RNA interference targeting cathepsin B of the carcinogenic liver fluke, Opisthorchis viverrini

Functional genomics have not been reported for Opisthorchis viverrini or the related fish-borne fluke, Clonorchis sinensis. Here we describe the introduction by square wave electroporation of Cy3-labeled small RNA into adult O. viverrini worms. Adult flukes were subjected to square wave electroporation employing a single pulse for 20 ms of 125 V in the presence of 50 μg/ml of Cy3-siRNA. The parasites tolerated this manipulation and, at 24 and 48 h after electroporation, fluorescence from the Cy3-siRNA was evident throughout the parenchyma of the worms, with strong fluorescence evident in the guts and reproductive organs of the adult worms. Second, other worms were treated using the same electroporation settings with double stranded RNA targeting an endogenous papain-like cysteine protease, cathepsin B. This manipulation resulted in a significant reduction in specific mRNA levels encoding cathepsin B, and a significant reduction in cathepsin B activity against the diagnostic peptide, Z-Arg-Arg-AMC. This appears to be the first report of introduction of reporter genes into O. viverrini and the first report of experimental RNA interference (RNAi) in this fluke. The findings indicated the presence of an intact RNAi pathway in these parasites which, in turn, provides an opportunity to probe gene functions in this neglected tropical disease pathogen.     

大肠杆菌poly(A)化mRNA基因的芯片制备

利用大肠杆菌mRNA中存在的一定程度的poly(A)现象,利用oligo(dT)与poly(A)特异结合的特性,纯化并逆转录mRNA,并应用RD-PCR双方法获得了170多条大肠杆菌poly(A)化mRNA的基因片段,利用这些片段打印成基因芯片,以供后续大肠杆菌的基因表达研究。     

Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus polyhedrin gene and its variant forms

The segments 10 (S10) of the 11 double stranded RNA genomes from Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) encoding a novel polyhedrin polypeptide was converted to cDNA, cloned, and sequenced. Three cDNA clones consisting of 1502 (AmCPV10-1), 1120 (AmCPV10-2), and 1415 (AmCPV10-3) nucleotides encoding polyhedrin of 254, 339, and 319 amino acids with molecular masses of 29, 39, and 37 kDa, respectively, were obtained, and verified by Northern analysis. These clones showed 70-94% sequence identity among them but none with any sequences in databases. The expression of AmCPV10-1 cDNA encoded polyhedrin in Sf-9 cells was detected by immunoblot analysis and formation of polyhedra by electron microscopy, as observed in AmCPV-infected gut cells, but no expression of AmCPV10-2 or AmCPV10-3 cDNA was detected, indicating that during AmCPV replication, along with functional S10 RNA, some defective variant forms of S10 RNAs are packaged in virion particles.     

The construction of a recombinant cDNA library representative of the poly(A)+ mRNA population from normal human lymphocytes.

A recombinant library has been constructed using the plasmid pAT153 and double stranded cDNA prepared from normal human lymphocyte poly(A)+ RNA. Transformation conditions were optimized to yield approximately 200,000 recombinants per microgram of double stranded cDNA. Statistical analysis as well as sequence complexity analysis of the inserted sequences indicates that the cDNA library is representative of > 99% of the poly(A)+ RNA present in the normal human lymphocyte.     

Development of methods for RNA interference in the sheep gastrointestinal parasite, Trichostrongylus colubriformis

The efficiency of RNA interference (RNAi) delivery to L1 through L3 stage worms of the sheep parasitic nematode Trichostrongylus colubriformis was investigated using several techniques. These were: (i) feeding of Escherichia coli expressing double stranded RNA (dsRNA); (ii) soaking of short interfering (synthetic) RNA oligonucleotides (siRNA) or in vitro transcribed dsRNA molecules; and (iii) electroporation of siRNA or in vitro transcribed dsRNA molecules. Ubiquitin and tropomyosin were used as a target gene because they are well conserved genes whose DNA sequences are available for several nematode parasite species. Ubiquitin siRNA or dsRNA delivered by soaking or electroporation inhibited development in T. colubriformis but with feeding as a delivery method, RNAi of ubiquitin was not successful. Feeding was, however, successful with tropomyosin as a target, suggesting that mode of delivery is an important parameter of RNAi. Electroporation is a particularly efficient means of inducing RNA in nematodes with either short dsRNA oligonucleotides or with long in vitro transcribed dsRNA molecules. These methods permit routine delivery of dsRNA for RNAi in T. colubriformis larval stage parasites and should be applicable to moderate to high-throughput screening.     

TRIP: a novel double stranded RNA binding protein which interacts with the leucine rich repeat of flightless I.

A northwestern screen of a CHO-K1 cell line cDNA library with radiolabelled HIV-1 TAR RNA identified a novel TAR RNA interacting protein, TRIP. The human trip cDNA was also cloned and its expression is induced by phorbol esters. The N-terminus of TRIP shows high homology to the coiled coil domain of FLAP, a protein which binds the leucine-rich repeat (LRR) of Flightless I (FLI) and the interaction of TRIP with the FLI LRR has been confirmed in vitro . TRIP does not bind single stranded DNA or RNA significantly and binds double stranded DNA weakly. In contrast, TRIP binds double stranded RNA with high affinity and two molecules of TRIP bind the TAR stem. The RNA binding domain has been identified and encompasses a lysine-rich motif. A TRIP-GFP fusion is localised in the cytoplasm and excluded from the nucleus. FLI has a C-terminal gelsolin-like domain which binds actin and therefore the association of TRIP with the FLI LRR may provide a link between the actin cytoskeleton and RNA in mammalian cells.     

