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PubMed2005

Binding specificity and mRNA targets of a C. elegans PUF protein, FBF-1.

Sequence-specific RNA-protein interactions underlie regulation of many mRNAs. Here we analyze the RNA sequence specificity of Caenorhabditis elegans FBF-1, a founding member of the PUF protein family. Like other PUF proteins, FBF-1 binds to the 3' UTR of target mRNAs and decreases expression of those target genes. Here, we show that FBF-1 and its close relative, FBF-2, bind with similar affinity to multiple RNA sites. We use mutagenesis and in vivo selection experiments to identify nucleotides that are essential for FBF-1 binding. The binding elements comprise a "core" central region and flanking sequences. The core region is similar but distinct from the binding sites of other PUF proteins. We combine the identification of binding elements with informatics to predict new FBF-1 binding sites in a C. elegans 3' UTR database. These data identify a set of new candidate mRNA targets of FBF-1 and FBF-2.

Amino Acid Sequence↗
PubMed2004

Tissue-specific modification of gld-2 mRNA in C. elegans: likely C-to-U editing.

Seventeen years after the discovery of tissue-specific apoB mRNA editing, only three nucleus-encoded mRNAs have been shown to undergo C-to-U editing. All three mRNAs occur in mammals. apoB mRNA editing is tissue-specific and occurs normally, whereas NF1 and NAT1 mRNA editing is found largely in tumors. Here we report the first example of C-to-U RNA editing in Caenorhabditis elegans. The gld-2 gene encodes an atypical poly(A) polymerase that governs the mitosis/meiosis decision in the germ line as well as progression through meiosis and early embryogenesis. At least two of its alternatively spliced transcripts are germline-specific. We find that most and perhaps all germline-specific transcripts generated by the gld-2 gene undergo C-to-U editing, but that somatic transcripts show no detectable editing. The gld-2 C-to-U editing event changes the codon from CCG to CUG, which is predicted to cause a proline to leucine substitution in the protein sequence. Our findings suggest the presence of a sequence- and tissue-specific cytidine deaminase acting on RNA, or CDAR. This CDAR modifies a specific base in gld-2 mRNA, and acts only in the germline.

Animals↗
PubMed2004

Nematode m7GpppG and m3(2,2,7)GpppG decapping: activities in Ascaris embryos and characterization of C. elegans scavenger DcpS.

A spliced leader contributes the mature 5'ends of many mRNAs in trans-splicing organisms. Trans-spliced metazoan mRNAs acquire an m3(2,2,7)GpppN cap from the added spliced leader exon. The presence of these caps, along with the typical m7GpppN cap on non-trans-spliced mRNAs, requires that cellular mRNA cap-binding proteins and mRNA metabolism deal with different cap structures. We have developed and used an in vitro system to examine mRNA degradation and decapping activities in nematode embryo extracts. The predominant pathway of mRNA decay is a 3' to 5' pathway with exoribonuclease degradation of the RNA followed by hydrolysis of resulting mRNA cap by a scavenger (DcpS-like) decapping activity. Direct decapping of mRNA by a Dcp1/Dcp2-like activity does occur, but is approximately 15-fold less active than the 3' to 5' pathway. The DcpS-like activity in nematode embryo extracts hydrolyzes both m7GpppG and m3(2,2,7)GpppG dinucleoside triphosphates. The Dcp1/Dcp2-like activity in extracts also hydrolyzes these two cap structures at the 5' ends of RNAs. Interestingly, recombinant nematode DcpS differs from its human ortholog in its substrate length requirement and in its capacity to hydrolyze m3(2,2,7)GpppG.

Animals↗
PubMed2000

Sequence and secondary structure variation in the Gyrodactylus (Platyhelminthes: Monogenea) ribosomal RNA gene array.

Nucleotide sequences were determined for the rRNA internal transcribed spacers 1 and 2 (ITS1 and 2) and the 5' terminus of the large subunit rRNA in selected Gyrodactylus species. Examination of primary sequence variation and secondary structure models in ITS2 and variable region V4 of the small subunit rRNA revealed that structure was largely conserved despite significant variation in sequence. ITS1 sequences were highly variable, and models of structure were unreliable but, despite this, show some resemblance to structures predicted in Digenea. ITS2 models demonstrated binding of the 3' end of 5.8S rRNA to the 5' end of the large subunit rRNA and enabled the termini of these genes to be defined with greater confidence than previously. The structure model shown here may prove useful in future phylogenetic analyses.

Animals↗
PubMed2003

Differential gene expression during desiccation stress in the insect-killing nematode Steinernema feltiae IS-6.

