Here we are testing the specific primers NEM06FWD2/NEM06REV2 and nem06FWD1/ nem06REV1 for the R6m-1 resistance gene to root-knot nematodes Meloidogyne spp. in breeding samples of sugar beet. Sugar beet plants of domestic and foreign breeding lines were the object of the study. To identify the relationship between R6m-1 gene, which is localized on the chromosome 1 and controls the stable level of the kinase activity signal, with sugar beet resistance to phytopathogens, PCR-analysis of 10 sugar beet samples were carried out using 2 pairs of molecular genetic markers. DNA amplification revealed a fragments ~500 bp and ~100 bp in length and as a result of sequencing of nucleotide sequences of R6m-1 gene region with subsequent alignment by Geneious Prime program, 3 single nucleotide substitutions (A/G, G/C, and G/A) in the resistant MS11018 genotype and one nucleotide substitution (A/G) and 3 deletions in a foreign hybrid Humber were identified. It can be assumed that these SNPs can form resistance by amino acid substitutions in the polypeptide chain. Finally, possibility to differentiate homozygous and heterozygous genotypes for this allele was shown.

Aim of the work was specific primers (FusA1F/FusA1R) testing to study the SE2 gene controlling acid chitinase effect under stress conditions. Plants of domestic and foreign sugar beet were the material for the investigation. To confirm relationship between the SE2 gene (localized to the chromosome 3) controlling steady effect of acid chitinase and sugar beet resistance to root rot, 10 sugar beet samples were genotyped using the FusA1 molecular marker. DNA-fragments of 600 and 400 b.p.in length were revealed in each of the investigated genotypes except plants of wild beet ( Beta corolliflora Zoss .). As a result of molecular-genetic studies of the SE2 gene, eight single nucleotide polymorphism (3 T/C, 2 C/G, A/G, G/A, C/T) and 3 single nucleotide inserts (nucleotide A) were identified in plants of the breeding sample No. 9 (Sh.1). Plants of this genotype showed symptoms of fusariose infection under field conditions as well. It can be concluded that the SNPs data lead to overcoming of resistance (by changing an amino-acid unit in a polypeptide), and, as consequence, to decrease of plant adaptability.

The goal of this study was a molecular genetic passportization of the initial breeding material of sugar beet (male sterile lines, synanthous pollinators, and their hybrids). The following SSR primers were used: Unigene 24 552, Unigene 2305, Unigene 17 623, Unigene 14 805, and Unigene 62 524. The length range of the obtained DNA fragments varied within 100–3000 bp, and up to 11 polymorphic bands per genotype were amplified. The maximum Polymorphic Information Content (PIC) value was revealed for the Unigene 17 623 (PIC = 0.88), Unigene 2305 (PIC = 0.84), and Unigene 14 805 (PIC = 0.85) loci that provided a possibility to differentiate sugar beet breeding material. Using five of the tested SSR markers, the molecular genetic passportization of 26 genotypes of valuable breeding samples of this crop was carried out that allowed their identification and certification for further use in a marker-assisted selection. The calculated genetic distances between male sterile forms and synanthous pollinators varied from 2.236 to 4.796. Parent samples located at considerable genetic distances from each other were recommended for use in creating heterotic hybrids.

This paper presents the results from molecular genetic studies on breeding material of domestic and foreign sugar beet hybrids for the presence of resistance genes to root-knot nematode, rhizomania, and powdery mildew. Testing of plants was carried out using the polymerase chain reaction method. The genes R6m-1, Rz1 and Rz2, and Pm were identified with the help of five single-chain RAPD and four allele-specific primers. As a result of molecular screening of the sugar beet cultivar samples, the presence of these resistance genes was revealed.

The genetic polymorphism of 12 microsatellite loci was revealed and unique DNA-profiles were obtained for initial lines of sugar beet. Two loci were monomorphic, other loci exhibited polymorphism. The genetic distances between the breeding materials were determined; the dendrogram of cluster analysis reflecting the probable divergence of samples was constructed. The data about the distance of the selection material will be used in the selection of pairs in heterotic breeding.

PCR analysis of eight microsatellite loci was performed to obtain individual genetic profiles for four transgenic sugar beet lines with mf2 gene, which encodes bacterial protein from Bacillus thuringiensis. Total DNA extractions from the vegetative tissue were carried out by the modified phenol-chloroform method. The primers Bvv15, 21, 23, 30, 32, 43, 53, and 64 were used (Smulders et al., 2010). The efficiency of using RAPD-markers (PawS 6, 16, and 17) for assessing the polymorphism of given plants and their genotyping was considered.

An optimized method for total DNA extraction from genetically modified sugar beet plants has been presented. Uses of high-toxic organic compounds and high-cost extraction kits for nucleic acid isolation are excluded. A comparative analysis of polymerase chain reaction products was performed with amplification of DNA obtained by two methods.

