Mar Biotechnol
DOI 10.1007/s10126-008-9107-8
ORIGINAL ARTICLE
Preparation and Analysis of an Expressed Sequence Tag
Library from the Toxic Dinoflagellate Alexandrium catenella
Paulina Uribe & Daniela Fuentes & Jorge Valdés &
Amir Shmaryahu & Alicia Zúñiga & David Holmes &
Pablo D. T. Valenzuela
Received: 12 June 2007 / Accepted: 10 April 2008
# Springer Science + Business Media, LLC 2008
Abstract Dinoflagellates of the genus Alexandrium are
photosynthetic microalgae that have an extreme importance
due to the impact of some toxic species on shellfish
aquaculture industry. Alexandrium catenella is the species
responsible for the production of paralytic shellfish poison-
ing in Chile and other geographical areas. We have
constructed a cDNA library from midexponential cells of
A. catenella grown in culture free of associated bacteria and
sequenced 10,850 expressed sequence tags (ESTs) that
were assembled into 1,021 contigs and 5,475 singletons for
a total of 6,496 unigenes. Approximately 41.6% of the
unigenes showed similarity to genes with predicted func-
tion. A significant number of unigenes showed similarity
with genes from other dinoflagellates, plants, and other
protists. Among the identified genes, the most expressed
correspond to those coding for proteins of luminescence,
carbohydrate metabolism, and photosynthesis. The sequen-
ces of 9,847 ESTs have been deposited in Gene Bank
(accession numbers EX 454357–464203).
P. Uribe : D. Fuentes : A. Zúñiga : P. D. T. Valenzuela (*)
Fundación Ciencia para la Vida,
Av. Zañartu 1482, Ñuñoa,
Santiago, Chile
e-mail: pvalenzu@bionova.cl
J. Valdés : A. Shmaryahu : D. Holmes : P. D. T. Valenzuela
Instituto MIFAB,
Zañartu 1482, Ñuñoa,
Santiago, Chile
J. Valdés : A. Shmaryahu : D. Holmes
Center for Bioinformatics and Genome Biology,
Zañartu 1482, Ñuñoa,
Santiago, Chile
Keywords Alexandrium catenella . cDNA sequencing .
Red tide microalgae . Toxic dinoflagellate
Introduction
Dinoflagellates represent a unique and important group of
organisms in the marine environment in terms of their
numbers and diversity, as well as their ecological and
physiological significance. They commonly occur as free-
living, photosynthetic, and marine unicellular algae, but also
include endosymbiotic, parasitic, heterotrophic, and freshwa-
ter taxa. Some species are responsible for the production of
potent toxins that can be accumulated by shellfish and affect
humans and marine mammals. They form harmful blooms or
“red tides,” in which cell numbers reach more than one million
cells per liter of seawater, producing a significant economic
impact and public health concern on different geographical
areas throughout the world (Scholin et al. 1995; Hallegraeff
1993). Dinoflagellates are the only photosynthetic organisms
capable of bioluminescence (Sweeney 1987). Dinoflagellates
are also unique among eukaryotes in many other biological
and morphological characteristics. Their DNA content is
higher than other eukayotes [from 3 to 250 pg/cell, or
approximately 3,000–215,000 megabases (Spector 1984;
Triplett et al. 1993; Santos and Coffroth 2003)]. This is up
to over 80 times the size of the human genome (Lin 2006).
Their chromosomes consist of permanently condensed,
genetically inactive central regions with peripheral loops of
B-DNA that protrude from this core and comprise the
actively transcribed DNA (Sigee 1984; Anderson et al. 1992;
Bhaud et al. 1999).
Dinoflagellates of the genus Alexandrium such as Alexan-
drium catenella cause paralytic shellfish poisoning through
saxitoxin production in Chile and in many geographical

areas of the world. In Chile, the first documented toxic
bloom was reported in 1972 in Magallanes (Guzmán and
Campodónico 1975). Since then, the dominant toxic dino-
flagellate species in Chilean southern coastal areas and
estuaries during toxic bloom events has been identified to be
A. catenella (Guzmán and Campodónico 1978).
The study of the molecular mechanisms that regulate
growth, toxicity, photosynthesis, luminescence, and of other
circadian-controlled expressed genes of A. catenella, is of
critical importance for understanding the physiological
mechanisms and bloom formation capacity of Alexandrium
species. However, there have been few studies regarding
the molecular biology of A. catenella. Sequencing of
complementary DNA libraries to generate expressed se-
quence tags (ESTs) is a reasonable approach for discover-
ing expressed genes. ESTs can be used as markers for genes
expressed under specific conditions, for predicting protein
families, and for the development of expression systems for
new proteins and their functions. Here, we have developed
an EST library of A. catenella strain ACC07, isolated from
Chilean waters, and have carried out large-scale sequencing
to yield an EST database containing 10,850 ESTs and 6,496
unique genes. This database provides an important genomic
resource for scientists working on the genus Alexandrium
and related dinoflagellates.
