The long non-coding RNA Paupar regulates the expression of both local and distal genes.

Although some long noncoding RNAs (lncRNAs) have been shown to regulate gene expression in cis, it remains unclear whether lncRNAs can directly regulate transcription in trans by interacting with chromatin genome-wide independently of their sites of synthesis. Here, we describe the genomically local and more distal functions of Paupar, a vertebrate-conserved and central nervous system-expressed lncRNA transcribed from a locus upstream of the gene encoding the PAX6 transcription factor. Knockdown of Paupar disrupts the normal cell cycle profile of neuroblastoma cells and induces neural differentiation. Paupar acts in a transcript-dependent manner both locally, to regulate Pax6, as well as distally by binding and regulating genes on multiple chromosomes, in part through physical association with PAX6 protein. Paupar binding sites are enriched near promoters and can function as transcriptional regulatory elements whose activity is modulated by Paupar transcript levels. Our findings demonstrate that a lncRNA can function in trans at transcriptional regulatory elements distinct from its site of synthesis to control large-scale transcriptional programmes.

The genome of the green anole lizard and a comparative analysis with birds and mammals.

The evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments. Among amniotes, genome sequences are available for mammals and birds, but not for non-avian reptiles. Here we report the genome sequence of the North American green anole lizard, Anolis carolinensis. We find that A. carolinensis microchromosomes are highly syntenic with chicken microchromosomes, yet do not exhibit the high GC and low repeat content that are characteristic of avian microchromosomes. Also, A. carolinensis mobile elements are very young and diverse-more so than in any other sequenced amniote genome. The GC content of this lizard genome is also unusual in its homogeneity, unlike the regionally variable GC content found in mammals and birds. We describe and assign sequence to the previously unknown A. carolinensis X chromosome. Comparative gene analysis shows that amniote egg proteins have evolved significantly more rapidly than other proteins. An anole phylogeny resolves basal branches to illuminate the history of their repeated adaptive radiations.

The genome of a songbird.

The zebra finch is an important model organism in several fields with unique relevance to human neuroscience. Like other songbirds, the zebra finch communicates through learned vocalizations, an ability otherwise documented only in humans and a few other animals and lacking in the chicken-the only bird with a sequenced genome until now. Here we present a structural, functional and comparative analysis of the genome sequence of the zebra finch (Taeniopygia guttata), which is a songbird belonging to the large avian order Passeriformes. We find that the overall structures of the genomes are similar in zebra finch and chicken, but they differ in many intrachromosomal rearrangements, lineage-specific gene family expansions, the number of long-terminal-repeat-based retrotransposons, and mechanisms of sex chromosome dosage compensation. We show that song behaviour engages gene regulatory networks in the zebra finch brain, altering the expression of long non-coding RNAs, microRNAs, transcription factors and their targets. We also show evidence for rapid molecular evolution in the songbird lineage of genes that are regulated during song experience. These results indicate an active involvement of the genome in neural processes underlying vocal communication and identify potential genetic substrates for the evolution and regulation of this behaviour.

Insights into the evolution of Darwin's finches from comparative analysis of the Geospiza magnirostris genome sequence.

BACKGROUND: A classical example of repeated speciation coupled with ecological diversification is the evolution of 14 closely related species of Darwin's (Galápagos) finches (Thraupidae, Passeriformes). Their adaptive radiation in the Galápagos archipelago took place in the last 2-3 million years and some of the molecular mechanisms that led to their diversification are now being elucidated. Here we report evolutionary analyses of genome of the large ground finch, Geospiza magnirostris. RESULTS: 13,291 protein-coding genes were predicted from a 991.0 Mb G. magnirostris genome assembly. We then defined gene orthology relationships and constructed whole genome alignments between the G. magnirostris and other vertebrate genomes. We estimate that 15% of genomic sequence is functionally constrained between G. magnirostris and zebra finch. Genic evolutionary rate comparisons indicate that similar selective pressures acted along the G. magnirostris and zebra finch lineages suggesting that historical effective population size values have been similar in both lineages. 21 otherwise highly conserved genes were identified that each show evidence for positive selection on amino acid changes in the Darwin's finch lineage. Two of these genes (Igf2r and Pou1f1) have been implicated in beak morphology changes in Darwin's finches. Five of 47 genes showing evidence of positive selection in early passerine evolution have cilia related functions, and may be examples of adaptively evolving reproductive proteins. CONCLUSIONS: These results provide insights into past evolutionary processes that have shaped G. magnirostris genes and its genome, and provide the necessary foundation upon which to build population genomics resources that will shed light on more contemporaneous adaptive and non-adaptive processes that have contributed to the evolution of the Darwin's finches.

