Evolutionary Sequence Conservation Research Articles

Networks of intergenic long-range enhancers and snpRNAs drive castration-resistant phenotype of prostate cancer and contribute to pathogenesis of multiple common human disorders

AbstractBiological and mechanistic relevance of intergenic disease-associated genetic loci (IDAGL) containing highly statistically significant disease-linked SNPs remains unknown. Here we present the experimental and clinical evidence revealing important role of IDAGL in human diseases. Targeted RT-PCR screen coupled with sequencing of purified PCR products detects widespread transcription at multiple intergenic disease-associated genomic loci (IDAGL) and identifies 96 small non-coding trans-regulatory RNAs of ~ 100-300 nt in length containing SNPs associated with 21 common human disorders (snpRNAs). Functionality of snpRNAs is supported by multiple independent lines of experimental evidence demonstrating their cell-type-specific expression and evolutionary conservation of sequences, genomic coordinates, and biological effects. Analysis of chromatin state signatures, expression profiling experiments using microarray and Q-PCR technologies, and luciferase reporter assays indicate that many IDAGL are Polycomb-regulated long-range enhancers. Expression of snpRNAs in human and mouse cells markedly affects cellular behavior and induces allele-specific clinically-relevant phenotypic changes: NLRP1-locus snpRNAs exert regulatory effects on monocyte/macrophage trans-differentiation, induce prostate cancer (PC) susceptibility snpRNAs, and transform low-malignancy hormone-dependent human PC cells into highly malignant androgen-independent PC. Q-PCR analysis and luciferase reporter assays demonstrate that snpRNA sequences represent allele-specific “decoy” targets of microRNAs which function as SNP-allele-specific modifiers of microRNA expression and activity. We demonstrate that trans-acting RNA molecules facilitating androgen depletion-independent growth (ADIG) in vitro and castration-resistant (CR) phenotype in vivo of PC contain intergenic 8q24-locus SNP variants which were recently linked with increased risk of developing PC. Expression level of 8q24-locus PC susceptibility snpRNAs is regulated by NLRP1-locus snpRNAs, which are transcribed from the intergenic long-range enhancer sequence located in 17p13 region at ~ 30 kb distance from the NLRP1 gene. Q-PCR analysis of clinical PC samples reveals markedly increased snpRNA expression levels in tumor tissues compared to the adjacent normal prostate [122-fold and 45-fold in Gleason 7 tumors (p = 0.03); 370-fold and 127-fold in Gleason 8 tumors (p = 0.0001); for NLRP1-locus and 8q24-locus SnpRNAs, respectively]. Highly concordant expression profiles of the NLRP1-locus snpRNAs and 8q24 CR-locus snpRNAs (r = 0.896; p < 0.0001) in clinical PC samples and experimental evidence of trans-regulatory effects of NLRP1-locus snpRNAs on expression of 8q24-locus SnpRNAs indicate that ADIG and CR phenotype of human PC cells can be triggered by RNA molecules transcribed from the NLRP1-locus intergenic enhancer and down-stream activation of the 8q24-locus snpRNAs. Our results define the intergenic NLRP1 and 8q24 regions as regulatory loci of ADIG and CR phenotype of human PC, reveal previously unknown molecular links between the innate immunity/inflammasome system and development of hormone-independent PC, and identify novel diagnostic and therapeutic targets exploration of which should be highly beneficial for clinical management of PC.

The long pentraxin PTX3 at the crossroads between innate immunity and tissue remodelling

Innate immunity represents the first line of defence against pathogens and plays key roles in the activation and orientation of the adaptive immune response. The innate immune system comprises both a cellular and a humoral arm. Components of the humoral arm include soluble pattern recognition molecules that recognize pathogen-associated molecular patterns and initiate the immune response in coordination with the cellular arm, therefore acting as functional ancestors of antibodies. Pentraxins are essential constituents of the humoral arm of innate immunity and represent a superfamily of highly conserved acute phase proteins, traditionally classified into short and long pentraxins. Pentraxin 3 (PTX3) is the prototypic member of the long pentraxins subfamily. As opposed to C-reactive protein, whose sequence and regulation have not been conserved during evolution from mouse to man, the evolutionary conservation of sequence, gene organization and regulation of PTX3 has allowed addressing its pathophysiological roles in genetically modified mice, in diverse conditions, ranging from infections to sterile inflammation, angiogenesis and female fertility. Despite this conservation, a number of predominantly non-coding polymorphisms have been identified in the PTX3 gene which, when associated in particular haplotypes, have been shown to be relevant in clinical conditions including infection and fertility. Here we review the studies on PTX3, with emphasis on pathogen recognition, tissue remodelling and crosstalk with other components of the innate immune system.

