ABSTRACT
A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was developed for the molecular identification of 2 introduced bucephalid trematodes, Prosorhynchoides ozakii and Parabucephalopsis parasiluri . The method was applied for sporocysts and cercariae obtained from the golden mussel Limnoperna fortunei collected in the Uji River, Japan. The PCR-RFLP method showed that L. fortunei is the intermediate host of both trematode species. The present study thus recognizes the risk of L. fortunei , an invasive molluscan species, as a potential host for pathogenic trematodes.
Subject(s)
Amplified Fragment Length Polymorphism Analysis/methods , Bivalvia/parasitology , Trematoda/isolation & purification , Animals , Base Sequence , DNA, Helminth/chemistry , DNA, Helminth/metabolism , DNA, Ribosomal/chemistry , Deoxyribonucleases, Type II Site-Specific , Electrophoresis, Agar Gel , Molecular Sequence Data , Polymorphism, Genetic , Polymorphism, Restriction Fragment Length , Sequence Alignment , Trematoda/classification , Trematoda/geneticsABSTRACT
A modified base at the first (wobble) position of some tRNA anticodons is critical for deciphering the genetic code. In eukaryotes and eubacteria, AUA codons are decoded by tRNAsIle with modified bases pseudouridine (and/or inosine) and lysidine, respectively. The mechanism by which archaeal species translate AUA codons is unclear. We describe a polyamine-conjugated modified base, 2-agmatinylcytidine (agm(2)C or agmatidine), at the wobble position of archaeal tRNA(Ile) that decodes AUA codons specifically. We demonstrate that archaeal cells use agmatine to synthesize agm(2)C of tRNA(Ile). We also identified a new enzyme, tRNA(Ile)-agm(2)C synthetase (TiaS), that catalyzes agm(2)C formation in the presence of agmatine and ATP. Although agm(2)C is chemically similar to lysidine, TiaS constitutes a distinct class of enzyme from tRNA(Ile)-lysidine synthetase (TilS), suggesting that the decoding systems evolved convergently across domains.
Subject(s)
Agmatine/chemistry , Anticodon/chemistry , Anticodon/genetics , Archaea/genetics , Archaea/metabolism , Cytidine/chemistry , RNA, Transfer/chemistry , RNA, Transfer/genetics , Agmatine/metabolism , Anticodon/metabolism , Archaea/cytology , Catalysis , Cytidine/metabolism , Ligases/metabolism , RNA, Transfer/metabolismABSTRACT
We found that both tetramethylammonium chloride (TMA-Cl) and tetra-ethylammonium chloride (TEA-Cl), which are used as monovalent cations for northern hybridization, drastically destabilized the tertiary structures of tRNAs and enhanced the formation of tRNA*oligoDNA hybrids. These effects are of great advantage for the hybridization-based method for purification of specific tRNAs from unfractionated tRNA mixtures through the use of an immobilized oligoDNA complementary to the target tRNA. Replacement of NaCl by TMA-Cl or TEA-Cl in the hybridization buffer greatly improved the recovery of a specific tRNA, even from unfractionated tRNAs derived from a thermophile. Since TEA-Cl destabilized tRNAs more strongly than TMA-Cl, it was necessary to lower the hybridization temperature at the sacrifice of the purity of the recovered tRNA when using TEA-Cl. Therefore, we propose two alternative protocols, depending on the desired properties of the tRNA to be purified. When the total recovery of the tRNA is important, hybridization should be carried out in the presence of TEA-Cl. However, if the purity of the recovered tRNA is important, TMA-Cl should be used for the hybridization. In principle, this procedure for tRNA purification should be applicable to any small-size RNA whose gene sequence is already known.
