STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF PUTATIVE REGULATORY DNA SEQUENCES OF FCP GENES IN THE CENTRIC DIATOM CYCLOTELLA CRYPTICA

Abstract

In order to investigate the transcriptional regulation of light harvesting proteins in diatoms we cloned and sequenced the regions encoding the N-termini together with the respective upstream DNA stretches of 11 different fucoxanthin chlorophyll a/c binding polypeptides (FCP) of Cyclotella cryptica. The deduced N-terminal amino acid sequences almost completely matched those deduced earlier from cDNA clones. No introns were found in the regions coding for the N-terminal plastid targeting sequences or shortly downstream of them.

A PLACE search for cis-acting DNA elements which are known from higher plants to be involved in the light-dependent or circadian expression of Lhc genes encoding chlorophyll a/b binding light-harvesting polypeptides in green plants resulted in the detection of several motifs. A comparative analysis with DNA stretches, upstream of the Lhc, LhcaR and Fcp genes of the green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae and the diatom Thalassiosira pseudonana, however, did not show any conservation of these elements. This indicates that green plants, red algae and diatoms most probably do not share the same conserved elements for light-regulated gene expression.

Furthermore a 924 bp DNA fragment habouring a stretch of 292 bp upstream DNA and the entire Fcp6 gene was amplified from genomic DNA of Cyclotella cryptica, cloned into a modified derivative of the transformation vector pPha-T1 and expressed in the pennate diatom Phaeodactylum tricornutum. Reverse transcriptase polymerase chain reaction using RNA of the transformants as template showed that the introduced Fcp6 gene indeed was transcribed, indicating that the regulatory sequence may be generally functional in diatoms.