Materials and Methods
Strains and Media Alexandrium catenella clone ACC07
isolated in Aysén, Chile, in 1994 was used. Cells were grown
in f/2 medium (Guillard 1995) at 16°C in a 10:14 light/dark
photoperiod. Axenic cultures were obtained according to
Uribe and Espejo (2003). Briefly, cells were subjected to
sequential washing and filtration through an 11-μm pore-size
nylon mesh with 0.05 mg/ml gentamicin and 0.2 mg/ml
penicillin G. Bacterial presence was determined by direct or
epifluorescence microscopy after staining with acridine
orange (Imai 1987; Kuwae and Hosokawa 1999). This
procedure allowed the detection of less than one bacterium
per dinoflagellate. The cell extracts of A. catenella strain
ACCO7 contained approximately 5–8 femtomoles of saxi-
toxin equivalents/cell.
cDNA library preparation Approximately 6×106 cells
from an exponential phase culture were collected by
centrifugation at 1,000×g for 5 min during the light phase
and were broken by four successive cycles of freezing,
grinding, and thawing. Approximately 400 μg of total RNA
was extracted using Trizol (Gibco BRL, Life Technologies,
Gaithsburg, MD, USA), according to the manufacturer’s
directions, and quantified spectrophotometrically. Poly A+
mRNA was isolated with the Poly (A) Quick mRNA
Mar Biotechnol
isolation kit (Stratagene, La Jolla, CA, USA). cDNA was
prepared from approximately 5 μg of polyA+ mRNA and
cloned using the vector pExpress 1, exploiting the Not I and
Eco RV restriction endonuclease sites. Double-strand cDNA
synthesis was performed according to manufacturer’s
directions and quantified spectrophotometrically. The
cDNA libraries were not normalized. Sequencing reactions
were carried out from the 5′ end of the cDNA insert using
the universal primers M13FWD (5′-gtaaaacgacggccagt-3′)
and M13REV (5′-caggaaacagctatgac-3′).
Computational Sequence Analysis and ESTs Assembly Vec-
tor-derived, ribosomal, and ambiguous sequences were
removed from the collected EST sequences. EST sequences
were assembled in clusters with a minimum value of 95%
identity for at least a 50-bp region of overlap using the
CAP3 program (Huang and Madan 1999). Clusters and
singletons generated were designed as unigenes and were
then subjected to similarity searches against the National
Center for Biotechnology Information nonredundant pro-
tein database, using the BLASTX algorithm (Altschul et al.
1990). Initially, sequence similarities were considered to be
significant when the E value was below e−5 at the
aminoacid sequence level. However, a stricter criterion
with a cut-off E value of e−20 or less was also used in the
analysis. The InterProScan (Mulder et al. 2007), gene
ontology (the gene ontology consortium 2007), and clusters
of orthologous groups (COGs) (Tatusov et al. 2003)
databases were used to infer the functional classification
of the predicted proteins.
Results and Discussion
Characteristics of the A. catenella cDNA Library
The cDNA library obtained had a titer of 1.1×106 colony
forming units per milliliter for a total of 1×107 primary
recombinants. Blue/white plaque identification following
plating of an aliquot of the library revealed 99% recombi-
nant plaques. The quality of the library was assessed by
examining the insert size of 768 (2×384 well plates)
randomly selected recombinant plaques. The average insert
size was 1.7 Kb, a value similar to that of a recent cDNA
library of Karenia brevis (Lidie et al. 2005). The average
size of the sequenced clones was 763 base pairs, and about
83% of the sequenced cDNA clones contained inserts that
were longer than the single sequence read. The global G+C
content for these ESTs was 56.8%. This value is similar to
that obtained for the coding regions of Alexandrium
tamarense (60.8%) (Hackett et al. 2005) and in the range
of the values obtained for other dinoflagellates such as K.
brevis (51%), Lingulodinium polyedrum (59.0%), Amphidi-
Mar Biotechnol
rium carterae (50.4%), and Crypthecodinium cohnii (50%)
(Lidie et al. 2005 and references therein).