A common ancestry for BAP1 and Uch37 regulators.

SUMMARY: To reveal how the polycomb repressive-deubiquitinase (PR-DUB) complex controls substrate selection specificity, we undertook a detailed computational sequence analysis of its components: additional sex combs like 1 (ASXL1) and BRCA1-associated protein 1 (BAP1) proteins. This led to the discovery of two previously unrecognized domains in ASXL1: a forkhead (winged-helix) DNA-binding domain and a deubiquitinase adaptor domain shared with two regulators of ubiquitin carboxyl-terminal hydrolase 37 (Uch37), namely adhesion regulating molecule 1 (ADRM1) and nuclear factor related to kappaB (NFRKB). Our analysis demonstrates a common ancestry for BAP1 and Uch37 regulators in PR-DUB, INO80 chromatin remodelling and proteosome complexes. CONTACT: luis.sanchezpulido@dpag.ox.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Accelerated evolution of PAK3- and PIM1-like kinase gene families in the zebra finch, Taeniopygia guttata.

Genes encoding protein kinases tend to evolve slowly over evolutionary time, and only rarely do they appear as recent duplications in sequenced vertebrate genomes. Consequently, it was a surprise to find two families of kinase genes that have greatly and recently expanded in the zebra finch (Taeniopygia guttata) lineage. In contrast to other amniotic genomes (including chicken) that harbor only single copies of p21-activated serine/threonine kinase 3 (PAK3) and proviral integration site 1 (PIM1) genes, the zebra finch genome appeared at first to additionally contain 67 PAK3-like (PAK3L) and 51 PIM1-like (PIM1L) protein kinase genes. An exhaustive analysis of these gene models, however, revealed most to be incomplete, owing to the absence of terminal exons. After reprediction, 31 PAK3L genes and 10 PIM1L genes remain, and all but three are predicted, from the retention of functional sites and open reading frames, to be enzymatically active. PAK3L, but not PIM1L, gene sequences show evidence of recurrent episodes of positive selection, concentrated within structures spatially adjacent to N- and C-terminal protein regions that have been discarded from zebra finch PAK3L genes. At least seven zebra finch PAK3L genes were observed to be expressed in testis, whereas two sequences were found transcribed in the brain, one broadly including the song nuclei and the other in the ventricular zone and in cells resembling Bergmann's glia in the cerebellar Purkinje cell layer. Two PIM1L sequences were also observed to be expressed with broad distributions in the zebra finch brain, one in both the ventricular zone and the cerebellum and apparently associated with glial cells and the other showing neuronal cell expression and marked enrichment in midbrain/thalamic nuclei. These expression patterns do not correlate with zebra finch-specific features such as vocal learning. Nevertheless, our results show how ancient and conserved intracellular signaling molecules can be co-opted, following duplication, thereby resulting in lineage-specific functions, presumably affecting the zebra finch testis and brain.

Tandem duplication, circular permutation, molecular adaptation: how Solanaceae resist pests via inhibitors.