By a polymerase chain reaction (PCR), eight individual genetically modified lines of a sugar beet were identified. As a result of the polymorphism analysis of 12 microsatellite loci, the genetic profiles for each line were received.

This study aimed to estimate the prevalence, patterns and risk factors of tobacco consumption among a stratified random sample of students at University of Sharjah, United Arab Emirates (UAE), during 2005. The overall reported smoking prevalence among 1057 sampled students was 15.1%; cigarette smokers were 9.4% and waterpipe smokers 5.6%. While women comprised only 8.9% of cigarette smokers, they were 26.2% of waterpipe smokers. Multiple linear regression analysis showed that the most important predictors for smoking among the students were: male sex, having a smoking friend, having a smoking family member (father/mother/both), non-UAE nationality and older age. There is a need to intensify efforts to prevent young people from starting smoking and to help young smokers to stop.

Nematodes are unusual in that diverse molecular forms of acetylcholinesterase are the product of distinct genes. This is best characterised in the free living organism Caenorhabditis elegans, in which 3 genes are known to give rise to distinct enzymes, with a fourth likely to be non-functional. ACE-1 is an amphiphilic tetramer associated with a hydrophobic non-catalytic subunit, analogous to vertebrate T enzymes, whereas ACE-2 and ACE-3 are glycosylphosphatidylinositol-linked amphiphilic dimers. The different ace genes show distinct anatomical patterns of expression in muscles, sensory neurons and motor neurons, with only a few examples of coordinated expression. Clear homologues of ace-1 and ace-2 have now been isolated from a variety of parasitic nematodes, and the predicted proteins have very similar C-terminal amino acid sequences, implying an analogous means of anchorage to membranes. In addition to these membrane-bound enzymes, many parasitic nematodes which colonise mucosal surfaces secrete acetylcholinesterases to the external (host) environment. These hydrophilic enzymes are separately encoded in the genome, so that some parasites may thus have a total complement of six ace genes. The secretory enzymes have been characterised from the intestinal nematode Nippostrongylus brasiliensis and the lungworm Dictyocaulus viviparus. These show a number of common features, including a truncated C-terminus and an insertion at the molecular surface, when compared to other nematode acetylcholinesterases. Although the function of these enzymes has not been determined, they most likely alter host physiological responses to promote survival of the parasite.

Infective larvae and adult stage Trichinella spiralis secrete a protein homologous to prosaposin, the precursor of sphingolipid activator proteins (saposins) A–D originally defined in vertebrates. The protein contains four saposin domains, with the six cysteine residues which form the three intramolecular disulphide bonds in close register in each case. It differs substantially from vertebrate prosaposins in the N-terminal prodomain, the region separating saposins A and B, and completely lacks the C-terminal domain which has been demonstrated to be essential for lysosomal targetting in these organisms. The protein is secreted in unprocessed form with an estimated mass of 56 kDa, and contains a single N-linked glycan which is bound by the monoclonal antibody NIM-M1, characteristic of the TSL-1 antigens which are capped by tyvelose (3,6-dideoxy-d-arabinohexose). Immuno-electron microscopy localised the protein to membrane-bound vesicles and more complex multi-lamellar organelles in diverse tissues including the hypodermis, intestine and stichosomes, although it was absent from the dense-core secretory granules typical of the latter. Possible functions of a secreted prosaposin are discussed.

We describe the molecular cloning, expression and biochemical characterisation of recombinant forms of two secreted acetylcholinesterases from adult Dictyocaulus viviparus. The two variants (designated Dv-ACE-1 and Dv-ACE-2) were 613 and 615 amino acids long and showed 94.7% identity to one another. The highest level of identity to other cholinesterases was with ACE-2 of Caenorhabditis elegans. Dv-ACE-1 and Dv-ACE-2 showed 48.0 and 47.7% identity to C. elegans ACE-2 over 577 amino acids, respectively. The primary structure of both enzymes showed conservation of the catalytic triad and of a tryptophan residue known to be critical for the choline-binding site, but differed in the number of potential glycosylation sites and at one amino acid in the peripheral anionic site. Southern blotting and PCR experiments indicated that the genes encoding these enzymes are distinct. When expressed in Pichia pastoris, the enzymes were active, but differed subtly in their biochemical characteristics. Both enzymes exhibited a preference for acetylcholine as substrate, but differed in the extent of excess substrate inhibition and in their optimal pH for activity. The lack of an obvious carboxy-terminal membrane anchor and the presence of an insertion at the molecular surface were other features which, thus far, appear to be characteristic of parasite secreted acetylcholinesterases.