Analysis of the codon usage revealed a major use of G
(35.1%) and C (44.8%) at the third position similar to that
obtained for A. tamarense (37.2% and 40.7%, respectively).
The most frequent stop codon was TGA (72.7%), compared
to TAA and TAG (6.5% and 20.7%, respectively).
Generation and Annotation of Expressed Sequences Tags
EST sequences were produced from the cDNA library and
scanned visually to confirm overall quality of peak shape and
correspondence with base identification. After the cleaning
process, the average length per EST of the remaining
sequences (9,847) was 736 base pairs and the Phred quality
value was larger than 20. The sequences were assembled into
1,021 contigs (clusters of assembled ESTs) and 5,475
singletons (sequences found only once) (Table 1). The
sequences of 9,847 ESTs have been deposited in Gene Bank
with accession numbers EX454357–464203.
Contigs were composed of multiple ESTs ranging from 2
to 438. The percentage of unigene sequences with
similarity to GenBank database was 41.6%. This EST
collection constitutes one of the largest dinoflagellate
libraries deposited (Lidie et al. 2005; Hackett et al. 2005;
Tanikawa et al. 2004). The total number of unigenes was
6,496, corresponding to less than half of the total sequences
obtained (Table 1). The ratio of sequenced ESTs to the
number of unigenes is similar to that reported for other
dinoflagellate EST libraries.
Using a cut-off E value of e−5 or less, a total of
5,443 ESTs corresponding to 2,700 unigenes, were found to
have similarity to previously identified genes from a wide
variety of organisms. Alexandrium catenella sequences
were classified according to the organism with the best
protein sequence hit. A significant proportion of the ESTs
show similarity to genes of dinoflagellates (32%), plants
(15%), and other protista (protozoa, ciliates, and other
microalgae, 13%). Different percentages were found in a
recent EST library analysis of the dinoflagellate L. poly-
edrum, where the groups most frequently found were land
Table 1 Overview of the results from the A. catenella genomic
library
Number of sequences
Total ESTs sequenced 10,859
Total valid ESTs 9,847
Average length per EST 736 bp
Number of contigs 1,021
Number of singletons 5,475
Total unigenes 6,496
Percentage known unigenes 41.6
plants and animals (21% and 16%, respectively), whereas
similarities with prokaryotes, flagellates, and protozoa were
14%, 14%, and 13%, respectively (Tanikawa et al. 2004). A
similar analysis was carried out using an E value of e−20 or
less. In this case, a total of 3,460 ESTs corresponding to
1,546 unigenes were found to have similarity to previously
identified genes. As shown in Fig. 1, a large proportion of
the ESTs show high level of similarity to genes of
dinoflagellates (47%), plants (18.1%), and other protista
(protozoa, ciliates, and other microalgae, 13.4%) (Fig. 1).
The unigenes could be assigned to known COGs. The
most represented group of proteins in ESTs corresponding
to cellular processes are those related to luminescence
(14.5%), carbohydrate metabolism (13.6%), aminoacid
metabolism (12.6%), protein modification (10.9%), and
photosynthesis (8.3%). Using an E value of e−20 or less, the
most represented group of proteins in ESTs corresponding
to cellular processes are those related to luminescence
(18.4%), carbohydrate metabolism (14.5%), aminoacid
metabolism (15%), protein modification (11.7%), and
photosynthesis (8.4%) (Fig. 2).
Among the first two categories, the majority of the
predicted proteins corresponded to those from dinoflagel-
lates. Proteins from plants were the most represented in
categories such as aminoacid and nucleic acid metabolism.
On the other hand, proteins from protozoa were the most
represented in translation and cellular cycle categories.
Some categories of proteins such as transport and those
with noncharacterized function were similarly distributed
among different taxonomic groups of eukaryotes and
prokaryotes. This distribution of categories of proteins
among different taxonomic groups was similar when E
values of e−30 or less were considered. In summary, by
using an E value of e−20 as cut off, a higher degree of
specificity was obtained resulting in an increased percent of
proteins from dinoflagellate, protozoa, plants, and other
microalgae.
