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Insights into a dinoflagellate genome through expressed sequence tag analysis.

Hackett JD, Scheetz TE, Yoon HS, Soares MB, Bonaldo MF, Casavant TL, Bhattacharya D - BMC Genomics (2005)

Bottom Line: We also identified rare transcripts encoding a predicted protein highly similar to histone H2A.X.We speculate this histone may be retained for its role in DNA double-strand break repair.These data will be instrumental to future research to understand the unique and complex cell biology of these organisms and for potentially identifying the genes involved in toxin production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences and Roy J, Carver Center for Comparative Genomics, University of Iowa, Iowa City, IA 52242, USA. jeremiah-hackett@uiowa.edu

ABSTRACT

Background: Dinoflagellates are important marine primary producers and grazers and cause toxic "red tides". These taxa are characterized by many unique features such as immense genomes, the absence of nucleosomes, and photosynthetic organelles (plastids) that have been gained and lost multiple times. We generated EST sequences from non-normalized and normalized cDNA libraries from a culture of the toxic species Alexandrium tamarense to elucidate dinoflagellate evolution. Previous analyses of these data have clarified plastid origin and here we study the gene content, annotate the ESTs, and analyze the genes that are putatively involved in DNA packaging.

Results: Approximately 20% of the 6,723 unique (11,171 total 3'-reads) ESTs data could be annotated using Blast searches against GenBank. Several putative dinoflagellate-specific mRNAs were identified, including one novel plastid protein. Dinoflagellate genes, similar to other eukaryotes, have a high GC-content that is reflected in the amino acid codon usage. Highly represented transcripts include histone-like (HLP) and luciferin binding proteins and several genes occur in families that encode nearly identical proteins. We also identified rare transcripts encoding a predicted protein highly similar to histone H2A.X. We speculate this histone may be retained for its role in DNA double-strand break repair.

Conclusion: This is the most extensive collection to date of ESTs from a toxic dinoflagellate. These data will be instrumental to future research to understand the unique and complex cell biology of these organisms and for potentially identifying the genes involved in toxin production.

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Analyses of A. tamarense histone H2A.X. A) Alignment of A. tamarense H2A.X with eukaryotic homologs. The alignment is shaded according to the level of conservation. The symbols above the alignment indicate the location of functional residues (T = trypsin cleavage site, ^ = arginines that contact the DNA helix, * = H2A-H2B interaction sites, U = ubiquitination site). The annotation below the alignment indicates conserved structural features including the α-helices, loops, and the SQ(E/D)Φgotif. B) A ML tree of H2A and H2A.X. The numbers above and below the branches are the results of ML and NJ bootstrap analyses, respectively. The thick branches indicate > 0.95 posterior probability from Bayesian inference. Only bootstrap values ≥ 50% are shown. Branch lengths are proportional to the number of substitutions per site (see scale bar).
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Figure 3: Analyses of A. tamarense histone H2A.X. A) Alignment of A. tamarense H2A.X with eukaryotic homologs. The alignment is shaded according to the level of conservation. The symbols above the alignment indicate the location of functional residues (T = trypsin cleavage site, ^ = arginines that contact the DNA helix, * = H2A-H2B interaction sites, U = ubiquitination site). The annotation below the alignment indicates conserved structural features including the α-helices, loops, and the SQ(E/D)Φgotif. B) A ML tree of H2A and H2A.X. The numbers above and below the branches are the results of ML and NJ bootstrap analyses, respectively. The thick branches indicate > 0.95 posterior probability from Bayesian inference. Only bootstrap values ≥ 50% are shown. Branch lengths are proportional to the number of substitutions per site (see scale bar).

Mentions: A significant finding of this study is the identification of two rare (2/11,171) ESTs that encode a partial histone H2A.X. The longest cDNA isolated from the library using PCR was predicted to encoded a protein of 169 amino acids that shares high sequence identity to eukaryotic histone H2A.X (Figure 3A). This clone putatively lacked only the start codon at the N-terminus. The divergent N-terminus of A. tamarense H2A.X is somewhat longer than in other homologs but the remainder of the sequence is conserved (in particular the α-helices of the histone fold). Several functional residues from the known crystal structure are also present in A. tamarense H2A.X including the lysine at the trypsin cleavage site, the arginines in the loops that interact with the DNA α-helix, and the lysine ubiquitination site [24]. The sites of interaction with histone H2B are also present.


