Pharmacological MEK inhibition promotes polyclonal T-cell reconstitution and suppresses xenogeneic GVHD.

Rapid immune reconstitution without developing graft-versus-host disease (GVHD) is required for the success of allogeneic hematopoietic stem cell transplantation. Here, we analyzed the effects of pharmacological MEK inhibition on human polyclonal T-cell reconstitution in a humanized mouse GVHD model utilizing deep sequencing-based T-cell receptor (TCR) repertoire analysis. GVHD mice exhibited a skewed TCR repertoire with a common clone within target organs. The MEK inhibitor trametinib ameliorated GVHD and enabled engraftment of diverse T-cell clones. Furthermore, trametinib also ameliorated GVHD sparing diverse T cell repertoire, even when it was given from day 15 through 28. Although tacrolimus also reduced development of GVHD, it disturbed diverse T cell reconstitution and resulted in skewed TCR repertoire. Thus, trametinib not only suppresses GVHD-inducing T cells but also promotes human T cell reconstitution in vivo, providing a novel rationale for translational studies targeting human GVHD.

Centromere-targeted de novo integrations of an LTR retrotransposon of Arabidopsis lyrata.

The plant genome evolves with rapid proliferation of LTR-type retrotransposons, which is associated with their clustered accumulation in gene-poor regions, such as centromeres. Despite their major role for plant genome evolution, no mobile LTR element with targeted integration into gene-poor regions has been identified in plants. Here, we report such targeted integrations de novo. We and others have previously shown that an ATCOPIA93 family retrotransposon in Arabidopsis thaliana is mobilized when the DNA methylation machinery is compromised. Although ATCOPIA93 family elements are low copy number in the wild-type A. thaliana genome, high-copy-number related elements are found in the wild-type Arabidopsis lyrata genome, and they show centromere-specific localization. To understand the mechanisms for the clustered accumulation of the A. lyrata elements directly, we introduced one of them, named Tal1 (Transposon of Arabidopsis lyrata 1), into A. thaliana by transformation. The introduced Tal1 was retrotransposed in A. thaliana, and most of the retrotransposed copies were found in centromeric repeats of A. thaliana, suggesting targeted integration. The targeted integration is especially surprising because the centromeric repeat sequences differ considerably between A. lyrata and A. thaliana. Our results revealed unexpectedly dynamic controls for evolution of the transposon-rich heterochromatic regions.

The ancestor of extant Japanese fancy mice contributed to the mosaic genomes of classical inbred strains.

Commonly used classical inbred mouse strains have mosaic genomes with sequences from different subspecific origins. Their genomes are derived predominantly from the Western European subspecies Mus musculus domesticus, with the remaining sequences derived mostly from the Japanese subspecies Mus musculus molossinus. However, it remains unknown how this intersubspecific genome introgression occurred during the establishment of classical inbred strains. In this study, we resequenced the genomes of two M. m. molossinus-derived inbred strains, MSM/Ms and JF1/Ms. MSM/Ms originated from Japanese wild mice, and the ancestry of JF1/Ms was originally found in Europe and then transferred to Japan. We compared the characteristics of these sequences to those of the C57BL/6J reference sequence and the recent data sets from the resequencing of 17 inbred strains in the Mouse Genome Project (MGP), and the results unequivocally show that genome introgression from M. m. molossinus into M. m. domesticus provided the primary framework for the mosaic genomes of classical inbred strains. Furthermore, the genomes of C57BL/6J and other classical inbred strains have long consecutive segments with extremely high similarity (>99.998%) to the JF1/Ms strain. In the early 20th century, Japanese waltzing mice with a morphological phenotype resembling that of JF1/Ms mice were often crossed with European fancy mice for early studies of "Mendelism," which suggests that the ancestor of the extant JF1/Ms strain provided the origin of the M. m. molossinus genome in classical inbred strains and largely contributed to its intersubspecific genome diversity.

Comparative genome and transcriptome analyses of the social amoeba Acytostelium subglobosum that accomplishes multicellular development without germ-soma differentiation.

