Home > Major Projects > Karenia brevis EST project

Community Sequencing Program - 2004

A genomics resource for the toxic dinoflagellate
Karenia brevis

     
  PI: Debashish Bhattacharya
  Co-PI: Frances Van Dolah
  JGI contact: Jan-Fang Cheng

Abstract: Karenia brevis ( Davis ) Hanson and Moestrup is a unicellular dinoflagellate protist that causes harmful algal blooms (HABs) that occur annually in the Gulf of Mexico . These "red tides" cause extensive marine animal mortalities and human illness through the production of highly potent neurotoxins known as brevetoxins. Insights into the molecular mechanisms that control the growth and persistence of K. brevis blooms is critical to understanding the formation of HABS and is a prerequisite for the development of control strategies. Karenia spp. (and other fucoxanthin-containing dinoflagellates) also occupy a critical position among algae with regard to plastid (photosynthetic organelle) evolution. These taxa have undergone a remarkable genomic transition from an ancestral condition in which their plastid genome was comprised of a small number (ca. 16) of single-gene minicircles (with the remaining plastid genes being nuclear-encoded) to having reverted to a putatively typical plastid through tertiary endosymbiosis. Here, we seek support to generate a comprehensive unigene EST set for K. brevis using serially subtracted cDNA libraries enriched for rare mRNAs, to facilitate comparative genomic and gene expression analyses of this species. In addition, we wish to generate draft sequences of the plastid and mitochondrial genomes of K. brevis and its sister Karlodinium micrum to gain insights into plastid endosymbiosis and organellar gene transfer.  
Scope of Work: The PI and Co-PI will provide the CSP with non-normalized, normalized, and subtracted K. brevis plasmid cDNA libraries and shotgun plasmid libraries of the organellar genomes for direct sequencing. For the EST work, we request the CSP to generate 10,000 3' reads of a normalized cDNA library of K. brevis. We already have a pool of 2,673 clones encoding unique cDNAs based on 5' reads of a non-normalized library of this species derived from log phase culture harvested during the dark. We also request that 1,000 ESTs be sequenced at the 3' terminus from non-normalized libraries of K. brevis grown under five different culture conditions - 1) nitrate-depletion, 2) phosphate-depletion, 3) in log phase under replete conditions, harvested during the light phase 4) in the presence of oxidative metals, 5) undergoing heat stress - to identify highly expressed stress-related or nutrient condition-specific genes. Thereafter, the unique cDNA clones that have already been generated from the 5' reads plus those identified with the 3' reads will be combined and used a driver to subtract from the complex mixture consisting of the 5 non-normalized cDNA libraries. We request that a total of 5,000 cDNAs in the subtracted library be sequenced at the 3' terminus. This aim requires, therefore, a total of 20,000 3' EST reads of K. brevis cDNA . Regarding the organellar genomes, red algal plastid genomes normally range between 120,000 - 190,000 bp in size and protist mitochondrial genomes are ca. 40,000 bp. We recently generated the complete sequence of the plastid genome in the red alga Gracilaria tenuistipitata and it was 183,803 bp (Hagopian et al. in review). For this work, we will generate shotgun libraries with fragments (1-3 kb) of nebulized, purified K. brevis and K. micrum plastid and mitochondrial DNA cloned into pBluescript. We propose high-throughput sequencing by the CSP of these clones either as separate or as a pooled library. Assuming maximum plastid and mitochondrial genome sizes of 200,000 and 40,000 bp, respectively, and an average read of 500 bp, we propose that 9,600 reads be done to provide approximately 10-fold coverage for each genome. Taken together, our project entails a grand total of 29,600 sequencing reads resulting in about 10.48 Mb of sequence (i.e., 10,000,000 + 200,000 + 200,000 + 40,000 + 40,000 bp).

 
Technical Information: K. brevis vegetative cells are haploid, with 121 chromosomes (Walker 1982) containing 50-100 pg DNA/cell, or a haploid genome of approximately 5 x 10 10 bp (Kim and Martin 1974; Rizzo et al. 1982; Sigee 1986; Kamykowski et al. 1998). This makes unfeasible the generation of a complete genome sequence for this species. To date 9,728 5' reads have been generated from a dark-grown K. brevis cDNA library, yielding 5,280 non-redundant clusters (we have 2,673 unique clones in hand). Of these, 69% are singletons and only 1,636 were found in more than one copy. The most highly expressed gene accounts for only 1% of the total ESTs. Twenty-nine percent of the ESTs were found have homology to known genes in the GenBank non-redundant protein database using an E-value cutoff of p<10 -4 . Fifty ESTs that were analyzed have a GC content of 51%, with a preference for G/C at third codon positions (53.5%).

