Efficient 5' ETS walking from conserved 18S rDNA sequences of the dinoflagellates <Emphasis Type="Italic">Alexandrium</Emphasis> and <Emphasis Type="Italic">Akashiwo sanguinea</Emphasis> (Dinophyceae) |
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Authors: | Email author" target="_blank">Jang-Seu?KiEmail author Myung-Soo?Han |
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Institution: | (1) Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, 133-791, Korea |
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Abstract: | An external transcribed spacer (ETS) walking PCR technique was developed for the isolation of unknown sequences adjacent to
the 18S rDNA. This strategy relied on four "walking primers", which were designed to bind unknown sequences upstream from
the 18S rDNA, and a specially programmed series of thermocycles. This method was successful in the isolation of the 5' ETS
regions from harmful dinoflagellates, including Alexandrium affine, A. catenella, A. minutum, A. tamarense, and Akashiwo sanguinea. Mono-directional sequencing reactions revealed the PCR products to be 392–962 nucleotides in length, and the 5' ETS in these
products were longer than 362 bp. These are the first such sequences available for A. sanguinea and the Alexandrium. In comparisons of the ETS sequences, genetic distance was considerably high within the Alexandrium. Furthermore, the sequences were significantly variable among the different strains of identical species: genetic distance
was recorded at 0.0420 for A. tamarense strains and as less than 0.7841 within strains of A. sanguinea. The 5'-start nucleotide of 18S rDNA was variable between the two genera: the five species of Alexandrium contained a T base, and A. sanguinea contained an A base. These results demonstrate the effectiveness of the ETS walking PCR method. This method will be valuable
in directional ETS walking from known regions to unknown regions, particularly concerning the boundary sequences of rRNA genes. |
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Keywords: | Akashiwo sanguinea Alexandrium ETS walking PCR external transcribed spacer ribosomal DNA |
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