Dinoflagellate Gene Structure and Intron Splice Sites in a Genomic Tandem Array.

Dinoflagellates are one of the last major lineages of eukaryotes for which little is known about genome structure and organization. We report here the sequence and gene structure of a clone isolated from a cosmid library which, to our knowledge, represents the largest contiguously sequenced, dinoflagellate genomic, tandem gene array. These data, combined with information from a large transcriptomic library, allowed a high level of confidence of every base pair call. This degree of confidence is not possible with PCR-based contigs. The sequence contains an intron-rich set of five highly expressed gene repeats arranged in tandem. One of the tandem repeat gene members contains an intron 26,372 bp long. This study characterizes a splice site consensus sequence for dinoflagellate introns. Two to nine base pairs around the 3' splice site are repeated by an identical two to nine base pairs around the 5' splice site. The 5' and 3' splice sites are in the same locations within each repeat so that the repeat is found only once in the mature mRNA. This identically repeated intron boundary sequence might be useful in gene modeling and annotation of genomes.

Plastid genes in a non-photosynthetic dinoflagellate.

Dinoflagellates are a diverse group of protists, comprising photosynthetic and heterotrophic free-living species, as well as parasitic ones. About half of them are photosynthetic with peridinin-containing plastids being the most common. It is uncertain whether non-photosynthetic dinoflagellates are primitively so, or have lost photosynthesis. Studies of heterotrophic species from this lineage may increase our understanding of plastid evolution. We analyzed an EST project of the early-diverging heterotrophic dinoflagellate Crypthecodinium cohnii looking for evidence of past endosymbiosis. A large number of putative genes of cyanobacterial or algal origin were identified using BLAST, and later screened by metabolic function. Phylogenetic analyses suggest that several proteins could have been acquired from a photosynthetic endosymbiont, arguing for an earlier plastid acquisition in dinoflagellates. In addition, intact N-terminal plastid-targeting peptides were detected, indicating that C. cohnii may contain a reduced plastid and that some of these proteins are imported into this organelle. A number of metabolic pathways, such as heme and isoprenoid biosynthesis, seem to take place in the plastid. Overall, these data indicate that C. cohnii is derived from a photosynthetic ancestor and provide a model for loss of photosynthesis in dinoflagellates and their relatives. This represents the first extensive genomic analysis of a heterotrophic dinoflagellate.

CHARACTERIZATION OF A GENE ENCODING THE LIGHT-HARVESTING VIOLAXANTHIN-CHLOROPHYLL PROTEIN OF NANNOCHLOROPSIS SP. (EUSTIGMATOPHYCEAE).

In contrast to vascular plants, green algae, and diatoms, the major light-harvesting complex of the marine eustigmatophyte genus Nannochloropsis is a violaxanthin-chlorophyll a protein complex that lacks chlorophylls b and c. The isolation of a single polypeptide from the light-harvesting complex of Nannochloropsis sp. (IOLR strain) was previously reported (Sukenik et al. 1992). The NH2 -terminal amino acid sequence of this polypeptide was significantly similar to NH2 -terminal sequences of the light-harvesting fucoxanthin, chlorophyll a/c polypeptides from the diatom Phaeodactylum tricornutum Bohlin. Using polyclonal antibodies raised to the Nannochloropsis light-harvesting polypeptide, a gene encoding this polypeptide was isolated from a cDNA expression library. The deduced amino acid sequence of the Nannochloropsis violaxanthin-chlorophyll a polypeptide reveals a 36 amino acid presequence followed by 173 amino acids that constitute the mature polypeptide. The mature polypeptide has 30%-40% sequence identity to the diatom fucoxanthin-chlorophyll a/c polypeptides and less then 27% identity to the green algal and vascular plant light-harvesting chlorophyll polypeptides that bind both chlorophylls a and b. Its molecular mass, as deduced from the gene sequence, is 18.4 kDa with three putative transmembrane helices and several residues that may be involved in chlorophyll binding. The cDNA encoding the violaxanthin-chlorophyll a polypeptide was used to isolate and characterize a 10 kb genomic fragment containing the entire gene. The open reading frame was interrupted by five introns ranging in size from 123 to 449 bp. The intron borders have typical eukaryotic GT … AG sequences.

Characterization of both polyunsaturated fatty acid biosynthetic pathways in Schizochytrium sp.

Schizochytrium produces long chain polyunsaturated fatty acids (PUFAs) via a PUFA synthase. Targeted mutagenesis of one gene of this synthase was conducted to confirm PUFA synthase function and determine its metabolic necessity. The resulting mutants were auxotrophic and required supplementation with PUFAs. In vivo labeling experiments with radioactive fatty acids demonstrated the presence of several elongase and desaturase activities associated with the standard pathway of PUFA synthesis. However, this system was missing a critical Delta12 desaturase activity and was therefore not capable of synthesizing PUFAs from the 16- or 18-carbon saturated fatty acid products of the fatty acid synthase. Because Schizochytrium uses a PUFA synthase system for the production of PUFAs, the existence of a partial desaturase-elongase system (if not a simple vestige) is suggested to be either a scavenging mechanism for intermediate fatty acids prematurely released by the PUFA synthase or for PUFAs found in the organism's native environment.

In vivo characterization of diatom multipartite plastid targeting signals.

Plastids of diatoms and related algae are delineated by four membranes: the outermost membrane (CER) is continuous with the endoplasmic reticulum while the inner two membranes are homologous to plastid envelope membranes of vascular plants and green algae. Proteins are transported into these plastids by pre-sequences that have two recognizable domains. To characterize targeting of polypeptides within diatom cells, we generated constructs encoding green fluorecent protein (GFP) fused to leader sequences. A fusion of GFP to the pre-sequence of BiP [an endoplasmic reticulum (ER)-localized chaperone] resulted in accumulation of GFP within the ER; a construct encoding the pre-sequence of a plastid protein fused to GFP was directed into the plastids. Additional constructs demonstrated that the N-terminal region of the bipartite plastid targeting pre-sequence was necessary for transport of polypeptides to the lumen of the ER, while the C-terminal region was shown to enable the proteins to traverse the plastid double envelope membrane. Our data strongly support the hypothesis of a multi-step plastid targeting process in chromophytic algae and raises questions about the continuity of the ER and CER and the function of the latter in polypeptide trafficking.

The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism.

Diatoms are unicellular algae with plastids acquired by secondary endosymbiosis. They are responsible for approximately 20% of global carbon fixation. We report the 34 million-base pair draft nuclear genome of the marine diatom Thalassiosira pseudonana and its 129 thousand-base pair plastid and 44 thousand-base pair mitochondrial genomes. Sequence and optical restriction mapping revealed 24 diploid nuclear chromosomes. We identified novel genes for silicic acid transport and formation of silica-based cell walls, high-affinity iron uptake, biosynthetic enzymes for several types of polyunsaturated fatty acids, use of a range of nitrogenous compounds, and a complete urea cycle, all attributes that allow diatoms to prosper in aquatic environments.