Thermodynamic Stability of Histone H3 is a Necessary but not Sufficient Driving Force for its Evolutionary Conservation

Determining the forces that drive evolutionary conservation of amino acid sequences of proteins has been a fundamental pursuit of molecular evolution. In the absence of any other function, it is thought that the core domains of proteins are conserved to maintain the thermodynamic stability of the protein. We have sought to test this idea further using the strongly conserved buried core of histone proteins H3 and H4. Consistent with previous notions, we observed a strong correlation between survival fitness in yeast and nucleosome thermodynamic stability. However, in striking contrast to what is normally observed, we find that thermodynamic stability of buried histone H3 residues is not correlated with evolutionary conservation. Thus, H3 is fundamentally different in comparison to many proteins studied to date, including highly conserved proteins like actin, tubulin and histone H4. The discovery of the uniqueness in conservation of H3 goes against the conventional view that buried H3 residues are highly conserved just to maintain the stability of the nucleosome. This discovery thus points to either new additional functions of histone H3 that have not been uncovered or a unique conservation mechanism that goes beyond survival pressure.

Structure, Conformational Dynamics, and Evolutionary Conservation of Human Fascin-1

Cells express a range of actin-binding proteins to crosslink filamentous actin (F-actin) into compact ordered bundles ranging from stereocilia and microvilli to filopodia. Human fascin-1 is found nearly exclusively in filopodia, which protrude from the leading edge of migrating cells in normal and cancerous cells. Previously, we and others have found that fascin-1 confers unusual mechanical integrity to these actin bundles, which play a role in sensing the extracellular environment. Here, we examine the packing, conformational flexibility, and evolutionary sequence conservation of full length Homo sapiens fascin-1. Unlike the ubiquitous crosslinking proteins fimbrin and alpha-actinin that consist of dual calponin homology domain pairs, fascin-1 consists of four β-trefoil domains organized into a double-lobe-like structure. Sequence analysis suggests that these β-trefoil domains are stabilized by bulky hydrophobic residues in the core of each domain, and that interfacial residues between the two lobes of fascin play an important role in stabilizing the molecule. Conformational dynamics analysis reveals an allosteric coupling between highly conserved surface patches near the putative actin-binding sites of the molecule, with potential implications on its ability to crosslink F-actin tightly and confer unusual mechanical properties to F-actin bundles. Future mutational and structural studies motivated by this work will further elucidate the molecular basis for the unique function of fascin in human cells.

Prediction of regulatory networks in mouse abdominal wall

Sequence specific transcription factors are essential for pattern formation and cell differentiation processes in mammals. The formation of the abdominal wall depends on a flawless merge of several developmental fields in time and space. The absence of Pitx2 leads to an open abdominal wall in mice, while mutations in humans result in umbilical defects, suggesting that a single homeobox transcription factor coordinates the formation and patterning of this anatomical structure. Gene expression analysis from abdominal tissue including the abdominal wall after removal of the major organs, of wild type, Pitx2 heterozygote and mutant mice, at embryonic day 10.5, identified 275 genes with altered expression levels. Pitx2 target genes were clustered using the “David Bioinformatics Functional Annotation Tool” web application, which bins genes according to gene ontology (GO) key word enrichment. This provided a way to both narrow the target gene list and to start identifying potential gene families regulated by Pitx2. Target genes in the most enriched bins were further analyzed for the presence and the evolutionary conservation of Pitx2 consensus binding sequence, TAATCY, on the − 20 kb, intronic and coding gene sequences. Twenty Pitx2 target genes that passed all the above criteria were classified as genes involved in cell transport and growth. Data from these studies suggest that Pitx2 acts as an inhibitor of protein transport and cell apoptosis contributing to the open body wall phenotype. This work provides the framework to which the developmental network leading to abdominal wall syndromes can be built.

Muscleblind-like 1 (Mbnl1) Promotes Insulin Receptor Exon 11 Inclusion via Binding to a Downstream Evolutionarily Conserved Intronic Enhancer

The insulin receptor exists as two isoforms, IR-A and IR-B, which result from alternative splicing of exon 11 in the primary transcript. These two isoforms show a cell-specific distribution, and their relative proportions also vary during development, aging, and in different disease states. We have previously demonstrated that both intron 10 and the alternatively spliced exon 11 contain regulatory sequences that affect insulin receptor splicing both positively and negatively and that these sequences bind the serine/arginine-rich (SR) proteins SRp20 and SF2/ASF and the CELF protein CUG-BP1. In this study, we describe a new intronic splicing element within intron 11 that is highly conserved across species. Using minigenes carrying deletion mutations within intron 11, we demonstrated that this sequence functions as an intronic splicing enhancer. We subsequently used RNA affinity chromatography to identify Mbnl1 as a splicing factor that recognizes this enhancer. By ribonucleoprotein immunoprecipitation, we also established that Mbnl1 binds specifically to the INSR (insulin receptor gene) RNA. Overexpression or knockdown of Mbnl1 in hepatoma and embryonic kidney cells altered the levels of exon 11 inclusion. Finally, we showed that deletion of the intronic enhancer eliminates the ability of Mbnl1 to promote exon inclusion. Collectively, these findings demonstrate a role for Mbnl1 in controlling insulin receptor exon 11 inclusion via binding to a downstream intronic enhancer element.