BACKGROUND: Social amoebae are lower eukaryotes that inhabit the soil. They are characterized by the construction of a starvation-induced multicellular fruiting body with a spore ball and supportive stalk. In most species, the stalk is filled with motile stalk cells, as represented by the model organism Dictyostelium discoideum, whose developmental mechanisms have been well characterized. However, in the genus Acytostelium, the stalk is acellular and all aggregated cells become spores. Phylogenetic analyses have shown that it is not an ancestral genus but has lost the ability to undergo cell differentiation. RESULTS: We performed genome and transcriptome analyses of Acytostelium subglobosum and compared our findings to other available dictyostelid genome data. Although A. subglobosum adopts a qualitatively different developmental program from other dictyostelids, its gene repertoire was largely conserved. Yet, families of polyketide synthase and extracellular matrix proteins have not expanded and a serine protease and ABC transporter B family gene, tagA, and a few other developmental genes are missing in the A. subglobosum lineage. Temporal gene expression patterns are astonishingly dissimilar from those of D. discoideum, and only a limited fraction of the ortholog pairs shared the same expression patterns, so that some signaling cascades for development seem to be disabled in A. subglobosum. CONCLUSIONS: The absence of the ability to undergo cell differentiation in Acytostelium is accompanied by a small change in coding potential and extensive alterations in gene expression patterns.

The amphioxus genome and the evolution of the chordate karyotype.

Lancelets ('amphioxus') are the modern survivors of an ancient chordate lineage, with a fossil record dating back to the Cambrian period. Here we describe the structure and gene content of the highly polymorphic approximately 520-megabase genome of the Florida lancelet Branchiostoma floridae, and analyse it in the context of chordate evolution. Whole-genome comparisons illuminate the murky relationships among the three chordate groups (tunicates, lancelets and vertebrates), and allow not only reconstruction of the gene complement of the last common chordate ancestor but also partial reconstruction of its genomic organization, as well as a description of two genome-wide duplications and subsequent reorganizations in the vertebrate lineage. These genome-scale events shaped the vertebrate genome and provided additional genetic variation for exploitation during vertebrate evolution.

A cDNA resource for gene expression studies of a hemichordate, Ptychodera flava.

Recent investigations into the evolution of deuterostomes and the origin of chordates have paid considerable attention to hemichordates (acorn worms), as hemichordates and echinoderms are the closest chordate relatives. The present study prepared cDNA libraries from Ptychodera flava, to study expression and function of genes involved in development of the hemichordate body plan. Expressed sequence tag (EST) analyses of nine cDNA libraries yielded 18,832 cloned genes expressed in eggs, 18,739 in blastulae, 18,539 in gastrulae, 18,811 in larvae, 18,978 in juveniles, 11,802 in adult proboscis, 17,259 in stomochord, 11,886 in gills, and 11,580 in liver, respectively. A set of 34,159 uni-gene clones of P. flava was obtained. This cDNA resource will be valuable for studying temporal and spatial expression of acorn worm genes during development.

Pre-existing cross-reactive neutralizing activity against SARS-CoV-2 and seasonal coronaviruses prior to the COVID-19 pandemic (2014-2019) with limited immunity against recent emerging SARS-CoV-2 variants, Vietnam.

OBJECTIVES: SARS-CoV-2 transmission and epidemic potential is related to the population's immunity levels. As such, assessing different regions' preexisting immune responses to SARS-CoV-2 is important to understand the transmission potential of emerging SARS-CoV-2 variants. DESIGN: In 975 serum samples from Vietnam (2014 to 2019), anti-SARS-CoV-2 Immunoglobulin G levels were determined by enzyme-linked immunosorbent assay. Plaque reduction neutralization test (PRNT) was performed using Wuhan strain and variants of concern (VOCs). Cross-reactivity was confirmed by analyzing B-cell receptor (BCR) repertoire sequences and identifying BCR repertoire sequences-derived T-cell epitopes. RESULTS: Overall, 20.9% (n = 76/364) and 9.2% (n = 7) demonstrated SARS-CoV-2 neutralizing activity (PRNT50) against the Wuhan and Alpha strain, respectively. Neutralizing activity against Beta, Gamma, and Delta strains was absent (PRNT50<5) in all samples. Cross-reactive epitopes against SARS-CoV-2 and other coronavirus spike proteins were detected in the N-terminal domain, S2, and receptor-binding domain regions. CONCLUSIONS: Following BCR and major histocompatibility complex analysis, T-cell receptor-recognized epitope motif (TREM) among pathogenic coronaviruses and coronaviruses spike proteins were the top TREM peptide, suggesting that pre-existing immunity against SARS-CoV-2 in Vietnam was due to exposure to common cold coronaviruses. With limited immunity against emerging VOCs, further monitoring, and control of the epidemic, along with COVID-19 vaccine programs against VOCs, are necessary.

