Differential DNA rearrangements of plastid genes, psbA and psbD, in two species of the dinoflagellate Alexandrium

Plastomes of the peridinin-containing dinoflagellates are composed of a limited number of genes, which are carried individually on small circular molecules, termed 'minicircles'. Although the prevalent plastid chromosome of most algae and plants has only a single copy of each gene, our previous study showed that low copy numbers of multiple variants of the gene psbA co-exist with the 'ordinary' gene encoding the D1 protein in minicircles of Alexandrium tamarense. Although none of the psbA variants encoded the entire protein, they persisted in culture. In this study, we compared the distribution and structure of psbA and psbD variants in two species of Alexandrium to characterize DNA rearrangement within these genes. In addition to four previously reported psbA variants, three psbD variants were found in A. tamarense minicircles. The ordinary psbA and psbD genes also co-existed with variants in another species, A. catenella. The sequences of the ordinary genes were virtually identical in the two species. All the variants comprised insertion or deletion mutations, with no base substitutions being identified. Duplicated parts of the coding sequences were contained in most of the insertions. Short direct repeats (4-14?bp) and/or adenine?+?thymine-rich motifs were present in all mutation regions, although the position and/or the sequence of each DNA rearrangement was unique to each variant. The results indicated that replication-based repeat-mediated recombination was responsible for generation of the variants.     

Genetic Manipulation of the Cyanobacterium Synechocystis sp. PCC 6803 (Development of Strains Lacking Photosystem I for the Analysis of Mutations in Photosystem II)

We have taken a genetic approach to eliminating the presence of photosystem I (PSI) in site-directed mutants of photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. By selecting under light-activated heterotrophic conditions, we have inactivated the psaA-psaB operon encoding the PSI reaction center proteins in cells containing deletions of the three psbA genes. We have also introduced deletions into both copies of psbD in a strain containing a mutation that inactivates psaA (ADK9). These strains, designated D1-/PSI- and D2-/PSI-, may serve as recipient strains for the incorporation of site-directed mutations in either psbA2 or psbD1. The characterization of these cells, which lack both PSI and PSII, is described.     

A new psbA mutation yielding an amino-acid exchange at the lumen-exposed site of the D1 protein

In eight metribuzin-resistant photoautotrophic cell cultures of Chenopodium rubrum (Thiemann and Barz, 1994 a, b) sequence analyses of a part of the psbA gene coding for the photosystem-II D1 protein had revealed different double and triple mutations within the herbicide binding niche of the protein (Schwenger-Erger et al., 1993). Two pairs of the examined cell lines carried identical mutations within this part of the protein, although their growth performance and their herbicide resistance patterns were different, both at the cellular and the thylakoid level. Starting from the known part of the psbA gene we have amplified the remaining psbA sequences using inverse polymerase chain reaction. Thus the complete sequence of the coding part of the gene was elucidated. After sequence analyses we found an additional amino acid exchange at the position 184 (ile-->asn) of the D1 protein in cell line L1. Metabolic consequences of this mutation are discussed. Partial sequence analyses of the psbD gene of the herbicide resistant cell culture lines revealed no mutation within that part of the D2 protein, which is in direct contact with the D1 protein.