Arbitrarily primed PCR fingerprinting of RNA.

Fingerprinting of RNA populations was achieved using an arbitrarily selected primer at low stringency for first and second strand cDNA synthesis. PCR amplification was then used to amplify the products. The method required only a few nanograms of total RNA and was unaffected by low levels of genomic double stranded DNA contamination. A reproducible pattern of ten to twenty clearly visible PCR products was obtained from any one tissue. Differences in PCR fingerprints were detected for RNAs from the same tissue isolated from different mouse strains and for RNAs from different tissues from the same mouse. The strain-specific differences revealed are probably due to sequence polymorphisms and should be useful for genetic mapping of genes. The tissue-specific differences revealed may be useful for studying differential gene expression. Examples of tissue-specific differences were cloned. Differential expression was confirmed for these products by Northern analysis and DNA sequencing uncovered two new tissue-specific messages. The method should be applicable to the detection of differences between RNA populations in a wide variety of situations.     

Drosophila RNA polymerase II elongation factor DmS-II has homology to mouse S-II and sequence similarity to yeast PPR2.

DmSII is a Drosophila RNA polymerase II elongation factor which suppresses pausing by RNA polymerase II at specific sites on double stranded templates. Using antibodies produced against the purified protein, a Drosophila cDNA expression library was screened and a cDNA was isolated which encoded a portion of DmSII. When this cDNA was used to probe Kc cell mRNA the predominant species was found to be 1.4 kb in length. The original cDNA was used to screen a Drosophila Kc cell cDNA library resulting in the isolation of a 1.4 kb cDNA which was then sequenced. The deduced protein sequence for DmSII exhibited high similarity to mouse SII protein sequence. In addition, significant sequence similarity was found with the protein encoded by the yeast gene PPR2, which is involved in regulation of URA4 gene expression. The comparison of amino acid sequences suggests that DmSII is comprised of two domains homologous to mouse SII separated by a flexible, serine rich region of low homology. The shorter yeast protein has sequence similarity only to the carboxy terminal domain.     

The cathepsin B of Toxoplasma gondii,toxopain-1, is critical for parasite invasion and rhoptry protein processing

Cysteine proteinases play a major role in invasion and intracellular survival of a number of pathogenic parasites. We cloned a single copy gene, tgcp1, from Toxoplasma gondii and refolded recombinant enzyme to yield active proteinase. Substrate specificity of the enzyme and homology modeling identified the proteinase as a cathepsin B. Specific cysteine proteinase inhibitors interrupted invasion by tachyzoites. The T. gondii cathepsin B localized to rhoptries, secretory organelles required for parasite invasion into cells. Processing of the pro-rhoptry protein 2 to mature rhoptry proteins was delayed by incubation of extracellular parasites with a cathepsin B inhibitor prior to pulse-chase immunoprecipitation. Delivery of cathepsin B to mature rhoptries was impaired in organisms with disruptions in rhoptry formation by expression of a dominant negative micro1-adaptin. Similar disruption of rhoptry formation was observed when infected fibroblasts were treated with a specific inhibitor of cathepsin B, generating small and poorly developed rhoptries. This first evidence for localization of a cysteine proteinase to the unusual rhoptry secretory organelle of an apicomplexan parasite suggests that the rhoptries may be a prototype of a lysosome-related organelle and provides a critical link between cysteine proteinases and parasite invasion for this class of organism.     

RNA干扰研究进展

RNA干扰(RNA interference,RNAi)是指由双链RNA(double-strandedRNA,dsRNA)启动的序列特异的转录后基因沉默现象,广泛存在于真菌、植物和动物中。它是细胞内由双链RNA诱导降解与其配对的特定mRNA的过程。细胞内双链RNA在酶的作用下,形成20-25碱基大小的小干扰RNA(siRNAs),由siRNAs进一步掺入多组分核酸酶并使其激活,从而精确降解与siRNAs序列相同的mRNA,抑制该基因在细胞内的翻译表达。RNAi技术是近年来迅速发展起来的高效、特异、易操作的基因沉默技术。与反义寡核苷酸等传统方法相比,RNAi技术有着无可比拟的优势。本文就其近年的研究进展作一综述。     

RNA interference: towards a functional genomics in mammalian cells?

The discovery of the induction of RNA degradation by double stranded RNA in C. elegans, "RNA interference", makes it possible to envision systematic studies of gene function in mammalian cells. Indeed, in spite of the existence in mammals of the interferon response to double stranded RNA, the introduction of small interfering RNA can induce a sequence specific inhibition of gene expression either through RNA degradation or by blocking translation. Although the inhibition is transient and usually not complete, strategies have been developed to achieve long term gene silencing. The issue of target specificity is still not completely clear and will probably constitute a limitation of this approach. However, because of the unprecedented ease with which large scale screens can be performed, RNA interference has already established itself as the tool of choice to initiate functional genomics in mammalian cells.     

Cloning of the novel putative apoptosis-related gene of Spirometra erinacei (Order Pseudophyllidae)

We postulated that apolysis was processed in accordance with apoptotic changes occurring in a cestode, Spirometra erinacei (Pseudophyllidea). We cloned the novel putative apoptosis-associated gene from S. erinacei via screening of a S. erinacei cDNA library with a ced-3 gene (activator of apoptosis) probe from Caenorhabditis elegans. We identified a 261-bp cDNA sequence, which encodes for an 86-amino acid protein. The cloned gene expression was observed in the neck and gravid proglottids via Northern blotting, using cloned cDNA inserts as probes, but the clone was not expressed in any of other tissues. We suggest that this gene may be involved in the apolysis of S. erinacei during normal tissue development and differentiation in cestode parasites.