Molecular cloning and characterization of the repetitive DNA sequences that comprise the constitutive heterochromatin of the W chromosomes of medaka fishes.

Among the medaka fishes of the genus Oryzias, most species have homomorphic sex chromosomes, while some species, such as Oryzias hubbsi and Oryzias javanicus, have heteromorphic ZW sex chromosomes. In this study, a novel family of repetitive sequence was molecularly cloned from O. hubbsi and characterized by chromosome in situ and filter hybridization, respectively. This repetitive element, which we designated as a BstNI family element, localized at heterochromatin regions on the W chromosome, as well as on two pairs of autosomes. Homologous sequences to this element were found only in O. javanicus, which is a sister species of O. hubbsi, suggesting that this repeated element originated in the common ancestor of these two species. However, the intensity of the hybridization signals was lower in O. javanicus than in O. hubbsi, and the chromosomal location of this element in O. javanicus was confined to heterochromatin regions on one pair of autosomes. Thus, we hypothesize that this repetitive element was extensively amplified in the O. hubbsi lineage, especially on its W chromosome, after the separation of the O. javanicus lineage. In addition, we also found the W chromosomal location of the 18S-28S ribosomal RNA genes in both O. hubbsi and O. javanicus. Our previous studies showed no linkage homology of the sex chromosomes in these species, indicating that the RNA genes were shared between W chromosomes of different origins. This situation may be explained by a translocation of the sex-determining region with the ribosomal RNA genes in either species or an independent accumulation of the RNA genes as a convergent process during W chromosome degeneration.

Axial patterning in cephalochordates and the evolution of the organizer.

The organizer of the vertebrate gastrula is an important signalling centre that induces and patterns dorsal axial structures. Although a topic of long-standing interest, the evolutionary origin of the organizer remains unclear. Here we show that the gastrula of the cephalochordate amphioxus expresses dorsal/ventral (D/V) patterning genes (for example, bone morphogenetic proteins (BMPs), Nodal and their antagonists) in patterns reminiscent of those of their vertebrate orthlogues, and that amphioxus embryos, like those of vertebrates, are ventralized by exogenous BMP protein. In addition, Wnt-antagonists (for example, Dkks and sFRP2-like) are expressed anteriorly, whereas Wnt genes themselves are expressed posteriorly, consistent with a role for Wnt signalling in anterior/posterior (A/P) patterning. These results suggest evolutionary conservation of the mechanisms for both D/V and A/P patterning of the early gastrula. In light of recent phylogenetic analyses placing cephalochordates basally in the chordate lineage, we propose that separate signalling centres for patterning the D/V and A/P axes may be an ancestral chordate character.

The medaka draft genome and insights into vertebrate genome evolution.

Teleosts comprise more than half of all vertebrate species and have adapted to a variety of marine and freshwater habitats. Their genome evolution and diversification are important subjects for the understanding of vertebrate evolution. Although draft genome sequences of two pufferfishes have been published, analysis of more fish genomes is desirable. Here we report a high-quality draft genome sequence of a small egg-laying freshwater teleost, medaka (Oryzias latipes). Medaka is native to East Asia and an excellent model system for a wide range of biology, including ecotoxicology, carcinogenesis, sex determination and developmental genetics. In the assembled medaka genome (700 megabases), which is less than half of the zebrafish genome, we predicted 20,141 genes, including approximately 2,900 new genes, using 5'-end serial analysis of gene expression tag information. We found single nucleotide polymorphisms (SNPs) at an average rate of 3.42% between the two inbred strains derived from two regional populations; this is the highest SNP rate seen in any vertebrate species. Analyses based on the dense SNP information show a strict genetic separation of 4 million years (Myr) between the two populations, and suggest that differential selective pressures acted on specific gene categories. Four-way comparisons with the human, pufferfish (Tetraodon), zebrafish and medaka genomes revealed that eight major interchromosomal rearrangements took place in a remarkably short period of approximately 50 Myr after the whole-genome duplication event in the teleost ancestor and afterwards, intriguingly, the medaka genome preserved its ancestral karyotype for more than 300 Myr.

Idiotope-Driven T-Cell/B-Cell Collaboration-Based T-Cell Epitope Prediction Using B-Cell Receptor Repertoire Sequences in Infectious Diseases.