BACKGROUND: The Potato type II (Pot II) family of proteinase inhibitors plays critical roles in the defense system of plants from Solanaceae family against pests. To better understand the evolution of this family, we investigated the correlation between sequence and structural repeats within this family and the evolution and molecular adaptation of Pot II genes through computational analysis, using the putative ancestral domain sequence as the basic repeat unit. RESULTS: Our analysis discovered the following interesting findings in Pot II family. (1) We classified the structural domains in Pot II family into three types (original repeat domain, circularly permuted domain, the two-chain domain) according to the existence of two linkers between the two domain components, which clearly show the circular permutation relationship between the original repeat domain and circularly permuted domain. (2) The permuted domains appear more stable than original repeat domain, from available structural information. Therefore, we proposed a multiple-repeat sequence is likely to adopt the permuted domain from contiguous sequence segments, with the N- and C-termini forming a single non-contiguous structural domain, linking the bracelet of tandem repeats. (3) The analysis of nonsynonymous/synonymous substitution rates ratio in Pot II domain revealed heterogeneous selective pressures among amino acid sites: the reactive site is under positive Darwinian selection (providing different specificity to target varieties of proteinases) while the cysteine scaffold is under purifying selection (essential for maintaining the fold). (4) For multi-repeat Pot II genes from Nicotiana genus, the proteolytic processing site is under positive Darwinian selection (which may improve the cleavage efficiency). CONCLUSION: This paper provides comprehensive analysis and characterization of Pot II family, and enlightens our understanding on the strategies (Gene and domain duplication, structural circular permutation and molecular adaptation) of Solanaceae plants for defending pathogenic attacks through the evolution of Pot II genes.

MPID-T: database for sequence-structure-function information on T-cell receptor/peptide/MHC interactions.

UNLABELLED: Normal adaptive immune responses operate under major histocompatibility complex (MHC) restriction by binding to specific, short antigenic peptides and presenting them to appropriate T-cell receptors (TcRs). Sequence-structure-function information is critical in understanding the principles governing peptide/MHC (pMHC) and TcR/pMHC recognition and binding. A new database for sequence-structure-function information on TcR/pMHC interactions, MHC-Peptide Interaction Database version T (MPID-T), is now available with the latest available Protein Data Bank (PDB) data and interaction parameters on TcR/pMHC complexes. MPID-T is a manually curated MySQL database containing experimentally determined structures of 187 pMHC complexes and 16 TcR/pMHC complexes available in the PDB. Each structure is manually verified, classified, and analysed for intermolecular interactions (i) between the MHC and its corresponding bound peptide and (ii) between TcR and its bound pMHC complex where TcR structural information is available. The MPID-T database retrieval system has precomputed interaction parameters that include solvent accessibility, hydrogen bonds, gap volume and gap index. Structural visualisation of the TcR/pMHC complex, pMHC complex, MHC or the bound peptide can be performed using freely available graphics applications such as MDL Chime or RasMol, while structural alignment (based on MHC class and peptide length) can be viewed using the Jmol molecular viewer or an MDL Chime-compatible web browser client. MPID-T contains structural descriptors for in-depth characterisation of TcR/pMHC and pMHC interactions. The ultimate purpose of MPID-T is to enhance the understanding of the binding mechanism underlying TcR/pMHC and pMHC interactions by mapping the TcR footprint on the MHC and its bound peptide, as this eventually determines T-cell recognition and binding. AVAILABILITY: The MPID-T database retrieval system is available at http://surya.bic.nus.edu.sg/mpidt CONTACT: Joo Chuan Tong (jctong@i2r.a-star.edu.sg).

SDPMOD: an automated comparative modeling server for small disulfide-bonded proteins.

Small disulfide-bonded proteins (SDPs) are rich sources for therapeutic drugs. Designing drugs from these proteins requires three-dimensional structural information, which is only available for a subset of these proteins. SDPMOD addresses this deficit in structural information by providing a freely available automated comparative modeling service to the research community. For expert users, SDPMOD offers a manual mode that permits the selection of a desired template as well as a semi-automated mode that allows users to select the template from a suggested list. Besides the selection of templates, expert users can edit the target-template alignment, thus allowing further customization of the modeling process. Furthermore, the web service provides model stereochemical quality evaluation using PROCHECK. SDPMOD is freely accessible to academic users via the web interface at http://proline.bic.nus.edu.sg/sdpmod.