The developmentally arrested life stage of the entomopathogenic nematode Steinernema feltiae is exposed to threats of survival, including desiccation. We adopted a comprehensive approach to the study of the molecular mechanisms of desiccation stress tolerance in S. feltiae IS-6. We identified, expressed sequence tags (ESTs) that are differentially expressed during desiccation stress in S. feltiae IS-6 infective juveniles using DNA subtractive hybridization. These ESTs included genes that are known to be stress related, genes that are homologous to hypothetical Caenorhabditis elegans proteins, and novel genes that may be involved in traits specific to S. feltiae. Expression pattern characterization revealed that all analyzed ESTs were induced during 8 and 24 hr of dehydration of S. feltiae IS-6. Our results unveiled some of the components of the genetic networks that are activated in S. feltiae IS-6 during dehydration and suggested a differing pattern of temporal regulation during nematode dehydration.

Animals↗
PubMed1997

PCR-RFLP pattern of three kinds of Metagonimus in Korea.

We tried to compare the three kinds of Metagonimus species, M. yokogawai, Metagonimus Miyata type, and M. takahashii, which were known to be distributed in Korea with polymerase chain reaction based-restriction fragment length polymorphism (PCR-RFLP) patterns. We amplified the internal transcribed spacer 1 (ITS1) site of ribosomal RNA and mitochondrial cytochrome c oxidase I (mCOI) gene. The restriction patterns of ITS1 gene with Rsa I, Alu I and Msp I showed multiple fragmented bands of different sizes between three kinds of Metagonimus. In case of mCOI gene, Rsa I and Alu I enzymes produced differentially fragmented band patterns. According to the parsimony analysis of PCR-RFLP patterns, the estimated genetic divergence between M. yokogawai and Metagonimus Miyata type was 0.034880, between Metagonimus Miyata type and M. takahashii was 0.028098, between M. yokogawai and M. takahashii was 0.018179. It is suggested that Metagonimus Miyata type may be separate species and evolutionize at the older time than the other two species.

Animals↗
PubMed2000

Identification of abundant mRNAs from the third stage larvae of the parasitic nematode, ostertagia ostertagi.

Reverse transcriptase-PCR (RT-PCR) was carried out on total RNA prepared from the third-stage larvae (L3) of Ostertagia ostertagi in order to clone and characterize the major transcripts expressed in this larval stage, as an initial investigation of arrested larval development in the parasite. Distinct bands were visible on an agarose gel and four of these were cloned and sequenced. Three of the bands represented multiple transcripts, while the fourth band encoded the enzyme GTP cyclohydrolase I (GTP-CH), which catalyses the first and rate-limiting step in pteridine biosynthesis. Northern blot analysis and RT-PCR demonstrated that GTP-CH is highly up-regulated in the L3 stage and undetectable in either the L2 or adult stages. Using immunogold electron microscopy, GTP-CH was shown to be predominantly localized to the cell body of the body wall muscles and the cells of the intestine in the L3.

Amino Acid Sequence↗
PubMedDate not supplied

[Comparative morphological and gene systematics analysis of the Proteocephalus thymalli (Cestoda: Proteocephalidae), a parasite of graylings in lakes Hovsogol and Baikal].

Results of comparative morphological and gene analyses of the cestode Proteocephalus thymalli, an intestine parasite of the lakes Hovsogol (Thymallus arcticus nigrescens) and Lake Baikal graylings (Thymallus arcticus baicalensis) are presented for the first time. The results indicate that representatives of the P. thymalli from these fishes are really two different species.

5' Untranslated Regions↗
PubMed2006

The liver profile in patients with hepatitis C virus and/or fascioliasis.