Highly Represented Genes
The contigs containing the highest number of ESTs
(analyzed with an E value of e−20) are listed in Table 2.
The sequence coding for luciferin-binding protein (LBP) was
the most abundant transcript in the library with 80 unigenes
(3%) representing 539 ESTs (15.6% of the total ESTs).
This gene was also highly expressed in L. polyedrum
(Machabée et al. 1994) and also highly expressed (4%) in a
previous study of a normalized EST library of this
dinoflagellate during the night phase (Tanikawa et al.
2004). Similar results were reported in A. tamarense
(Hackett et al. 2005). Recently, in a global transcriptional
profiling of the toxic dinoflagellate Alexandrium fundyense,
four of the 15 signature sequences matched with the LBP
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Fig. 1 Taxonomic group distri-

bution of targets with the best
hit by A. catenella ESTs con-
sidering an E value of less than
10−20 to the National Center for
Biotechnology Information pro-
tein nonredundant database

Fig. 2 Distribution of A. cate-

nella ESTs into the GO catego-
ries of cellular processes
Mar Biotechnol

Table 2 Most highly represented ESTs in A. catenella cDNA library
Protein Number of % species (Liu et al. 2004). Internal regions of each domain are
ESTs
LBP 539 15.6
S-adenosyl-L-homocysteine hydrolase 169 4.9
Glyceraldehyde-3-phosphate dehydrogenase 128 3.7
isoform 2 (GPDH)
S-adenosylmethionine synthetase 2 105
Actin 87
EF-1 alpha-like protein 80
Fumarate reductase 71
Peridinin chl a binding protein 62
Hsp90 62
Phosphoglycerate kinase 59
Ribonucleoside-diphosphate reductase R2 56
Hsp70 54
Chloroplast phosphoribulokinase 49
Polyubiquitin 42
Chloroplast light harvesting complex protein 40
LCF 35
Light-harvesting polyprotein precursor 30
gene (Edner and Anderson 2006). In the present study, the
sequences of the two luminescence proteins of A. catenella
were subjected to a more detailed analysis.
Luciferin-binding Protein
The complete sequence of the LBP coding region of A.
catenella ACCO7 was obtained. It comprises 2,194
nucleotides, corresponding to 663 aminoacids. Sequencing
the genomic coding region indicated the lack of introns, and
after expression in bacteria, an 80-kDa protein was obtained
(data not shown). The LBP contains four domains with low
identity (15%) between them. The highest similarity in the
EST database was found with A. tamarense (Table 3). At
the aminoacid level, the highest similarity (76%) was found
with L. polyedrum. As found previously in Lingulodinium,
the amino terminal region of approximately 100 aminoacids
of LBP of A. catenella is similar (50%) to the equivalent
region of luciferase (LCF). This is the first complete
sequence of LBP reported in a toxic strain of the genus
Alexandrium (accession number EU236684).
Luciferase
Another highly expressed luminescence protein was the LCF.
Complete sequence analysis of the 3,476 nucleotides coding
for the A. catenella enzyme showed that the most closely
related were those from A. tamarense and A. affine (94%
identity) (Liu et al. 2004). The sequence contains no introns
and presents three domains with an identity of 76% between
them, a significantly lower value than the identity obtained
when these domains are compared to other dinoflagellate
the most conserved, corresponding to the probable catalytic
site of this enzyme. Four conserved histidines are present, at
the following positions within each domain: first domain
(D1): H138, H148, H163, and H169; second domain (D2):
H512, H525, H540, and H546; and third domain (D3):
3.0 H891, H901, H916, and H922. These histidines were
2.5 previously reported in L. polyendrum and are probably
2.3 related to the pH regulation of the activity of this enzyme (Li
2.1 et al. 1997). The first and the third domains of the LCF were
1.8 expressed in bacteria and the products were 60 and 45 kDa,
1.8 respectively. The three domains of this protein have shown
1.7
to be functional in L. polyedrum (Li et al. 1997).
1.6
1.6
The synthesis of the two luminescence proteins LCF and
1.4 LBP of Lingulodinium is regulated translationally; their
1.2 mRNA and protein levels remain constant over the
1.2 circadian cycle (Machabée et al. 1994). Remarkably, both
1.0 LCF and LBP in L. polyedrum are destroyed at the end of
0.9 the night phase and then resynthesized in the next cycle.