A genome-wide CRISPR library for high-throughput genetic screening in Drosophila cells.

The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines, enabling screening for cellular phenotypes resulting from genetic aberrations. Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi. This is in part due to the lower degree of redundancy between genes in this organism, whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes. The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques, but allows analysis over longer periods of time which can be critical for certain phenotypes. In this study, we have designed and built a genome-wide CRISPR library covering 13,501 genes, among which 8989 genes are targeted by three or more independent single guide RNAs (sgRNAs). Moreover, we describe strategies to monitor the population of guide RNAs by high throughput sequencing (HTS). We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes, and as a source of guide RNA designs for future studies.

The long non-coding RNA Dali is an epigenetic regulator of neural differentiation.

Many intergenic long noncoding RNA (lncRNA) loci regulate the expression of adjacent protein coding genes. Less clear is whether intergenic lncRNAs commonly regulate transcription by modulating chromatin at genomically distant loci. Here, we report both genomically local and distal RNA-dependent roles of Dali, a conserved central nervous system expressed intergenic lncRNA. Dali is transcribed downstream of the Pou3f3 transcription factor gene and its depletion disrupts the differentiation of neuroblastoma cells. Locally, Dali transcript regulates transcription of the Pou3f3 locus. Distally, it preferentially targets active promoters and regulates expression of neural differentiation genes, in part through physical association with the POU3F3 protein. Dali interacts with the DNMT1 DNA methyltransferase in mouse and human and regulates DNA methylation status of CpG island-associated promoters in trans. These results demonstrate, for the first time, that a single intergenic lncRNA controls the activity and methylation of genomically distal regulatory elements to modulate large-scale transcriptional programmes.

Two Antarctic penguin genomes reveal insights into their evolutionary history and molecular changes related to the Antarctic environment.

BACKGROUND: Penguins are flightless aquatic birds widely distributed in the Southern Hemisphere. The distinctive morphological and physiological features of penguins allow them to live an aquatic life, and some of them have successfully adapted to the hostile environments in Antarctica. To study the phylogenetic and population history of penguins and the molecular basis of their adaptations to Antarctica, we sequenced the genomes of the two Antarctic dwelling penguin species, the Adélie penguin [Pygoscelis adeliae] and emperor penguin [Aptenodytes forsteri]. RESULTS: Phylogenetic dating suggests that early penguins arose ~60 million years ago, coinciding with a period of global warming. Analysis of effective population sizes reveals that the two penguin species experienced population expansions from ~1 million years ago to ~100 thousand years ago, but responded differently to the climatic cooling of the last glacial period. Comparative genomic analyses with other available avian genomes identified molecular changes in genes related to epidermal structure, phototransduction, lipid metabolism, and forelimb morphology. CONCLUSIONS: Our sequencing and initial analyses of the first two penguin genomes provide insights into the timing of penguin origin, fluctuations in effective population sizes of the two penguin species over the past 10 million years, and the potential associations between these biological patterns and global climate change. The molecular changes compared with other avian genomes reflect both shared and diverse adaptations of the two penguin species to the Antarctic environment.

Rapid evolution of Beta-keratin genes contribute to phenotypic differences that distinguish turtles and birds from other reptiles.

Sequencing of vertebrate genomes permits changes in distinct protein families, including gene gains and losses, to be ascribed to lineage-specific phenotypes. A prominent example of this is the large-scale duplication of beta-keratin genes in the ancestors of birds, which was crucial to the subsequent evolution of their beaks, claws, and feathers. Evidence suggests that the shell of Pseudomys nelsoni contains at least 16 beta-keratins proteins, but it is unknown whether this is a complete set and whether their corresponding genes are orthologous to avian beak, claw, or feather beta-keratin genes. To address these issues and to better understand the evolution of the turtle shell at a molecular level, we surveyed the diversity of beta-keratin genes from the genome assemblies of three turtles, Chrysemys picta, Pelodiscus sinensis, and Chelonia mydas, which together represent over 160 Myr of chelonian evolution. For these three turtles, we found 200 beta-keratins, which indicate that, as for birds, a large expansion of beta-keratin genes in turtles occurred concomitantly with the evolution of a unique phenotype, namely, their plastron and carapace. Phylogenetic reconstruction of beta-keratin gene evolution suggests that separate waves of gene duplication within a single genomic location gave rise to scales, claws, and feathers in birds, and independently the scutes of the shell in turtles.