T-cell recognition of antigen epitopes is a crucial step for the induction of adaptive immune responses, and the identification of such T-cell epitopes is, therefore, important for understanding diverse immune responses and controlling T-cell immunity. A number of bioinformatic tools exist that predict T-cell epitopes; however, many of these methods highly rely on evaluating conventional peptide presentation by major histocompatibility complex (MHC) molecules, but they ignore epitope sequences recognized by T-cell receptor (TCR). Immunogenic determinant idiotopes are present on the variable regions of immunoglobulin molecules expressed on and secreted by B-cells. In idiotope-driven T-cell/B-cell collaboration, B-cells present the idiotopes on MHC molecules for recognition by idiotope-specific T-cells. According to the idiotype network theory formulated by Niels Jerne, such idiotopes found on anti-idiotypic antibodies exhibit molecular mimicry of antigens. Here, by combining these concepts and defining the patterns of TCR-recognized epitope motifs (TREMs), we developed a T-cell epitope prediction method that identifies T-cell epitopes derived from antigen proteins by analyzing B-cell receptor (BCR) sequences. This method allowed us to identify T-cell epitopes that contain the same TREM patterns between BCR and viral antigen sequences in two different infectious diseases caused by dengue virus and SARS-CoV-2 infection. The identified epitopes were among the T-cell epitopes detected in previous studies, and T-cell stimulatory immunogenicity was confirmed. Thus, our data support this method as a powerful tool for the discovery of T-cell epitopes from BCR sequences.

A genomic overview of short genetic variations in a basal chordate, Ciona intestinalis.

BACKGROUND: Although the Ciona intestinalis genome contains many allelic polymorphisms, there is only limited data analyzed systematically. Establishing a dense map of genetic variations in C. intestinalis is necessary not only for linkage analysis, but also for other experimental biology including molecular developmental and evolutionary studies, because animals from natural populations are typically used for experiments. RESULTS: Here, we identified over three million candidate short genomic variations within a 110 Mb euchromatin region among five C. intestinalis individuals. The average nucleotide diversity was approximately 1.1%. Genetic variations were found at a similar density in intergenic and gene regions. Non-synonymous and nonsense nucleotide substitutions were found in 12,493 and 1,214 genes accounting for 81.9% and 8.0% of the entire gene set, respectively, and over 60% of genes in the single animal encode non-identical proteins between maternal and paternal alleles. CONCLUSIONS: Our results provide a framework for studying evolution of the animal genome, as well as a useful resource for a wide range of C. intestinalis researchers.

Mass identification of transcriptional units expressed from the Bombyx mori nucleopolyhedrovirus genome.

In order to identify the transcriptional units expressed from an entire nucleopolyhedrovirus (NPV) genome during infection, we constructed a full-length-enriched cDNA library from Bombyx mori NPV (BmNPV)-infected BmN cells. We randomly sequenced 11,520 clones from both ends to obtain a total of 4679 BmNPV-derived transcriptional units. The data revealed a number of novel transcripts, including putative non-coding RNAs, most of which are expressed from recognized baculovirus early or late promoter motifs. These findings provide new insights into the complex transcriptional regulation of an NPV genome and suggest roles for as-yet-uncharacterized transcripts.

Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum) cultivar Micro-Tom, a reference system for the Solanaceae genomics.

BACKGROUND: The Solanaceae family includes several economically important vegetable crops. The tomato (Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. Recently, a number of tomato resources have been developed in parallel with the ongoing tomato genome sequencing project. In particular, a miniature cultivar, Micro-Tom, is regarded as a model system in tomato genomics, and a number of genomics resources in the Micro-Tom-background, such as ESTs and mutagenized lines, have been established by an international alliance. RESULTS: To accelerate the progress in tomato genomics, we developed a collection of fully-sequenced 13,227 Micro-Tom full-length cDNAs. By checking redundant sequences, coding sequences, and chimeric sequences, a set of 11,502 non-redundant full-length cDNAs (nrFLcDNAs) was generated. Analysis of untranslated regions demonstrated that tomato has longer 5'- and 3'-untranslated regions than most other plants but rice. Classification of functions of proteins predicted from the coding sequences demonstrated that nrFLcDNAs covered a broad range of functions. A comparison of nrFLcDNAs with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants, most of which did not have known protein domains. Mapping of the nrFLcDNAs onto currently available tomato genome sequences facilitated prediction of exon-intron structure. Introns of tomato genes were longer than those of Arabidopsis and rice. According to a comparison of exon sequences between the nrFLcDNAs and the tomato genome sequences, the frequency of nucleotide mismatch in exons between Micro-Tom and the genome-sequencing cultivar (Heinz 1706) was estimated to be 0.061%. CONCLUSION: The collection of Micro-Tom nrFLcDNAs generated in this study will serve as a valuable genomic tool for plant biologists to bridge the gap between basic and applied studies. The nrFLcDNA sequences will help annotation of the tomato whole-genome sequence and aid in tomato functional genomics and molecular breeding. Full-length cDNA sequences and their annotations are provided in the database KaFTom http://www.pgb.kazusa.or.jp/kaftom/ via the website of the National Bioresource Project Tomato http://tomato.nbrp.jp.