Moreover, the scintillons themselves are broken down and
reformed each day (Machabée et al. 1994).
Although the ecological function of the luminescence in
dinoflagellates has not been determined, it is probably
related to predation avoidance and communication (Esaias
and Curl 1972; Abrahams and Townsend 1993). Taken into
account that the luminescence proteins are among the most
expressed in A. catenella, probably a high proportion of the
energy of this dinoflagellate is dedicated to this particular
physiological response. Taken together, the specific char-
acteristics of the luminescent proteins and their expression
patterns in a paralytic shellfish poisoning producing
dinoflagellate such as A. catenella are of special relevance
to unveil the mechanisms of bloom formation in a toxin-
producing species. These specific features could be useful
for the development of new tools for the detection and
localization of this toxic species using bio-optical instru-
ments (Seliger et al. 1961; Widder et al. 1993).
Also highly expressed are transcripts that show a very high
similarity to the enzymes S-adenosyl-L-homocysteine hydro-
lase and the S-adenosylmethionine synthetase 2 (E values of
e−129 and e−112, respectively). These enzymes are involved in
methylation reactions that play a major role in the
modification of a large variety of acceptor molecules, such
as lipids, polysaccharides, nucleic acids, proteins, and
secondary plant products (reviewed by Giovanelli 1987). In
eukaryotes, DNA methylation has been implicated in the
control of several cellular processes, including differentia-
tion, gene regulation, and embryonic development (Cheng
1995). The high expression level of genes that matched with
the two heat shock proteins HSP90 and HSP70 sequences
was also remarkable. These proteins participate in various
cellular processes including signal transduction, protein
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Table 3 Photosynthesis and light harvesting proteins of A. catenella EST library

GenBank access number Function E value % Identity Organism

Photosynthesis
EX456598 Chloroplast photosystem II 12 kDa extrinsic 6.00E-64 6.00E-64 Alexandrium tamarense
protein (PsbU)
EX455192 Photosystem II 23 kDa polypeptide (PsbP) E-121 86 Phakopsora pachyrhizi
EX458868 PSII cytochrome c550 oxygen-evolving (PsbV) E-108 91 Alexandrium tamarense
EX455275 Plastid oxygen evolving enhancer 1 precursor (PsbO) 4.00E-71 94 Alexandrium tamarense
EX456053 Chloroplast cytochrome f (PetA) E-116 90 Alexandrium tamarense
EX457854 Chloroplast ferredoxin (PetF) 9.00E-90 83 Alexandrium tamarense
EX455467 Chloroplast ferredoxin-NADP{+) reductase (PetH) E-127 90 Heterocapsa triquetra
EX455749 Rieske iron–sulfur protein precursor (PetC) E-125 94 Alexandrium tamarense
EX463406 Photosystem I iron–sulfur center (PsaC) 3.00E-87 98 Alexandrium tamarense
EX456301 Chloroplast photosystem I subunit XI (PsaL) 3.00E-40 74 Heterocapsa triquetra
EX456206 PSI, ferredoxin-binding protein II (PsaD) 3.00E-51 90 Symbiodinium sp.
EX462386 Chloroplast photosystem I, subunit III (PsaF) E-101 93 Alexandrium tamarense
EX459236 Chloroplast ATP synthase gamma subunit (AtpC) 1.00E-91 89 Alexandrium tamarense
EX462908 Chloroplast ATP synthase subunit C (AtpH) 4.00E-76 76 Alexandrium tamarense
EX462123 Chloroplast light harvesting complex protein 2.00E-84 85 Lingulodinium polyedrum
EX460350 Peridinin-chlorophyll a-binding protein (PCP) 4.00E-75 89 Lingulodinium polyedrum
EX463946 Chloroplast phosphoribulokinase E-133 89 Amphidinium carterae
EX463746 Chloroplast transketolase E-114 80 Euglena gracilis
EX462931 Cytosolic class II fructose bisphosphate aldolase E-130 94 Heterocapsa triquetra
EX462518 Glyceraldehyde-3-phosphate dehydrogenase isoform 2 E-159 91 Symbiodinium sp.