The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage.

BACKGROUND: We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing. RESULTS: Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented. CONCLUSIONS: Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.

Evidence for conserved post-transcriptional roles of unitary pseudogenes and for frequent bifunctionality of mRNAs.

BACKGROUND: Recent reports have highlighted instances of mRNAs that, in addition to coding for protein, regulate the abundance of related transcripts by altering microRNA availability. These two mRNA roles - one mediated by RNA and the other by protein - are inter-dependent and hence cannot easily be separated. Whether the RNA-mediated role of transcripts is important, per se, or whether it is a relatively innocuous consequence of competition by different transcripts for microRNA binding remains unknown. RESULTS: Here we took advantage of 48 loci that encoded proteins in the earliest eutherian ancestor, but whose protein-coding capability has since been lost specifically during rodent evolution. Sixty-five percent of such loci, which we term 'unitary pseudogenes', have retained their expression in mouse and their transcripts exhibit conserved tissue expression profiles. The maintenance of these unitary pseudogenes' spatial expression profiles is associated with conservation of their microRNA response elements and these appear to preserve the post-transcriptional roles of their protein-coding ancestor. We used mouse Pbcas4, an exemplar of these transcribed unitary pseudogenes, to experimentally test our genome-wide predictions. We demonstrate that the role of Pbcas4 as a competitive endogenous RNA has been conserved and has outlived its ancestral gene's loss of protein-coding potential. CONCLUSIONS: These results show that post-transcriptional regulation by bifunctional mRNAs can persist over long evolutionary time periods even after their protein coding ability has been lost.

REPORT OF A BLOOM-FORMING DINOFLAGELLATE TAKAYAMAACROTROCHA FROM TROPICAL COASTAL WATERS OF SINGAPORE(1).

Four clonal cultures of the unarmored dinoflagellate Takayama acrotrocha (J. Larsen) de Salas, Bolch et Hallegraeff were established from Singapore coastal water on October 20, 2004, and January 1, 2007, for a HAB monitoring project. LM and SEM observations demonstrated that the isolates were not consistent with the other five species within this genus in position of nucleus, shape of the apical groove, and number and shape of chloroplasts. New morphological observations of the Singapore isolates that were not in the type description of T. acrotrocha include a narrow and shallow slit located above the entire anterior edge of the cingulum, a tube-like structure in the sulcus, numerous multilateral plate-like surface vesicles, a sulcal intrusion into the epicone, and possibly a peduncle in between the two emerging points of flagella. The presence of sulcal intrusion into the epicone was not consistent with the type description but is prominent in SEM micrographs. Phylogenetic analysis of the partial LSU rDNA sequences indicated Singapore strains of T. acrotrocha are conspecific with two isolates from Italy, but less homologous to T. helix, T. tasmanica, and T. tuberculata. Laboratory fish bioassays using Asian sea bass (Lates calcarifer) and sheepshead minnows (Cyprinodon variegates) did not indicate fish-killing activity by this species, and to our knowledge, there were no reports of fish-kills occurring during blooms of this species in Singapore and Italy. This is the first report of T. acrotrocha from tropical waters and indicates a likely cosmopolitan distribution of the species.

Morphological variation and phylogenetic analysis of the dinoflagellate Gymnodinium aureolum from a tributary of Chesapeake Bay.

