Abstract
Dinoflagellate genomes present unique challenges including large size, modified DNA bases, lack of nucleosomes, and condensed chromosomes. EST sequencing has shown that many genes are found as many slightly different variants implying that many copies are present in the genome. As a preliminary survey of the genome our goal was to obtain genomic sequences for 47 genes from the dinoflagellate Amphidinium carterae. A PCR approach was used to avoid problems with large insert libraries. One primer set was oriented inward to amplify the genomic complement of the cDNA and a second primer set would amplify outward between tandem repeats of the same gene. Each gene was also tested for a spliced leader using cDNA as template. Almost all (14/15) of the highly expressed genes (i.e. those with high representation in the cDNA pool) were shown to be in tandem arrays with short intergenic spacers, and most were trans-spliced. Only two moderately expressed genes were found in tandem arrays. A polyadenylation signal was found in genomic copies containing the sequence AAAAG/C at the exact polyadenylation site and was conserved between species. Four genes were found to have a high intron density (>5 introns) while most either lacked introns, or had only one to three. Actin was selected for deeper sequencing of both genomic and cDNA copies. Two clusters of actin copies were found, separated from each other by many non-coding features such as intron size and sequence. One intron-rich gene was selected for genomic walking using inverse PCR, and was not shown to be in a tandem repeat. The first glimpse of dinoflagellate genome indicates two general categories of genes in dinoflagellates, a highly expressed tandem repeat class and an intron rich less expressed class. This combination of features appears to be unique among eukaryotes.
Highlights
Taken together dinoflagellates are important primary producers in oceans, estuaries, and lakes, and create environmental problems with disruptive and destructive blooms
The intergenic region can be divided into three components (Figure 1): first the stop codon to the polyadenylation site, or genomic complement of the 39 UTR of the mRNA, second an intergenic region between the polyadenylation site and the transsplicing site, and the splicing site to the start codon, or the 59 UTR
Most striking is that highly expressed genes are present at a high copy number in repeated tandem arrays, and transsplicing of these genes was readily detected
Summary
Taken together dinoflagellates are important primary producers in oceans, estuaries, and lakes, and create environmental problems with disruptive and destructive blooms. A combination of EST surveys and targeted gene sequencing suggest high copy number and tandem gene arrangement for many dinoflagellate genes. For example in Lingulodinium polyedrum, the peridinin-chlorophyll a-protein gene is present as ,5000 identical copies per genome with the copies linked by short intergenic spacers [15]. When other abundantly expressed genes are compared, a large number of synonymous substitutions were noted between different ESTs in L. polyedrum, Amphidinium carterae and Karenia brevis [10,17]. These results suggest high copy number for many genes and a certain degree of diversity between these copies
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