In this study, twenty HCV/PCR-RNA positive patients with neither infection nor infestation were 15 males and five females with ages ranging between 17-78 years old. Liver function tests: S. albumin was decreased in 15 patients (75%), total protein decreased in 3 patients (15%), total bilirubin increased in 7 patients (35%), AST/ALT increased in 3 patients (15%). Globulin value increased in 15 patients (75%). A/G ratio decreased in 12 (60%). Ten normal individuals (five males & five females), gave normal findings. However, a 19 years-old female had non significant elevation (0.26 mg/dl) in direct bilirubin. Liver function tests: one patient had increased AST/ALT, Globulin value decreesed (-0.2) in another one (10%) and A/G ratio increased (+0.3 to +0.6) in three (30%) individuals. Twenty randomly selected patients (15 males and five females) HCV/3rd generation ELISA showed ALT elevation in 17 (85%), AST in all (100%), S. albumin decreased in 9 (45%), and increased in 1 (5%). Total protein decreased in 3 (15%), total bilirubin increased in 7 (35%) and direct bilirubin in 4 (20%), AST/ALT value increased in 3 patients (15%), Globulin value increased in 15 patients (75%). A/G ratio decreased in 12 (60%). Three patients had schistosomiasis, one 30 years old male had increased AST/ALT, normal globulin, increased A/G and positive HBs-Ag. The second one was a 33-year-old male had normal AST/ALT, normal globulin, increased A/G and positive HBs-Ag. The third patient was a 19-year-old female with normal AST/ALT and normal globulin, increased A/G and positive HBs-Ag. Two patients had fascioliasis, one was a 20-year-old male with increased AST/ALT, globulin normal, A/G normal and negative HBs-Ag. The second one was a 26-year-old female with normal AST/ALT and normal globulin, increased A/G and positive HBs-Ag. The other four positive HBs-Ag patients were parasite-free. Also, the other HCV/ELISA positive were negative for HBs-Ag. HCV/ELISA may have cross-reacted with HBs-Ag and/or with elevated ALT and gave false positive HCV. The 3rd generation ELISA in detection of HCV was not as sensitive as PCR/RNA. Out of 41 fascioliasis patients (26 males & 15 females), 14 were positive HCV/ELISA (34.1%), but only six were positive HCV/PCR (14.6%). ALT increased in 18 fascioliasis patients (43.9%), AST in 23 patients (56.1%) but, albumin decreased in 7 patients (17.1%), total protein decreased in 5 (12.2%), but total bilirubin increased in 14 (34.1%) and direct bilirubin increased in 2 (4.9%). Liver function tests of 14 fascioliasis and ELISA positive HCV showed AST/ALT increased in 6 (42.9%), globulin increased in 3 (21.4%) and decreased in 6 (42.8%). A/G decreased in 4 (28.6%) and increased in 8 (57.2%). Liver function tests of pure 27 fascioliasis patients showed that AST /ALT increased in 8 (29.6%), globulin increased in one patient (3.7%) but decreased in 10 (37.0%) and A/G ratio increased in 13 (48.1%).

Adolescent↗
PubMed2006

HCV/PCR positivity in bile and doudenal aspiration of fascioliasis and/or HCV patients.

In this study, three bile aspirates taken from 10 fascioliasis patients (30.0%) showed HCV positivity by PCR/RNA. Also, four duodenal aspirates (66.7%) taken from six HCV/PCR-RNA positive patients and three duodenal aspirates (20%) taken from 15 pure fascioliasis patients showed HCV positivity by PCR/ RNA. This is the first time to demonstrate HCV/PCR-RNA in the bile and duodenal aspirates of fascioliasis patients and in the duodenal aspirates of HCV patients. So, PCR can be used for the detection of HCV in the bile and/or duodenal aspirates of HCV suspected patient. On the other hand, this outcome results may incriminate HCV infection as a concomitant with fascioliasis or incriminate fascioliasis as paving the way to HCV.

Adult↗
PubMed1994

Detection of Fasciola hepatica in infected intermediate hosts using RT-PCR.

Fasciola hepatica, the common bile duct fluke, is an economically important parasite of domestic livestock. Current research interest is directed toward an understanding of the parasite's biology at the intermediate host level. To permit study of seasonal transmission patterns and parasite/intermediate host interactions, a fasciolid-specific assay has been developed to detect infected snail vectors. This assay uses the reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify specifically a region of F. hepatica small-subunit rRNA, followed by hybridization to an F. hepatica-specific probe. The assay does not cross-react with 2 trematodes outside of the Fasciolidae but does detect Fascioloides magna rRNA. Sequence alignment with additional small-subunit rRNAs shows Fasciolopsis buski would also cross-react with the assay. The detection limit of the assay is 10 fg of fluke total RNA with 5 micrograms of snail RNA added as background. Additionally, the assay detects individual infected snails immediately after miracidial exposure and throughout the parasite's development period.

Animals↗
PubMed1996

Direct analysis of nematode cis- and trans-spliceosomes: a functional role for U5 snRNA in spliced leader addition trans-splicing and the identification of novel Sm snRNPs.

Most nuclear pre-mRNAs in nematodes are processed by both cis- and trans-splicing. In trans-splicing, the 5' terminal exon, the spliced leader sequence (SL), is derived from a trans-splicing specific Sm snRNP, the SL RNP. Because U snRNPs are required cofactors for trans-splicing, and because this processing reaction proceeds via a two-step reaction pathway identical to that of cis-splicing, it has long been assumed that trans-splicing is catalyzed in a complex analogous to the cis-spliceosome. However, similarities or differences between cis- and trans-spliceosomes have not been established. In particular, the role of U5 snRNP in trans-splicing has been unclear. Here, we have used affinity selection to analyze the U snRNA constituents of nematode cis- and trans-spliceosomes. We find that U5 snRNP is an integral component of the trans-spliceosome and, using site-specific crosslinking, we show that U5 snRNP establishes specific Interactions with the SL RNA exon. We also identify two novel Sm snRNPs that are enriched in both cis- and trans-spliceosomes. Finally, we provide evidence that a SL RNP-containing multi-snRNP (SL, U4, U5, and U6 RNPs) may be a functional precursor in trans-spliceosome assembly.