 
Available Genomic Resources: We have a cDNA library for K. brevis from which we have identified 5,280 non-redundant gene clusters. This library was generated from clonal cultures grown in replete medium and harvested during the dark phase of the diel cycle to maximize the likelihood of acquiring cell cycle regulatory genes. We are in the process of normalizing this library for the proposed 3' reads and are developing cDNA libraries harvested from cells grown under the five different culture conditions. No organellar genomes have been sequenced thus far from fucoxanthin dinoflagellates but DB has considerable experience in plastid phylogenetics and genomics (e.g., Yoon et al. 2002a, b; Bhattacharya et al. 2004; Hagopian et al. in review) and shotgun libraries are currently being generated in his lab for K. brevis and K. micrum using CsCl-gradient purified organellar DNA. Our preliminary CsCl runs show a clear separation of the putative plastid and mitochondrial fractions from the nuclear DNA in K. brevis (Nosenko and Bhattacharya unpubl. data).  

 Scientific Importance: Karenia brevis is responsible for red tides (Fig. 1) that cause extensive marine animal mortalities and human illness through their production of brevetoxins. In addition to their adverse effects on human and environmental health, K. brevis red tides have a substantial economic impact on the west coast of Florida, costing an estimated $20M in cleanup and lost tourism revenue annually (Anderson et al. 2000). Understanding the mechanisms that control growth and cell division in K. brevis and mechanisms by which it adapts to stressful environmental conditions encountered in coastal waters is critical to understanding the formation and persistence of red tides and is a prerequisite to the development of control strategies.

Fig. 1.
A) The HAB species, Karenia brevis. B) A red tide in Florida .
From: http://www.learner.org/jnorth/tm/manatee/RedTide.html

 

Analyses of the K. brevis cell cycle:
The dinoflagellate nucleus has a number of characteristics that are unique among eukaryotes, including the absence of nucleosomes, chromatin that remains permanently condensed throughout interphase and mitosis, and a unique form of mitosis in which the nuclear envelope remains intact and chromosomal segregation is achieved via an extranuclear spindle that traverses the nucleus through cytoplasmic channels.


Text Box: Fig. 2. Nuclear and cytoskeletal structures of K. brevis during different cell cycle stages. Chl chloroplast; n nucleus; ms mitotic spindle (Barbier et al. 2003).
Because the histones that make up nucleosomes and the nuclear lamins that maintain nuclear envelope integrity are key substrates of the universal cell cycle regulator, cyclin dependent kinase (CDK), the question arises as to what unique mechanisms of cell cycle control (Fig. 2) may exist in dinoflagellates that might be suitable targets for HAB control measures. We have identified CDK in K. brevis by western blot analysis and demonstrated its requirement for cell cycle progression at both G1/S and G2/M transitions using specific inhibitors of CDK activity (Van Dolah and Leighfield 1999). Its regulatory partner, cyclin, was also identified using immunological approaches (Barbier et al. 2003). In eukaryotes, the activity of CDK is regulated in part through the transcriptional activation cyclin, which is absent in early G1, and is expressed in late G1, when it becomes available to partner with CDK. However, cyclin expression in K. brevis is somewhat unusual relative to other eukaryotes in that it is expressed throughout the cell cycle, including early G1 and stationary phase cells. ESTs for a number of additional cell cycle genes have now been identified in the K. brevis cDNA library. Among these are S-phase specific genes that in higher eukaryotes are coordinately regulated by transcriptional activation at the G1/S transition. We are currently studying the expression of these genes over the cell cycle, and to date have not observed transcriptional activation associated with S-phase entry by RT-PCR (Van Dolah et al. unpubl.) . Development of microarrays containing 4,000 oligonucleotides specific to unique ESTs from the library is underway which will provide preliminary assessment of changes in gene expression over the cell cycle. We anticipate that these studies will yield insights not only into coordinately regulated cell cycle genes, but also the transcription factors and upstream signaling pathways responsible for their coordinate expression. However, the additional ESTs acquired through the proposed sequencing project will be essential to produce microrrays that fully represent the expressed genome of K. brevis .


Fucoxanthin dinoflagellates and their remarkable plastid genome evolution: Tertiary endosymbiosis entails the engulfment of an alga with a secondary plastid (as are found in chromist, euglenid, and chlorarachniophyte algae; Bhattacharya et al. 2004). Tertiary endosymbiosis has, until now, been limited to the dinoflagellates and in this group involves the replacement of the existing red algal secondary plastid (shared with the chromists) with another of secondary origin. The putative ancestral (and most common) plastid in dinoflagellates contains the pigment peridinin as the main carotenoid. The peridinin plastid does not, however, contain a typical genome because its genes have been reduced to single- or two-gene minicircles (Zhang et al. 1999). Until now, only 16 plastid protein genes have been found in peridinin dinoflagellates, and the remainder, as DB's lab has recently reported (Hackett et al. 2004), have been transferred to the nuclear genome.

