drosophila embryos, but weakly by the catalytic CK2α-subunit alone, suggesting that this interaction requires the regulatory CK2β-subunit. Consistent with this, Hairy preferentially forms a direct complex with CK2-HoloE. Importantly, we demonstrate genetic interactions between CK2 and hairy (h). Thus, flies trans-heterozygous for alleles of CK2α and h display neural hyperplasia akin to homozygous hypomorphic h alleles. In addition, we show that similar phenotypes are elicited in wild-type flies upon expression of RNAi constructs against CK2α/β, and that these defects are sensitive to h gene dosage. Together, these studies suggest that CK2 contributes to repression by Hairy.     

pWormgatePro enables promoter-driven knockdown by hairpin RNA interference of muscle and neuronal gene products in Caenorhabditis elegans

Recent advances in genome research and RNA interference (RNAi) technology have accelerated the adoption of genome-wide experimental approaches for determining gene function in the model organism Caenorhabditis elegans. Despite recent successes, the application of RNAi is limited when gene knockdown causes complex phenotypes or embryonic lethality. Recently, the high-throughput pWormgate cloning system has been introduced as a tool to efficiently generate heat-shock-inducible hairpin RNA constructs using the Gateway recombination technology. We have modified pWormgate into a versatile hairpin cloning plasmid, pWormgatePro, which facilitates temporally and spatially inducible hairpin RNAi using constitutively active, tissue-specific promoters. To demonstrate its utility we knocked down unc-22 in body wall muscles as well as the axon guidance gene unc-5 in the nervous system indicating that promoter-driven hairpins can overcome the neuronal resistance to RNAi. Using pWormgatePro we also show that RNAi in the nervous system of C. elegans is non-autonomous and that spreading of the RNAi signal from neurons to muscle is substantially reduced but not abolished in spreading-defective sid-1 mutant animals. Our findings illustrate the effectiveness of pWormgatePro for gene silencing in muscle cells and neurons and bring forward the possibility of applying tissue-specific RNAi on a genome-wide scale.     

Use of RNA interference to dissect the roles of trans-acting factors in alternative pre-mRNA splicing

RNA interference (RNAi) is becoming a popular method for analyzing gene function in a variety of biological processes. We have used RNAi in cultured Drosophila cells to identify trans-acting factors that regulate the alternative splicing of endogenously transcribed pre-mRNAs. We have generated a dsRNA library comprising 70% of the Drosophila genes encoding RNA binding proteins and assessed the function of each protein in the regulation of alternative splicing. This approach not only identifies trans-acting factors regulating specific alternative splicing events, but also can provide insight into the alternative splicing regulatory networks of Drosophila. Here, we describe this RNAi approach to identify alternative splicing regulatory proteins in detail.     

PGE(2) induces oenocytoid cell lysis via a G protein-coupled receptor in the beet armyworm, Spodoptera exigua

Eicosanoids mediate cellular and humoral immune responses in the beet armyworm, Spodoptera exigua, including activation of prophenoloxidase (PPO). PPO activation begins with release of its inactive zymogen, PPO, from oenocytoids in response to prostaglandins (PGs). Based on the biomedical literature, we hypothesized that PGs exert their actions via specific G protein-coupled receptor(s) in S. exigua. This study reports a G protein-coupled receptor (Se-hcPGGPCR1) gene, which is expressed in the hemocytes of S. exigua. The Se-hcPGGPCR1 consists of 420 amino acids and belongs to rhodopsin-type GPCRs. The high content of hydrophobic amino acid residues within the Se-hcPGGPCR1 protein is explained by prediction of seven-transmembrane domains that are characteristic of these GPCRs. Except for the eggs, Se-hcPGGPCR1 was expressed in all life stages. During the larval stage, it was expressed in hemocytes and gut, but not in fat body nor in epidermis. Real time quantitative RT-PCR showed that bacterial challenge induced more than 20-fold increases in its expression level. Fluorescence in situ hybridization showed that Se-hcPGGPCR1 was expressed in a specific hemocyte type, the oenocytoids. A specific eicosanoid, PGE₂, significantly induced oenocytoid lysis and increased PO activity in the plasma. In contrast, when Se-hcPGGPCR1 expression was suppressed by RNA interference (RNAi), the oenocytoid lysis and the PO activation in response to PGE₂ were not elevated above basal levels. A binding assay using intracellular calcium mobilization showed that the RNAi-treated hemocytes were significantly less responsive to PGE₂ than the control hemocytes. These results support our hypothesis with the specific finding that PGE₂ acts through Se-hcPGGPCR1 to activate PPO by lysing oenocytoids.     