Helminth parasites have been largely refractory to genetic analysis, and as a result, functional studies of gene products have been limited by available technologies, guided largely by predictions from primary structure. This has posed a considerable problem to research efforts in this area thus far, and will clearly be a limiting factor in addressing questions raised by the large number of expressed sequence tags (ESTs) currently being generated from these organisms. This is particularly acute in the case of sequences for which no clear function can be ascribed. Moreover, even when a specific function appears evident from the primary structure, proteins may often perform unexpected roles, particularly in the context of parasitism. Development of genetic knockout techniques have not been possible due to the intrinsic difficulty in genetic crosses and the lack of methods for heritable transformation, although some progress has been made in transient expression of DNA and RNA by ballistic transfer [1,2]. RNA interference (RNAi) offers an opportunity to address some of these problems. Initially performed on Caenorhabditis elegans by microinjection [3,4], delivery of double stranded RNA (dsRNA) through the intestine was subsequently achieved via ingestion of transfected Escherichia coli [5], and by direct soaking of worms in dsRNA [6]. Dissemination from the nematode intestine to other somatic tissues and the germ line has been demonstrated, and somewhat surprisingly, the RNAi effect can be remarkably long-lived. Thus for many genes, interference may persist for several days postinjection and may in some cases be inherited in subsequent generations [6]. We therefore decided to assay whether RNAi could be applied to the parasitic nematode Nippostrongylus brasiliensis , utilising secreted acetylcholinesterases (AChEs) as targets. These enzymes are encoded by three separate genes expressed by fourth stage larvae (L4) and adult stages of this parasite. AChE B and AChE C are 90% identical in amino acid sequence, whereas AChE A is 63 /64% identical to the others [7,8]. In addition, N. brasiliensis expresses at least one other gene encoding a non-secreted (neuromuscular) AChE. Although these have yet to be cloned, the protein(s) can be discriminated from secreted variants by their characteristic slow migration in non-denaturing polyacrylamide gels stained for AChE activity [9]. Adult worms were recovered from the jejunum of rats at different times post-infection and soaked overnight in dsRNA. This was produced by T3 and T7 RNA polymerase following insertion of cDNA fragments into pBluescript, and in vitro transcription with T3 and T7 RNA polymerase (RiboMAX, Promega). Optimal results were obtained with dsRNA concentrations of 1 mg ml 1 or greater, and incubation was generally carried out in a volume of less than 200 ml of DEPCtreated phosphate buffered saline (PBS) at 37 8C. Following overnight (16 h) incubation, worms were washed 3 / in PBS and cultured in vitro for up to 6 days as previously described [10]. Culture medium was changed daily, passed through 0.2 mM filters and assayed for protein and AChE content [11]. Initial experiments were carried out with dsRNA derived from a full length cDNA for AChE B, i.e. 1799 bp [7]. Fig. 1A demonstrates that incubation in this dsRNA Abbreviations: AChE, acetylcholinesterase; BuChE, butyrylcholinesterase; ACh, acetylcholine; ASCh, acetylthiocholine; BuSCh, butyrylthiocholine; RNAi, RNA interference. * Corresponding author. Tel.: /44-207-594-5214; fax: /44-207225-0960. E-mail address: m.selkirk@ic.ac.uk (M.E. Selkirk). Molecular & Biochemical Parasitology 122 (2002) 91 /94

A third variant of acetylcholinesterase (AChE A) secreted by the parasitic nematode Nippostrongylus brasiliensis has been isolated which shows 63–64% identity to AChE B and AChE C, with a truncated carboxyl terminus and a short internal insertion relative to AChEs from other species. Three of the fourteen aromatic residues which line the active site gorge in Torpedo AChE are substituted by non-aromatic residues (Y70T, W279D and F288M). All three enzymes have 8 cysteine residues in conserved positions, including 6 which have been implicated in disulphide bonds in other AChEs. Phylogenetic analysis suggests that these enzymes form a distinct group which evolved after speciation and are most closely related to ACE-2 of Caenorhabditis elegans. Recombinant AChE A secreted by Pichia pastoris was monomeric and hydrophilic, with a substrate preference for acetylthiocholine and negligible activity against butyrylthiocholine. A model structure of AChE A built from the coordinates of the Torpedo californica AChE suggests that W345 (F331 in Torpedo) limits the docking of butyrylcholine. This model is consistent with mutational analysis of the nematode enzymes. Expression of AChE A is regulated at the transcriptional level independently of the other 2 secreted variants, with maximal expression by fourth stage larvae and young adult worms. These enzymes thus appear to represent an unusual family of AChEs with conserved structural features which operate outside the normal boundaries of known functions in regulation of endogenous neurotransmitter activity.

The yeast Pichia pastoris is a convenient production system that enables expression of heterologous proteins in high amounts. As a fermentation method, shaking flasks are very popular because of their simplicity of handling and their low cost. We compared the expression level of the enzyme acetylcholinesterase in a transformed strain of P. pastoris grown in different flasks, presenting various designs but all with the same volume. A several-thousand-fold difference appeared in the expression levels; and the results could not be explained by differences between the flasks in the oxygenation of the medium. The data show that flask design is an important factor to consider for optimising fermentation processes.