Given the ancestral origin of the red algal secondary plastid in dinoflagellates, tertiary replacement explains the plastid in taxa such as Karenia spp. and K. micrum that contain chlorophylls c 1 + c 2 and 19'-hexanoyloxy-fucoxanthin and/or 19'-butanoyloxy-fucoxanthin but lack peridinin, similar to the haptophyte algae (Tengs et al. 2000). Amazingly, none of the 48 non-minicircle-encoded photosynthetic genes that were found in our EST data set of the peridinin dinoflagellate Alexandrium tamarense have yet been located in the 5,280 unique ESTs (albeit sampled during the dark-phase) of K. brevis (Hackett, Van Dolah, and Bhattacharya unpublished data). This potentially suggests that the nuclear-encoded genes have been lost when the haptophyte tertiary plastid replaced the ancestral chromalveolate "red" plastid in the dinoflagellate common ancestor. This unprecedented level of genomic transformation needs to be confirmed by generating the plastid genome sequences of K. brevis and K. micrum to verify their "normal" genome complement and by comprehensively searching the nuclear genome of K. brevis for remnant transferred photosynthetic genes from its past. Understanding these remarkable events is of fundamental importance to broadening our knowledge of genome evolution, organellar gene transfer, and endosymbiosis.

   
     

References

Anderson , D.M., P. Hoagland, Y.Kaoru, and A.W. White. 2000. Estimated annual economic impacts of harmful algal blooms (HABs) in the United States . Woods Hole Oceanographic Institution Technical Report WHOI-2000-11, 97 p.

Barbier, M, T.A. Leighfield, M-O. Soyer-Gobillard, , and F.M. Van Dolah. 2003. Expression of a cyclin B homologue in the cell cycle of a primitive dinoflagellate, Karenia brevis . J. Euk. Microbiol. 50(2):123-31

Bhattacharya, D., H. S. Yoon, and J. D. Hackett. 2004. Chromalveolates unite: endosymbiosis connects the dots. BioEssays 26:50-60.

Hackett, J.D., H.S. Yoon, M.B. Soares, M.F. Bonaldo, T.L. Casavant, T.E. Scheetz, T. Nosenko, and D. Bhattacharya. 2004. Migration of the plastid genome to the nucleus in a peridinin dinoflagellate. Curr. Biol. 14:213-218.

Hagopian, J.C., M. Reis, J.P. Kitajima, D. Bhattacharya, and M.C. de Oliveira. In review. Comparative analysis of the complete plastid genome sequence of the red alga Gracilaria tenuistipitata var. liui : insights on the evolution of rhodoplasts and their relationship to other plastids. J. Mol. Evol.

Kamykowski D, Milligan EJ, Reed RE. 1998. Biochemicsl relationships in the orientation of the autotrophic dinoflagellate Gymnodinium breve under nutrient replete conditions. Marine Ecol. Prog. Ser. 167: 105-117

Kim YS and Martin DF 1974. Effects of salinity on synthesis of DNA, acidic polysaccharide and ichthyotoxin in Gymnodinium breve . Phytochemistry 13: 533-538.

Rizzo P.D. 1982. Isolation and properties of isolated nuclei from the Florida red tide dinoflagellate Gymnodinium breve . J. Protozool. 29: 217-222.

Sigee , D.C. 1986. The dinoflagellate chromosome. Adv. Bot. Res. 12: 205-265. Tengs T., O.J. Dahlberg, K. Shalchian-Tabrizi, D. Klaveness, K. Rudi, C.F. Delwiche, and K.S. Jakobsen. 2000. Phylogenetic analyses indicate that the 19'Hexanoyloxy-fucoxanthin-containing dinoflagellates have tertiary plastids of haptophyte origin. Mol. Biol. Evol. 17:718-729.

Van Dolah F.M. and T.A. Leighfield. 1999. Diel phasing of the cell cycle in the Florida red tide dinoflagellate, Gymnodinium breve . J. Phycol . 35S: 1404-1411.

Walker, L. 1982. Evidence for a sexual cycle in the Florida red tide dinoflagellate, Ptychodiscus brevis (= Gymnodinium breve ). Trans. Am. Microbiol. Soc. 101: 287-293.

Yoon, H.S., J. Hackett, and D. Bhattacharya. 2002a. A single origin of the peridinin-, and fucoxanthin-containing plastids in dinoflagellates through tertiary endosymbiosis. Proc. Natl. Acad. Sci. USA 99:11724-11729.

Yoon, H.S., J. Hackett, G. Pinto, and D. Bhattacharya. 2002b. The single, ancient origin of chromist plastids. Proc. Natl. Acad. Sci. USA 99:15507-15512.

Yoon, H.S., J. Hackett, C. Ciniglia, G. Pinto, and D. Bhattacharya. 2004. A molecular timeline for the origin of photosynthetic eukaryotes. Mol. Biol. Evol. [Epub ahead of print].

Zhang, Z., B.R. Green, and T. Cavalier-Smith. 1999. Single gene circles in dinoflagellate chloroplast genomes. Nature 400 : 155-159.