Mechanism of entecavir resistance of hepatitis B virus with viral breakthrough as determined by long-term clinical assessment and molecular docking simulation.

The mechanism by which entecavir resistance (ETVr) substitutions of hepatitis B virus (HBV) can induce breakthrough (BT) during ETV therapy is largely unknown. We conducted a cross-sectional study of 49 lamivudine (LVD)-refractory patients and 59 naïve patients with chronic hepatitis B. BT was observed in 26.8% of the LVD-refractory group during weeks 60 to 144 of ETV therapy. A line probe assay revealed ETVr substitutions only in the LVD-refractory group, i.e., in 4.9% of patients at baseline, increasing to 14.6%, 24.4%, and 44.8% at weeks 48, 96, and 144, respectively. Multivariate logistic regression analysis adjusted for age, gender, HBV DNA levels, and LVD resistance (LVDr) (L180M and M204V, but not M204I) indicated that T184 substitutions and S202G (not S202C) were a significant factor for BT (adjusted odds ratio [OR], 141.12, and 95% confidence interval [CI], 6.94 to 2,870.20; OR, 201.25, and 95% CI, 11.22 to 3608.65, respectively). Modeling of HBV reverse transcriptase (RT) by docking simulation indicated that a combination of LVDr and ETVr (T184L or S202G) was characterized by a change in the direction of the D205 residue and steric conflict in the binding pocket of ETV triphosphate (ETV-TP), by significantly longer minimal distances (2.2 A and 2.1 A), and by higher potential energy (-117 and -99.8 Kcal/mol) for ETV-TP compared with the wild type (1.3 A; -178 Kcal/mol) and LVDr substitutions (1.5 A; -141 Kcal/mol). Our data suggest that the low binding affinity of ETV-TP for the HBV RT, involving conformational change of the binding pocket of HBV RT by L180M, M204V plus T184L, and S202G, could induce BT.

Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D.

Small, compact genomes of ultrasmall unicellular algae provide information on the basic and essential genes that support the lives of photosynthetic eukaryotes, including higher plants. Here we report the 16,520,305-base-pair sequence of the 20 chromosomes of the unicellular red alga Cyanidioschyzon merolae 10D as the first complete algal genome. We identified 5,331 genes in total, of which at least 86.3% were expressed. Unique characteristics of this genomic structure include: a lack of introns in all but 26 genes; only three copies of ribosomal DNA units that maintain the nucleolus; and two dynamin genes that are involved only in the division of mitochondria and plastids. The conserved mosaic origin of Calvin cycle enzymes in this red alga and in green plants supports the hypothesis of the existence of single primary plastid endosymbiosis. The lack of a myosin gene, in addition to the unexpressed actin gene, suggests a simpler system of cytokinesis. These results indicate that the C. merolae genome provides a model system with a simple gene composition for studying the origin, evolution and fundamental mechanisms of eukaryotic cells.

UTGB/medaka: genomic resource database for medaka biology.

Medaka (Oryzias latipes) is a small egg-laying freshwater teleost native to East Asia that has become an excellent model system for developmental genetics and evolutionary biology. The draft medaka genome sequence (700 Mb) was reported in June 2007, and its substantial genomic resources have been opened to the public through the University of Tokyo Genome Browser Medaka (UTGB/medaka) database. This database provides basic genomic information, such as predicted genes, expressed sequence tags (ESTs), guanine/cytosine (GC) content, repeats and comparative genomics, as well as unique data resources including (i) 2473 genetic markers and experimentally confirmed PCR primers that amplify these markers, (ii) 142,414 bacterial artificial chromosome (BAC) and 217,344 fosmid end sequences that amount to 15.0- and 11.1-fold clone coverage of the entire genome, respectively, and were used for draft genome assembly, (iii) 16,519,460 single nucleotide polymorphisms (SNPs), and 2 859 905 insertions/deletions detected between two medaka inbred strain genomes and (iv) 841 235 5'-end serial analyses of gene-expression (SAGE) tags that identified 344 266 transcription start sites on the genome. UTGB/medaka is available at: http://medaka.utgenome.org/.