Upregulation of PHLDA2 in Dicer knockdown HEK293 cells

It has been reported that RNAi-dependent chromatin silencing in vertebrates is not restricted to the centromeres. To address whether RNAi machinery could regulate the chromatin structure of imprinted genes, we knocked down Dicer in HEK293 cells and found that the expression of PHLDA2, one of the several genes in the imprinted gene domain of 11p15.5, was specifically upregulated. This was accompanied by a shift towards more activated chromatin at PHLDA2 locus as indicated by change in H3K9 acetylation, however, the methylation state at this locus was not affected. Furthermore, we found that PHLDA2 was downregulated in growth-arrested HEK293 cells induced by either serum deprivation or contact inhibition. This suggests that PHLDA2 upregulation might be a direct result of Dicer depletion rather than the consequence of growth arrest induced by Dicer knockdown. Considering the reports that there is consistent placental outgrowth in PHLDA2 knockout mice and that PHLDA2 overexpression in mice causes growth inhibition, we speculate that PHLDA2 may be a candidate for contributing to the reduced growth rate of Dicer-deficient cells and the very early embryonic lethality in Dicer knockout mice.     

Putative tumor suppressor Lats2 induces apoptosis through downregulation of Bcl-2 and Bcl-x(L)

Lats2, also known as Kpm, is the second mammalian member of the novel Lats tumor suppressor gene family. Recent studies have demonstrated that Lats2 negatively regulates the cell cycle by controlling G1/S and/or G2/M transition. To further understand the role of Lats2 in the control of human cancer development, we have expressed the protein in human lung cancer cells by transduction of a replication-deficient adenovirus expressing human Lats2 (Ad-Lats2). Using a variety of techniques, including Annexin V uptake, cleavage of PARP, and DNA laddering, we have demonstrated that the ectopic expression of human Lats2 induced apoptosis in two lung cancer cell lines, A549 and H1299. Caspases-3, 7, 8, and 9 were processed in the Ad-Lats2-transduced cells; however, it was active caspase-9, not caspase-8, that initiated the caspase cascade. Inhibitors specific to caspase-3 and 9 delayed the onset of Lats2-mediated apoptosis. Western blot analysis revealed that anti-apoptotic proteins, BCL-2 and BCL-x(L), but not the pro-apoptotic protein, BAX, were downregulated in Ad-Lats2-transduced human lung cancer cells. Overexpression of either Bcl-2 or Bcl-x(L) in these cells lead to the suppression of Lats2-mediated caspase cleavage and apoptosis. These results show that Lats2 induces apoptosis through downregulating anti-apoptotic proteins, BCL-2 and BCL-x(L), in human lung cancer cells.     

Differentiation-induced HL-60 cell apoptosis: A mechanism independent of mitochondrial disruption?

HL-60 cell differentiation into neutrophil like cells is associated with their induction of apoptosis. We investigated the cellular events that occur pre and post mitochondrial permeability transition to determine the role of the mitochondria in the induction of differentiation induced apoptosis. Pro-apoptotic Bax was translocated to and cleaved at the mitochondrial membrane in addition to t-Bid activation. These processes contributed to mitochondrial membrane disruption and the release of cytochrome c and Smac/DIABLO. The release of cytochrome c was caspase independent, as the caspase inhibitor Z-VAD.fmk, which inhibited apoptosis, did not block the release of cytochrome c. In contrast, the release of Smac/DIABLO was partially inhibited by caspase inhibition indicating differential release pathways for these mitochondrial pro-apoptotic factors. In addition to caspase inhibition we assessed the effects of the Bcl-2 anti-apoptotic family on differentiation induced apoptosis. BH4-Bcl-xl-TAT recombinant protein did not delay apoptosis, but did block the release of cytochrome c and Smac/DIABLO. Bcl-2 over-expression also inhibited differentiation induced apoptosis but was associated with the inhibition of the differentiation process. Differentiation mediated mitochondrial release of cytochrome c and Smac/DIABLO, may not trigger the induction of apoptosis, as BH4-Bclxl-TAT blocks the release of pro-apoptotic factors from the mitochondria, but does not prevent apoptosis.     