Animals↗
PubMed1996

Multiple requirements for nematode spliced leader RNP function in trans-splicing.

The 5' exon donor in nematode trans-splicing, the SL RNA, is a small (approximately 100 nt) RNA that resembles cis-spliceosomal U snRNAs. Extensive analyses of the RNA sequence requirements for SL RNA function have revealed four essential elements, the core Sm binding site, three nucleotides immediately downstream of this site, a region of Stem-loop II, and a 5' splice site. Although these elements are necessary and sufficient for SL RNA function in vitro, their respective roles in promoting SL RNA activity have not been elucidated. Furthermore, although it has been shown that assembly of the SL RNA into an Sm RNP is a prerequisite for function, the protein composition of the SL RNP has not been determined. Here, we have used oligoribonucleotide affinity to purify the SL RNP and find that it contains core Sm proteins as well as four specific proteins (175, 40, 30, and 28 kDa). Using in vitro assembly assays; we show that association of the 175- and 30-kDa SL-specific proteins correlates with SL RNP function in trans-splicing. Binding of these proteins depends upon the sequence of the core Sm binding site; SL RNAs containing the U1 snRNA Sm binding site assemble into Sm RNPs that contain core, but not SL-specific proteins. Furthermore, mutational and thiophosphate interference approaches reveal that both the primary nucleotide sequence and a specific phosphate oxygen within a segment of Stemloop II of the SL RNA are required for function. Finally, mutational activation of an unusual cryptic 5' splice site within the SL sequence itself suggests that U5 snRNA may play a primary role in selecting and specifying the 5' splice site in SL addition trans-splicing.

Animals↗
PubMed1996

Nematode spliced leaders--ubiquity, evolution and utility.

Most nematode messenger RNAs (mRNAs) have at their 5' end a common 22 nucleotide leader sequence, the trans-spliced leader or SL1. The presence of this leader on some but not all mRNAs raises several questions: What is the role of the spliced leader in mRNA maturation, stability and translation? Why do some genes have a spliced leader and others not? What is the evolutionary origin of this trans-splicing mechanism? Recently, additional trans-spliced leaders (SL2, 3, 4, 5) have been described. What role do these variants play in nematode gene expression? While definitive answers to these questions remain elusive, it is clear that the spliced leader will significantly facilitate the cloning and sequence analysis of most nematode mRNAs.

Animals↗
PubMed1997

Species variation within the internal transcribed spacer (ITS) region of Gyrodactylus (Monogenea:Gyrodactylidae) ribosomal RNA genes.

The ribosomal internal transcribed spacer (ITS) region from individual Gyrodactylus specimens was amplified by polymerase chain reaction (PCR). The reaction amplified the entire ITS1-5.8S-ITS2 region of the ribosomal RNA gene cluster using primers that hybridize to the 3' terminus of the small subunit and the 5' terminus of the large subunit ribosomal RNA genes. The PCR products from Gyrodactylus salaris and Gyrodactylus thymalli were cloned and sequenced. The Gyrodactylus 5.8S gene was identified following comparative alignment of the G. salaris sequence and a Schistosoma 5.8S gene sequence. The ITS regions from G. salaris, G. thymalli, Gyrodactylus derjavini, and Gyrodactylus truttae were compared by restriction enzyme analysis and interspecific restriction fragment length polymorphisms were found. Gyrodactylus salaris and G. thymalli restriction fragment sizes were confirmed from sequence data. ITS amplification followed by Sau3AI digestion enables rapid and clear differentiation of G. salaris, G. derjavini, and G. truttae.

Animals↗
PubMed2007

RNA interference in parasitic helminths: current situation, potential pitfalls and future prospects.

RNA interference (RNAi) has become an invaluable tool for the functional analysis of genes in a wide variety of organisms including the free-living nematode Caenorhabditis elegans. Recently, attempts have been made to apply this technology to parasitic helminths of animals and plants with variable success. Gene knockdown has been reported for Schistosoma mansoni by soaking or electroporating different life-stages in dsRNA. Similar approaches have been tested on parasitic nematodes which clearly showed that, under certain conditions, it was possible to interfere with gene expression. However, despite these successes, the current utility of this technology in parasite research is questionable. First, problems have arisen with the specificity of RNAi. Treatment of the parasites with dsRNA resulted, in many cases, in non-specific effects. Second, the current RNAi methods have a limited efficiency and effects are sometimes difficult to reproduce. This was especially the case in strongylid parasites where only a small number of genes were susceptible to RNAi-mediated gene knockdown. The future application of RNAi in parasite functional genomics will greatly depend on how we can overcome these difficulties. Optimization of the dsRNA delivery methods and in vitro culture conditions will be the major challenges.

Animals↗