Characterization of the chloroplast genome of selected dinoflagellates : mini- and digenic circles in adenoides eludens

• Main Author: Nelson, Martha
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/14149

DNA was isolated from three species of peridinin-containing dinoflagellate (Scrippsiella trochoidea, Prorocentrum micans and Adenoides eludens) and run through a CsCl density gradient to separate it into main (nuclear) and satellite (organellas) fractions. The fractions were hybridized with chloroplast gene probes. This showed that chloroplast genes are found on discrete pieces of satellite 2 DNA in two species: small pieces (~1.5 kb) in Prorocentrum micans, and larger pieces (5 kb, 9 kb and larger) in Adenoides eludens. These results were similar to those of Zhang et al. (1999), who discovered unigenic minicircles in the chloroplast DNA of the peridinin-containing dinoflagellate Heterocapsa triquetra. Sequence data for chloroplast genes psbA, psbC, and psbD were obtained from Adenoides eludens. Outward directed polymerase chain reaction (PCR) primers were designed for these genes to amplify minicircles, and products of 4.1 to 5.2 kb were obtained, indicating the likely presence of minicircles in the chloroplast DNA of this species. The outward PCR products from the psbA reaction were cloned. Sequencing revealed that clones of the psbA minicircle were different from one another, unlike the results of previous studies that showed that the sequence of minicircles containing the same gene are the same (Zhang et al. 1999, Zhang et al. 2002, Barbrook and Howe 2000, Barbrook et al. 2001, Hiller 2001). The clones did share some sequence identity, however, including some secondary structure, repeated motifs, and a 200 base pair conserved region. The length of the fully sequenced region was 4300 base pairs, making the minicircle 4489 base pairs in total. Outward and inward directed PCR primers from three chloroplast genes (psbA, psbC, and psbD) were used in all combinations to amplify many products ranging in size from 5.0 to 6.5 kb. The product resulting from the psbD Ro and psbA Fi primer pair was cloned. The sequence of the ends of three of these clones revealed that both of the gene ends (psbD and psbA) were present and that the non-coding regions of the clones were different from one another. Further experiments using nested PCR showed some similarities between these products and the psbA minicircles. This result implies that digenic circles may exist in Adenoides eludens.