EX455341 Phosphoglycerate kinase E-142 88 Karenia brevis
EX455303 Ribose-5-phosphate isomerase 5.00E-73 90 Phaeodactylum tricornutum
EX461668 RuBisCO form II E-153 96 Amphidinium carterae
EX461810 Triose-phosphate isomerase E-116 87 Isochrysis galbana
Chlorophyll synthesis
EX458222 NADPH protochlorophyllide reductase E-132 77 Phaeodactylum tricornutum
EX455862 Magnesium chelatase H-subunit E-114 88 Ostreococcus lucimarinus
EX455291 Mg-protoporhyrin IX (ChlI) 2.00E-68 88 Amphidinium carterae
EX455321 NADPH-protochlorophyllide oxidoreductase E-127 74 Phaeodactylum tricornutum
EX462775 Chloroplast geranylgeranyl reductase/hydrogenase 7.00E-50 81 Heterocapsa triquetra
EX456318 Glutamate 1-semialdehyde 2,1-aminomutase 7.00E-83 89 Amphidinium carterae
Luminescence
EX458318 LCF E-115 81 Lingulodinium polyedrum
EU236684 LBP 0 97 Alexandrium tamarense
Light receptors
EX456649 Cryptochrome dash 9.00E-47 65 Euglena gracilis
EX462564 Rhodopsin E-71 78 Branchiostoma floridae

folding, protein degradation, and morphological evolution
(Lindquist and Craig 1988). HSP70 proteins can be found in
different cellular compartments and have a role in the dis-
assembly of clathrin cages and also participate in the post-
translational transmembrane targeting of proteins to cellular
organelles (Craig 1989). The sequence coding for these pro-
teins have also been found in high frequency in the EST
library of the dinoflagellate A. tamarense (Hackett et al.
2005).
Photosynthesis and Light Harvesting Genes
None of the 15 known plastid-encoded genes from peridinin-
producing dinoflagellates were represented among the ESTs
of the library (Zhang et al. 1999). Thus, the 30 photosyn-
thesis unigenes represented in the A. catenella cDNA library
are probably encoded in the nucleus (Table 3). All these
plastid protein sequences contain tripartite N-terminal target-
ing signals that are shown to direct the trafficking of these
proteins through the different membranes of the dinoflagel-
late secondary plastids. The distribution of these signal
elements in A. catenella plastid protein sequences was equiv-
alent to those observed in the dinoflagellate Heterocapsa
triquera (Patron et al. 2005).
The origin of these nuclear encoded plastid protein
sequences is suggested by the relative high similarity with
those present in other peridinin-pigmented dinoflagellates
(Table 3). The nuclear location of these genes can be verified
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by using the spliced leader sequence recently found in the blue light (400–500 nm) and UV-A (320–400 nm) receptor,
nuclear-encoded mRNAs of dinoflagellates (Zhang et al. were found. This protein, which is involved in the light
2007). As expected, within this group, the majority regulation of growth and development in plants and other
(accession numbers: EX455192, EX455275, EX455467, cellular processes such as growth and the induction of
EX455749, EX456053, EX456206, EX456301, EX456598, sexual reproduction in algae (Liscum et al. 2003) shows
EX457854, EX458868, EX459236, EX462386, EX462908, 30% identity to those from K. brevis and Arabidopsis
and EX463406) belong to the related species A. tamarense thaliana. We consider that these light receptors are an
(from the A. cantenella–tamarense–fundyense species com- interesting subject of study in relation to the high level of
plex) (Scholin et al. 1995) followed by those from A. expression of blue light luminescence proteins in A.
carterae and H. triquera; L. polyedrum, and Symbiodinium catenella, considering the probable role of the lumines-
sp. Only few ESTs were similar to chloroplast sequences cence in the cellular communication of dinoflagellates.
from the fucoxantin pigmented K. brevis, or from other
organisms such as euglenoids, green algae, and strameno- Other Proteins
piles, which have a different but parallel origin of the plastid
proteins. Two A. catenella unigenes show a 100% identity with a
The most expressed transcripts with a high similarity to toxic strain-specific sequence of A. tamarense (AT-T1),
photosynthesis genes were those predicted to encode the previously identified as a biomarker of toxicity by Chan et
light harvesting complex, composed of a chlorophyll a-/c- al. (2006). Both A. catenella sequences also show similarity
and peridinin-binding protein and those corresponding to a to unknown proteins of the nontoxic dinoflagellate H.
number of proteins of the light phase of the photosynthesis, triquera. These sequences contain signal peptide sequences,
such as photosystems I and II, cytochrome b6f, and ATP suggestive of a plastid targeting protein (Patron et al. 2005).
