Viruses are considered key players in phytoplankton population control in oceans. However, mechanisms that control viral gene expression in prominent microalgae such as diatoms remain largely unknown. In this study, potential promoter regions isolated from several marine diatom-infecting viruses (DIVs) were linked to the egfp reporter gene and transformed into the Pennales diatom Phaeodactylum tricornutum. We analysed their activity in cells grown under different conditions. Compared to diatom endogenous promoters, novel DIV promoter (ClP1) mediated a significantly higher degree of reporter transcription and translation. Stable expression levels were observed in transformants grown under both light and dark conditions, and high levels of expression were reported in cells in the stationary phase compared to the exponential phase of growth. Conserved motifs in the sequence of DIV promoters were also found. These results allow the identification of novel regulatory regions that drive DIV gene expression and further examinations of the mechanisms that control virus-mediated bloom control in diatoms. Moreover, the identified ClP1 promoter can serve as a novel tool for metabolic engineering of diatoms. This is the first report describing a promoter of DIVs that may be of use in basic and applied diatom research.

We previously reported a method, termed enzyme-mediated activation of radical sources (EMARS) for analysis of co-clustered molecules with horseradish peroxidase (HRP) fusion proteins expressed in living cells. This method is featured by radical formation of labeling reagents by HRP. In the current study, we have employed another labeling reagent, fluorescein-conjugated tyramide (FT) instead of the original arylazide compounds. Although hydrogen peroxide is required for the activation of FT, the labeling efficiency by HRP and the nonspecific reactions by endogenous enzyme(s) have been dramatically improved compared with the original fluorescein arylazide. This revised EMARS method has enabled visualization of co-clustered molecules in the endoplasmic reticulum and Golgi membranes with confocal microscopy. By using this method, we have found that GPI-anchored proteins, decay accelerating factor (DAF) and Thy-1 are exclusively co-clustered with HRP-DAFGPI and HRP-Thy1GPI, in which GPI attachment signals of DAF and Thy-1 have been connected to HRP, respectively. Furthermore, the N-glycosylation types of DAF and Thy-1 have been found to correspond to those of HRP-DAFGPI and HRP-Thy1GPI, respectively. These results indicate that each GPI-anchored protein species forms a specific lipid raft depending on its GPI attachment signal, and that the EMARS method can segregate individual lipid rafts.

Lipid rafts that are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins serve as a platform for important biological events. To elucidate the molecular mechanisms of these events, identification of co-clustering molecules in individual raft domains is required. Here we describe an approach to this issue using the recently developed method termed enzyme-mediated activation of radical source (EMARS), by which molecules in the vicinity within 300 nm from horseradish peroxidase (HRP) set on the probed molecule are labeled. GPI-anchored HRP fusion proteins (HRP-GPIs), in which the GPI attachment signals derived from human decay accelerating factor and Thy-1 were separately connected to the C-terminus of HRP, were expressed in HeLa S3 cells, and the EMARS reaction was catalyzed by these expressed HRP-GPIs under a living condition. As a result, these different HRP-GPIs had differences in glycosylation and localization and formed distinct clusters. This novel approach distinguished molecular clusters associated with individual GPI-anchored proteins, suggesting that it can identify co-clustering molecules in individual raft domains.

A nuclear transformation system for the centric diatom Chaetoceros sp. has been established using two plasmids pTpfcp/nat and pTpNR/green fluorescent protein (GFP) that had been used for Thalassiosira pseudonana transformation. These contain the nourseothricin resistance gene (nat) with the fucoxanthin chlorophyll a/c binding protein (fcp) promoter/terminator from T. pseudonana and the enhanced green fluorescent protein gene (egfp), with the nitrate reductase (NR) promoter/terminator from T. pseudonana, respectively. Transformants were recovered in the presence of the antibiotic nourseothricin. One to four copies of both nat and egfp genes were integrated into genomic DNA of the transformants. Transformation efficiency was 1.5-6.0 transformants per 10 8 cells. This work is the first report of stable genetic transformation of Chaetoceros, which is important as not only a constituent member of marine ecosystem but also feed for aquaculture.

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Lipid rafts serve as a platform for important biological events such as signal transduction, cell adhesion, and protein trafficking. To elucidate the molecular mechanisms of these events, identification of interacting molecules in individual raft domains is required. We have developed a novel method termed enzymemediated activation of radical source (EMARS), by which molecules in the vicinity within 300 nm from horseradish peroxidase (HRP) set on the probed molecule are labeled. Recently, we have established a new version of the EMARS system, in which the EMARS reaction is catalyzed by HRP expressed by genetic engineering. In order to express HRP in lipid rafts, HRP was constructed as a GPI-anchored form (HRP-GPI). Two kinds of HRP-GPIs, in which GPI attachment signals of human decay accelerating factor and Thy-1 were separately connected to the Cterminus of HRP, were expressed in human HeLa S3 cells, and the EMARS reaction was catalyzed by these expressed HRP-GPIs under a living condition. As a result, these HRP-GPIs underwent different N-glycosylation and formed distinct molecular clusters.Thus, this novel approach is able to identify molecular clusters associated with particular GPI-anchored proteins, suggesting that it can segregate individual lipid raft domains.

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