Epstein-Barr virus-related diffuse large B-cell lymphoma in mogamulizumab-treated adult T-cell leukemia with incomplete T-cell reconstitution.

Adult T-cell leukemia (ATL) is an aggressive mature T-cell malignancy with a poor prognosis. The anti-C-C motif chemokine receptor 4 (CCR4) antibody mogamulizumab (moga) reduces ATL cells and induces reconstitution of polyclonal T cells; however, ATL cases often remain resistant and moga sometimes causes fatal immunopathology. Epstein-Barr virus (EBV)-related B-cell lymphoma develops in severely immunocompromised subjects, and is particularly associated with impaired T-cell immunity. Here, we report an ATL patient who had received conventional chemotherapy plus moga, and subsequently developed EBV-related diffuse large B-cell lymphoma (DLBCL) of the central nervous system. Next-generation sequencing-based T-cell receptor repertoire analyses identified residual abnormal clones and revealed that reconstitution of polyclonal T cells was incomplete, even after moga treatment. Furthermore, a skin rash that developed after moga treatment was found to contain ATL clones. This case suggests that the limited therapeutic effects of moga and incomplete T-cell reconstitution are associated with severely impaired T-cell immunity and subsequent development of EBV-related DLBCL.

Construction of complete Tupaia belangeri transcriptome database by whole-genome and comprehensive RNA sequencing.

The northern tree shrew (Tupaia belangeri) possesses high potential as an animal model of human diseases and biology, given its genetic similarity to primates. Although genetic information on the tree shrew has already been published, some of the entire coding sequences (CDSs) of tree shrew genes remained incomplete, and the reliability of these CDSs remained difficult to determine. To improve the determination of tree shrew CDSs, we performed sequencing of the whole-genome, mRNA, and total RNA and integrated the resulting data. Additionally, we established criteria for the selection of reliable CDSs and annotated these sequences by comparison to the human transcriptome, resulting in the identification of complete CDSs for 12,612 tree shrew genes and yielding a more accurate tree shrew genome database (TupaiaBase: http://tupaiabase.org ). Transcriptome profiles in hepatitis B virus infected tree shrew livers were analyzed for validation. Gene ontology analysis showed enriched transcriptional regulation at 1 day post-infection, namely in the "type I interferon signaling pathway". Moreover, a negative regulator of type I interferon, SOCS3, was induced. This work, which provides a tree shrew CDS database based on genomic DNA and RNA sequencing, is expected to serve as a powerful tool for further development of the tree shrew model.

A cDNA resource for the cephalochordate amphioxus Branchiostoma floridae.

Cephalochordates are the basal invertebrate chordates within the phylum Chordata. They are widely used as a model system for research in evolutionary developmental biology (EvoDevo) to understand the basic patterning mechanisms for the chordate body plan and the origin of vertebrates. Recently, the genome of the cephalochordate Branchiostoma floridae was sequenced, which further brings this organism to the front for comparative genomic studies. In this paper, we report the generation of large-scale 5'- and 3'-expressed sequence tags (ESTs) from B. floridae and the complementary deoxyribonucleic acid (cDNA) resource for this species. Both 5'- and 3'-ESTs were sequenced for approximately 140,000 cDNA clones derived from five developmental stages, and the cDNA clones were subsequently grouped into independent clusters using 3'-EST sequences. We identified 21,229 cDNA clusters, and each corresponds to a unique transcript species from B. floridae. We then chose 24,020 cDNA clones representing all of these 21,229 clusters to generate the "Branchiostoma floridae Gene Collection Release 1." We also constructed a database with a searchable interface for this EST dataset and the related information on "Branchiostoma floridae Gene Collection Release 1." This set of cDNA clones along with our cDNA database will serve as an important resource for future research in this basal chordate. This Gene Collection and the original 140,000 individual cDNA clones are available to the research community upon request.