Insights into a dinoflagellate genome through expressed sequence tag analysis.

Hackett JD, Scheetz TE, Yoon HS, Soares MB, Bonaldo MF, Casavant TL, Bhattacharya D - BMC Genomics (2005)

Analyses of A. tamarense histone H2A.X. A) Alignment of A. tamarense H2A.X with eukaryotic homologs. The alignment is shaded according to the level of conservation. The symbols above the alignment indicate the location of functional residues (T = trypsin cleavage site, ^ = arginines that contact the DNA helix, * = H2A-H2B interaction sites, U = ubiquitination site). The annotation below the alignment indicates conserved structural features including the α-helices, loops, and the SQ(E/D)Φgotif. B) A ML tree of H2A and H2A.X. The numbers above and below the branches are the results of ML and NJ bootstrap analyses, respectively. The thick branches indicate > 0.95 posterior probability from Bayesian inference. Only bootstrap values ≥ 50% are shown. Branch lengths are proportional to the number of substitutions per site (see scale bar).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1173104&req=5

Figure 3: Analyses of A. tamarense histone H2A.X. A) Alignment of A. tamarense H2A.X with eukaryotic homologs. The alignment is shaded according to the level of conservation. The symbols above the alignment indicate the location of functional residues (T = trypsin cleavage site, ^ = arginines that contact the DNA helix, * = H2A-H2B interaction sites, U = ubiquitination site). The annotation below the alignment indicates conserved structural features including the α-helices, loops, and the SQ(E/D)Φgotif. B) A ML tree of H2A and H2A.X. The numbers above and below the branches are the results of ML and NJ bootstrap analyses, respectively. The thick branches indicate > 0.95 posterior probability from Bayesian inference. Only bootstrap values ≥ 50% are shown. Branch lengths are proportional to the number of substitutions per site (see scale bar).
Mentions: A significant finding of this study is the identification of two rare (2/11,171) ESTs that encode a partial histone H2A.X. The longest cDNA isolated from the library using PCR was predicted to encoded a protein of 169 amino acids that shares high sequence identity to eukaryotic histone H2A.X (Figure 3A). This clone putatively lacked only the start codon at the N-terminus. The divergent N-terminus of A. tamarense H2A.X is somewhat longer than in other homologs but the remainder of the sequence is conserved (in particular the α-helices of the histone fold). Several functional residues from the known crystal structure are also present in A. tamarense H2A.X including the lysine at the trypsin cleavage site, the arginines in the loops that interact with the DNA α-helix, and the lysine ubiquitination site [24]. The sites of interaction with histone H2B are also present.

Bottom Line: We also identified rare transcripts encoding a predicted protein highly similar to histone H2A.X.We speculate this histone may be retained for its role in DNA double-strand break repair.These data will be instrumental to future research to understand the unique and complex cell biology of these organisms and for potentially identifying the genes involved in toxin production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences and Roy J, Carver Center for Comparative Genomics, University of Iowa, Iowa City, IA 52242, USA. jeremiah-hackett@uiowa.edu

ABSTRACT

Background: Dinoflagellates are important marine primary producers and grazers and cause toxic "red tides". These taxa are characterized by many unique features such as immense genomes, the absence of nucleosomes, and photosynthetic organelles (plastids) that have been gained and lost multiple times. We generated EST sequences from non-normalized and normalized cDNA libraries from a culture of the toxic species Alexandrium tamarense to elucidate dinoflagellate evolution. Previous analyses of these data have clarified plastid origin and here we study the gene content, annotate the ESTs, and analyze the genes that are putatively involved in DNA packaging.

Results: Approximately 20% of the 6,723 unique (11,171 total 3'-reads) ESTs data could be annotated using Blast searches against GenBank. Several putative dinoflagellate-specific mRNAs were identified, including one novel plastid protein. Dinoflagellate genes, similar to other eukaryotes, have a high GC-content that is reflected in the amino acid codon usage. Highly represented transcripts include histone-like (HLP) and luciferin binding proteins and several genes occur in families that encode nearly identical proteins. We also identified rare transcripts encoding a predicted protein highly similar to histone H2A.X. We speculate this histone may be retained for its role in DNA double-strand break repair.

Conclusion: This is the most extensive collection to date of ESTs from a toxic dinoflagellate. These data will be instrumental to future research to understand the unique and complex cell biology of these organisms and for potentially identifying the genes involved in toxin production.

Show MeSH
Related in: MedlinePlus