Cultures of four strains of the dinoflagellate Gymnodinium aureolum (Hulburt) G. Hansen were established from the Elizabeth River, a tidal tributary of the Chesapeake Bay, USA. Light microscopy, scanning electron microscopy, nuclear-encoded large sub-unit rDNA sequencing, and culturing observations were conducted to further characterize this species. Observations of morphology included: a multiple structured apical groove; a peduncle located between the emerging points of the two flagella; pentagonal and hexagonal vesicles on the amphiesma; production and germination of resting cysts; variation in the location of the nucleus within the center of the cell; a longitudinal ventral concavity; and considerable variation in cell width/length and overall cell size. A fish bioassay using juvenile sheepshead minnows detected no ichthyotoxicity from any of the strains over a 48-h period. Molecular analysis confirmed the dinoflagellate was conspecific with G. aureolum strains from around the world, and formed a cluster along with several other Gymnodinium species. Morphological evidence suggests that further research is necessary to examine the relationship between G. aureolum and a possibly closely related species Gymnodinium maguelonnense.

Mutation of a conserved asparagine in the I-like domain promotes constitutively active integrins alphaLbeta2 and alphaIIbbeta3.

The leukocyte beta2 integrins are heterodimeric adhesion receptors required for a functional immune system. Many leukocyte adhesion deficiency-1 (LAD-1) mutations disrupt the expression and function of beta2 integrins. Herein, we further characterized the LAD-1 mutation N329S in the beta2 inserted (I)-like domain. This mutation converted alphaLbeta2 from a resting into a high affinity conformer because alphaLbeta2N329S transfectants adhered avidly to ligand intercellular adhesion molecule (ICAM)-3 in the absence of additional activating agent. An extended open conformation is adopted by alphaLbeta2N329S because of its reactivity with the beta2 activation reporter monoclonal antibodies MEM148 and KIM127. A corresponding mutation in beta3 generated constitutively active alphaIIbbeta3 that adhered to fibrinogen. This Asn is conserved in all human beta subunits, and it resides before the last helix of the I-like domain, which is known to be important in activation signal propagation. By mutagenesis studies and review of existing integrin structures, we conjectured that this conserved Asn may have a primary role in shaping the I-like domain by stabilizing the conformation of the alpha7 helix and the beta6-alpha7 loop in the I-like domain.

Down-regulation of integrin alpha M beta 2 ligand-binding function by the urokinase-type plasminogen activator receptor.

The cell adhesion molecule integrin alphaMbeta2 associates with the urokinase-type plasminogen activator receptor (uPAR) on monocytes and neutrophils. uPAR also associates with members of the beta1 and beta3 integrins, and it modulates the ligand-binding function of these integrins. In this study, we showed that co-expressing uPAR with alphaMbeta2 in 293 transfectants down-regulated the ligand-binding capacity of alphaMbeta2 to denatured protein, fibrinogen, and intercellular adhesion molecule 1 (ICAM-1). Migration of transfectants on fibrinogen mediated by alphaMbeta2 was reduced in the presence of uPAR. In addition, the constitutive ligand-binding property of an alphaMbeta2 mutant was attenuated by its association with uPAR. Co-immunoprecipitation analyses using a panel of alphaMbeta2-specific mAbs suggest shielding of the ligand-recognition site of alphaMbeta2 by uPAR.

Delineation of modular proteins: domain boundary prediction from sequence information.

The delineation of domain boundaries of a given sequence in the absence of known 3D structures or detectable sequence homology to known domains benefits many areas in protein science, such as protein engineering, protein 3D structure determination and protein structure prediction. With the exponential growth of newly determined sequences, our ability to predict domain boundaries rapidly and accurately from sequence information alone is both essential and critical from the viewpoint of gene function annotation. Anyone attempting to predict domain boundaries for a single protein sequence is invariably confronted with a plethora of databases that contain boundary information available from the internet and a variety of methods for domain boundary prediction. How are these derived and how well do they work? What definition of 'domain' do they use? We will first clarify the different definitions of protein domains, and then describe the available public databases with domain boundary information. Finally, we will review existing domain boundary prediction methods and discuss their strengths and weaknesses.