Susan E. Douglas

The plastid genome of the cryptophyte alga, Guillardia theta: complete sequence and conserved synteny groups confirm its common ancestry with red algae.

Abstract. The plastid genome of the cryptophyte alga Guillardia theta (121,524 bp) has been completely sequenced. The genome is 33% G+C and contains a short, nonidentical inverted repeat (4.9 kb) encoding the two rRNA cistrons. The large and small single-copy regions are 96.3 and 15.4 kb, respectively. Forty-six genes encoding proteins for photosynthesis, 5 genes for biosynthetic function, 5 genes involved in replication and division, 30 tRNA genes, 44 ribosomal protein genes (26 large subunit and 18 small subunit), 3 translation factors, 8 genes encoding components of the transcriptional machinery including 3 ycfs (hypothetical chloroplast frames), and 26 additional ycfs have been identified. There are eight ORFs larger than 50 amino acids, 3 of which have homologues on the plastid genome of the rhodophyte, Porphyra purpurea (Reith and Munholland 1995) and/or the Synechocystis genome (Kaneko et al. 1996) and can be designated new ycfs. Intergenic spacers are very short, no introns have been detected, and several genes overlap, all resulting in a very compact genome. In addition, large clusters of genes (such as those for the ribosomal proteins) are organized into single transcriptional units (Wang et al. 1997), again resulting in an economically organized genome. The cryptophyte plastid genome is almost completely comprised of clusters of genes that are found on the rhodophyte Porphyra purpurea, confirming its common ancestry with red algae. Furthermore, recombination events involving both tRNA genes and the rRNA cistrons appear to have been responsible for the structure of the cryptophyte plastid genome, including the formation of the inverted repeat.

Cloning and developmental expression of a family of pleurocidin-like antimicrobial peptides from winter flounder, Pleuronectes americanus (Walbaum)

Low molecular weight antimicrobial peptides are an important component of the innate immune system in animals, yet they have not been examined widely in fish. Of particular interest is their expression during development and in response to environmental conditions and disease. Here, we report the isolation of four genomic sequences encoding putative antimicrobial peptides from the winter flounder, Pleuronectes americanus (Walbaum), as well as reverse transcription-PCR products from two tissues that form the first defensive barrier to microbes - skin and intestine. Alignment of the predicted polypeptide sequences shows a conserved hydrophobic signal peptide of 22 amino acids followed by 25 amino acids that are identical (WF2) or homologous to the amino acid sequence of pleurocidin, followed by a conserved acidic portion. Southern hybridisation analysis indicates that related peptides are encoded in the genomes of other flatfish species. Northern and RT-PCR analyses of RNA from multiple tissues show that two of the pleurocidin genes are expressed predominantly in the skin whereas two other genes are expressed mainly in the intestine. RT-PCR assays of total RNA from larvae of different ages provide the first evidence of developmental expression of antimicrobial peptides in fish and indicate that the pleurocidin gene is first expressed at 13 days post-hatch in winter flounder.

Identification of Immune-Relevant Genes from Atlantic Salmon Using Suppression Subtractive Hybridization

In order to probe the interaction between an invading microorganism and its host, we have investigated differential gene expression in Atlantic salmon (Salmo salar) experimentally infected with the pathogen Aeromonas salmonicida, the causative agent of furunculosis. Subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH) from 3 immune-relevant tissues at 2 time points during the infection process. Both forward- and reverse-subtracted libraries were generated, and approximately 200 clones were sequenced from each library, giving a total of 1778 expressed sequence tags (ESTs), which were annotated according to functional categories and deposited in GenBank (BQ035314–BQ037059). Numerous genes involved in signal transduction, innate immunity, and other processes have been uncovered in the subtractive libraries. These include known acute-phase reactants, along with more novel genes encoding proteins such as tachylectin, hepcidin, precerebellin-like protein, O-methyltransferase, a putative saxitoxin-binding protein, and others. A subset of genes that were represented in the subtracted libraries was further analyzed by virtual Northern, or reverse transcription–polymerase chain reaction (RT-PCR) assays to verify their differential expression as a result of infection.