Cell-free translation system from Drosophila S2 cells that recapitulates RNAi

RNA interference (RNAi) is a fundamental mechanism of gene regulation in a variety of organisms. In Drosophila cells, long double-stranded RNAs (dsRNAs) are processed into 21- to 23-nucleotide double-stranded fragments, termed short interfering RNAs (siRNAs). The siRNAs trigger sequence-specific mRNA degradation, which results in the inhibition of gene expression. These phenomena can be recapitulated in vitro in lysates of Drosophila syncytial blastoderm embryos. In the present work, we used the common Drosophila cell line, Schneider Line 2 (S2), as a source to establish a cell-free translation system. We demonstrate here that the S2 cell-free translation system can recapitulate RNAi. Both long dsRNAs and siRNAs can trigger RNAi in this system, and the silencing effects are significant. This system should provide an important tool for biochemical analyses of the RNAi mechanism.     

Caenorhabditis elegans WASP-interacting protein homologue WIP-1 is involved in morphogenesis through maintenance of WSP-1 protein levels

Mammalian WASP and N-WASP are involved in reorganization of the actin cytoskeleton through activation of the Arp2/3 complex and in regulation of cell motility or cell shape changes. In the present study, we identified WASP-interacting protein homologue (WIP)-1 in Caenorhabditis elegans. WIP-1 contains the domains and sequences conserved among mammalian WIP family proteins. Yeast two-hybrid analysis detected a physical interaction between WIP-1 and WSP-1, the sole homologue of WASP/N-WASP in C. elegans. Western analysis of embryo lysates showed that RNA interference (RNAi) treatment for wip-1 decreased levels of WSP-1 protein, and wsp-1(RNAi) treatment decreased levels of WIP-1 protein. However, wsp-1 mRNA levels were not decreased in wip-1(RNAi)-treated embryos, and wip-1 mRNA levels were not decreased in wsp-1(RNAi)-treated embryos. Furthermore, disruption of WIP-1 by RNAi resulted in embryonic lethality with morphologic defects in hypodermal cell migration, a process known as ventral enclosure. This phenotype was similar to that observed in RNAi experiments for wsp-1. Immunostaining showed that WIP-1 was expressed by migrating hypodermal cells, as was WSP-1. This expression during ventral enclosure was reduced in wip-1(RNAi)-treated embryos and wsp-1(RNAi)-treated embryos. Our results suggest that C. elegans WIP-1 may function in hypodermal cell migration during ventral enclosure by maintaining levels of WSP-1.     

The <Emphasis Type="Italic">transformer2</Emphasis> gene in <Emphasis Type="Italic">Musca domestica</Emphasis> is required for selecting and maintaining the female pathway of development

We present the isolation and functional analysis of a transformer2 homologue Mdtra2 in the housefly Musca domestica. Compromising the activity of this gene by injecting dsRNA into embryos causes complete sex reversal of genotypically female individuals into fertile males, revealing an essential function of Mdtra2 in female development of the housefly. Mdtra2 is required for female-specific splicing of Musca doublesex (Mddsx) which structurally and functionally corresponds to Drosophila dsx, the bottom-most regulator in the sex-determining pathway. Since Mdtra2 is expressed in males and females, we propose that Mdtra2 serves as an essential co-factor of F, the key sex-determining switch upstream of Mddsx. We also provide evidence that Mdtra2 acts upstream as a positive regulator of F supporting genetic data which suggest that F relies on an autocatalytic activity to select and maintain the female path of development. We further show that repression of male courtship behavior by F requires Mdtra2. This function of F and Mdtra2 appears not to be mediated by Mddsx, suggesting that bifurcation of the pathway at this level is a conserved feature in the genetic architecture of Musca and Drosophila.Edited by D. Tautz