The synaptic enzyme acetylcholinesterase (AChE), which is the target of many insecticides and potential warfare agents, is implied in Alzheimer's disease and is a good potential candidate to be used in biosensors. This promotes a strong demand for production of recombinant AChE to be used in various studies. A promising expression system is the yeast Pichia pastoris, but the expression efficiency needs to be improved. Optimization studies require a rapid and efficient screening test to detect positive yeast colonies after transformation. Using indoxylacetate as a substrate, we designed a chromogenic test that is not interfered with by the culture media background color and, thus, is suitable for microplate screening. Moreover, it was possible to adapt the test for direct on-plate detection of AChE-expressing colonies.

The tripeptide glutathione (GSH) plays an important role in the maintenance of the intracellular thiol redox state and in detoxification processes. The intracellular GSH level depends on glutathione reductase as well as on GSH synthesis. The first and rate limiting step in the synthetic pathway is catalysed by gamma-glutamylcysteine synthetase (gamma-GCS). The gamma-GCS was partially purified from the filarial parasite Onchocerca volvoulus and preliminary steady state kinetics were performed. The Ki-value for L-buthionine-S,R-sulphoximine (BSO), a specific inhibitor of gamma-GCS, was determined to be 0.13 microM, which is 54-fold lower than the Ki-value for the mammalian enzyme. Filarial gamma-GCS was also inhibited by cystamine with a Ki-value of 3.9 microM compared with 22.2 microM determined for the rat enzyme. Further, the cDNA and the gene of the O. volvulus gamma-GCS were cloned and sequenced. The gene of 5762 bp is composed of 14 exons and 13 introns. Southern blot analysis indicates that the gamma-GCS gene is present as a single-copy gene. In accordance with Northern blot analysis, the entire cDNA sequence encompasses 2377 bp. At its 5' end a nematode-specific spliced leader 130 bp upstream of the first in frame methionine was identified. The cDNA encodes a polypeptide of 652 amino acids with 50 and 69% sequence identity to the human and the Caenorhabditis elegans counterparts, respectively. The filarial gamma-GCS is proposed as a potential drug target.

We have previously determined that Nippostrongylus brasiliensis secretes three monomeric nonamphiphilic (G1na) variants of acetylcholinesterase (AChE) with broadly similar properties. In this study we have examined AChE expression in somatic extracts of N. brasiliensis and report the identification of an additional enzyme which is not secreted. The enzyme was resolved by sucrose density gradient centrifugation with a sedimentation coefficient of 10.2 S which was shifted to 9.4 S in the presence of Triton X-100, identifying the enzyme as a tetrameric amphiphilic (G4a) form. The amphiphilic properties of this enzyme were confirmed by charge-shift electrophoresis, in which migration was accelerated by interaction with sodium deoxycholate. The enzyme showed low activity with butyrylthiocholine, and a Michaelis constant of 91 +/- 13 microM for acetylthiocholine was determined. It was highly sensitive to the AChE-specific inhibitor bis (4-allyldimethylammoniumphenyl)pentan-3-one dibromide, with an IC50 of 6.5 +/- 0.4 microM, but was also inhibited by the butyrylcholinesterase-specific inhibitor tetramonoisopropylpyrophosphortetramide, albeit with a higher IC50 of 46.5 +/- 6.1 microM. This enzyme can therefore be distinguished from the secreted AChEs by its amphiphilic properties, sedimentation in sucrose gradients, and sensitivity to cholinesterase inhibitors.

We have isolated a full-length cDNA encoding an acetylcholinesterase secreted by the nematode parasite Nippostrongylus brasiliensis. The predicted protein is truncated in comparison with acetylcholinesterases from other organisms such that the carboxyl terminus aligns closely to the end of the catalytic domain of the vertebrate enzymes. The residues in the catalytic triad are conserved, as are the six cysteines which form the three intramolecular disulfide bonds. Three of the fourteen aromatic residues which line the active site gorge in the Torpedo enzyme are substituted by nonaromatic residues, corresponding to Tyr-70 (Thr), Trp-279 (Asn), and Phe-288 (Met). High level expression was obtained via secretion from Pichia pastoris. The purified enzyme behaved as a monomeric hydrophilic species. Although of invertebrate origin and possessing the above substitutions in the active site gorge residues, the enzyme efficiently hydrolyzed acetylthiocholine and showed minimal activity against butyrylthiocholine. It displayed excess substrate inhibition with acetylthiocholine at concentrations over 2. 5 mM and was highly sensitive to both active site and "peripheral" site inhibitors. Northern blot analysis indicated a progressive increase in mRNA for AChE B in parasites isolated from 6 days postinfection.