Use of human cDNA microarrays for identification of differentially expressed genes in Atlantic salmon liver during Aeromonas salmonicida infection.

Commercially available human complementary DNA microarrays were used to compare differential expression in the livers of Atlantic salmon (Salmo salar) infected with Aeromonas salmonicida and of healthy fish. Complementary DNA probes were prepared from total RNA isolated from livers of control salmon and infected salmon by reverse transcription in the presence of 33P-dCTP and independently hybridized to human GENEFILTERS GF211 microarrays. Of the 4131 known genes on the microarray, 241 spots gave clearly detectable signals using labeled RNA from the control salmon liver. Of these, 4 spots were consistently found to have a greater than 2-fold increase in infected salmon compared with controls when using the same pair of filters to generate hybridization data from triplicates. These up-regulated genes were ADP/ATP translocase (AAT2), Na+/K+ ATPase, acyloxyacyl hydrolase (AOAH), and platelet-derived growth factor (PDFG-A). A BlastN search revealed an AAT2 homolog from Atlantic salmon, and a reverse transcriptase polymerase chain reaction assay using primers based on this sequence confirmed its up-regulation (approx. 1.8-fold) during early infection. This work demonstrates the feasibility of using human microarrays to facilitate the discovery of differentially expressed genes in Atlantic salmon, for which no homologous microarrays are available.

Expressed Sequence Tags — A Snapshot of the Fish Genome

Expressed Sequence Tags (ESTs) represent the transcribed portion of an organism’s genome and as such provide a snapshot of the expression of that genome at a particular time. Since non-coding DNA such as introns, intergenic spacers, repeat elements, etc. which comprise the bulk of the genome, is not sequenced, ESTs provide an information-rich source of data that is relatively easily interpreted and managed. EST surveys of non-normalized libraries can also provide an estimate of the abundance of particular transcripts. At the Institute for Marine Biosciences, we have optimized inexpensive template preparation and automated sequencing methodologies to produce large numbers of ESTs in an efficient and cost-effective manner. Tissue-specific EST libraries for winter flounder, our model system for studying flatfish biology, have been generated both for gene discovery purposes and for comparative purposes with other organisms. We have also produced an EST database from liver, head kidney and spleen of Atlantic salmon that will aid in genome mapping, identification of duplicated genes and DNA microarray construction, as well as providing information on genes critical to the fish immune system. An EST approach is also being used with subtractive libraries constructed from salmon infected with the pathogen Aeromonas salmonicida in order to focus on transcripts that are differentially regulated in response to disease.

THE ATPA GENE CLUSTER OF GUILLARDIA THETA (CRYPTOPHYTA) : A PIECE IN THE PUZZLE OF CHLOROPLAST GENOME EVOLUTION

We cloned and sequenced the genes of the atpA (atp1) cluster, together with its upstream and downstream sequences, of the chloroplast genome of the cryptomonad Guillardia theta Hill et Wetherbee and used gene signatures, predicted amino acid sequences, and gene arrangements for phylogenetic inferences. The Guillardia atpA cluster contains the genes atpI,H,G,F,D,A in the given order, thus reflecting the cyanobacterial/red algal/chromophyte gene arrangement. In addition, the order of both the upstream sequences rpoB,C1,C2,rps2,tsf and the downstream sequences ycf16/24, which is found exclusively in red algal chloroplast genomes, is conserved in Guillardia. This gene order, which presumably is the result of the reduction process of the cyanobacterial genome following primary endosymbiosis, provides strong evidence for a red algal ancestry of the Guillardia chloroplast and supports the hypothesis of secondary endosymbioses giving rise to chl a+c‐containing algae. The close evolutionary relationship of the chloroplasts of Guillardia and red algae is furthermore supported by the lack of introns, high degrees of sequence similarities, and additional gene signatures, including spacers, gene overlaps, and inverted repeats. Gene cluster analysis, including the ATPase genes together with their upstream and downstream genes, is consistent with a single primary photosynthetic eukaryote that gave rise to all extant algal lineages and land plants by either direct filiation or secondary endosymbioses.