synthase (Patron et al. 2005) (Table 3). Highly expressed We have also found an A. catenella unigene coding for a
are the unigenes that are highly similar to the carbon protein with a high level of similarity to two interesting
fixation enzyme glyceraldehyde-3-phosphate dehydroge- conjugation-induced proteins, SPS19 from Saccharomyces
nase isoform 2 that was 86% identical to the one from L. cerevisiae and eIF-4A, an eukaryotic elongation factor that
polyedrum. This enzyme participates in the aldehyde was found recently to be induced during conjugation in the
formation during the Calvin cycle in the dark phase of dinoflagellates A. catenella and A. tamarense (Hosoi-
photosynthesis. Sequences coding for this enzyme were Tanabe et al. 2005).
also found among the highest expressed in other EST
libraries of different dinoflagellates such as L. polyedrum
(Bachvaroff et al. 2004), A. tamarense (Hackett et al. 2005),
K. brevis (Lidie et al. 2005), and A. fundyense (Taroncher-
Oldenburg and Anderson 2000). Other highly expressed
genes similar to sequences encoding carbon fixation
proteins were the phosphoglycerate kinase and the chloro-
plast phosphoribulokinase. The library contains the coding
sequences of six enzymes related to chlorophyll synthesis
and two enzymes involved in the synthesis of photo-
protective pigments (Table 3).
We have also found sequences with high similarity to
light receptors. One has 77% identity to the green light
receptor (450 and 500 nm) type 1 rhodopsin described in
Pyrocystis lunula, (Okamoto and Hastings 2003) and to
those from the marine chryptophyte Guillardia theta
(Sineshchekov et al. 2005) and Cryptomonas spp. (29%
and 26%, respectively). Type 1 rhodopsins have recently
been described in the green alga Chlamydomonas rein-
hardtii, where they function as receptors for phototaxis
responses (Sineshchekov et al. 2002). This photosensitive
protein is similar to γ-proteobacterial rhodopsins and more Fig. 3 Venn diagram of the comparison between the A. catenella
ESTs with the genomes of A. thaliana; T. pseudonana; C. merolae;
abundantly expressed during the early day hours (Okamoto Entoamoeba histolytica; and Plasmodium falciparum. The number
and Hastings 2003). Sequences that correspond to a second and percentage of the homologous sequences of A. catenella with each
photosensory receptor, the chryptochrome dash protein, a organism is referred in the intersection

Two sequences code for a protein with a cysteine-rich
region, which has similarity to the EhV_307 protein from
the Emiliania huxleyi virus (Wilson et al. 2005). Other viral
sequences from the Paramecium bursaria Chlorella virus
were also found but with a lower similarity.
Genes predicted to encode a diversity of proteins
involved in transport processes were detected; among them
were Na, K, Ca, phosphate, and ammonium channels, and
also antiporters; ABC-transporters; aminoacid transporters;
and the Sec61 and SecY translocases, involved in secretion
pathways in eukaryotes. Thirteen sequences that correspond
to transposable elements previously described by Armbrust
et al. (2004) in the Thalassiosira pseudonana genome were
found with a relatively low similarity.
Comparative Genomics
The A. catenella protein database was compared with ge-
nomes of the plant A. thaliana and to genomes of unicellular
eukaryotes of the protista kingdom, such as T. pseudonana,
Entoamoeba histolytica, Cryptosporidium hominis, and the
red algae Cyanidioschyzon merolae (Fig. 3). The Venn
diagram shows the highest similarity with A. thaliana
(19.3%), the diatom T. pseudonana (19.1%), and C. merolae
(18.3%) (Fig. 3). We observed a similar distribution of
functional groups (COGs) among the sequences in common
with those five organisms (not shown).
When the unigenes of this library were compared with
10,886 ESTs of the closely related species A. tamarense
(Hackett et al. 2005) present in the public database, we found
3,045 (46.9%) hits. From them, the 1,236 common unigenes
were classified into COGs, and the most represented
categories corresponded to carbohydrate metabolism
(11.8%), posttranslational modification and chaperones
(9.1%), energy production (7.4%), and luminescence (6.6%).
Acknowledgments This research has been partially funded by
CONICYT-FONDEF Project MR02I1003 and by a Microsoft Re-
search Joint R&D Program.
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