Effect of pseudouridylation on the structure and activity of the catalytically essential P6.1 hairpin in human telomerase RNA

Telomerase extends the 3′-ends of linear chromosomes by adding conserved telomeric DNA repeats and is essential for cell proliferation and genomic stability. Telomerases from all organisms contain a telomerase reverse transcriptase and a telomerase RNA (TER), which together provide the minimal functional elements for catalytic activity in vitro. The RNA component of many functional ribonucleoproteins contains modified nucleotides, including conserved pseudouridines (Ψs) that can have subtle effects on structure and activity. We have identified potential Ψ modification sites in human TER. Two of the predicted Ψs are located in the loop of the essential P6.1 hairpin from the CR4-CR5 domain that is critical for telomerase catalytic activity. We investigated the effect of P6.1 pseudouridylation on its solution NMR structure, thermodynamic stability of folding and telomerase activation in vitro. The pseudouridylated P6.1 has a significantly different loop structure and increase in stability compared to the unmodified P6.1. The extent of loop nucleotide interaction with adjacent residues more closely parallels the extent of loop nucleotide evolutionary sequence conservation in the Ψ-modified P6.1 structure. Pseudouridine-modification of P6.1 slightly attenuates telomerase activity but slightly increases processivity in vitro. Our results suggest that Ψs could have a subtle influence on human telomerase activity via impact on TER–TERT or TER–TER interactions.

Decoding accuracy in eRF1 mutants and its correlation with pleiotropic quantitative traits in yeast.

Translation termination in eukaryotes typically requires the decoding of one of three stop codons UAA, UAG or UGA by the eukaryotic release factor eRF1. The molecular mechanisms that allow eRF1 to decode either A or G in the second nucleotide, but to exclude UGG as a stop codon, are currently not well understood. Several models of stop codon recognition have been developed on the basis of evidence from mutagenesis studies, as well as studies on the evolutionary sequence conservation of eRF1. We show here that point mutants of Saccharomyces cerevisiae eRF1 display significant variability in their stop codon read-through phenotypes depending on the background genotype of the strain used, and that evolutionary conservation of amino acids in eRF1 is only a poor indicator of the functional importance of individual residues in translation termination. We further show that many phenotypes associated with eRF1 mutants are quantitatively unlinked with translation termination defects, suggesting that the evolutionary history of eRF1 was shaped by a complex set of molecular functions in addition to translation termination. We reassess current models of stop-codon recognition by eRF1 in the light of these new data.

Structure, Evolutionary Conservation, and Conformational Dynamics of Homo sapiens Fascin-1, an F-actin Crosslinking Protein

Eukaryotes have several highly conserved actin-binding proteins that crosslink filamentous actin into compact ordered bundles present in distinct cytoskeletal processes, including microvilli, stereocilia and filopodia. Fascin is an actin-binding protein that is present predominantly in filopodia, which are believed to play a central role in normal and aberrant cell migration. An important outstanding question regards the molecular basis for the unique localization and functional properties of fascin compared with other actin crosslinking proteins. Here, we present the crystal structure of full-length Homo sapiens fascin-1, and examine its packing, conformational flexibility, and evolutionary sequence conservation. The structure reveals a novel arrangement of four tandem β-trefoil domains that form a bi-lobed structure with approximate pseudo 2-fold symmetry. Each lobe has internal approximate pseudo 2-fold and pseudo 3-fold symmetry axes that are approximately perpendicular, with β-hairpin triplets located symmetrically on opposite sides of each lobe that mutational data suggest are actin-binding domains. Sequence conservation analysis confirms the importance of hydrophobic core residues that stabilize the β-trefoil fold, as well as interfacial residues that are likely to stabilize the overall fascin molecule. Sequence conservation also indicates highly conserved surface patches near the putative actin-binding domains of fascin, which conformational dynamics analysis suggests to be coupled via an allosteric mechanism that might have important functional implications for F-actin crosslinking by fascin.

Regulation of Synaptic Pumilio Function by an Aggregation-Prone Domain

We identified Pumilio (Pum), a Drosophila translational repressor, in a computational search for metazoan proteins whose activities might be regulated by assembly into ordered aggregates. The search algorithm was based on evolutionary sequence conservation patterns observed for yeast prion proteins, which contain aggregation-prone glutamine/asparagine (Q/N)-rich domains attached to functional domains of normal amino acid composition. We examined aggregation of Pum and its nematode ortholog PUF-9 by expression in yeast. A domain of Pum containing the Q/N-rich sequence, denoted as NQ1, the entire Pum N terminus, and the complete PUF-9 protein localize to macroscopic aggregates (foci) in yeast. NQ1 and PUF-9 can generate the yeast Pin+ trait, which is transmitted by a heritable aggregate. NQ1 also assembles into amyloid fibrils in vitro. In Drosophila, Pum regulates postsynaptic translation at neuromuscular junctions (NMJs). To assess whether NQ1 affects synaptic Pum activity in vivo, we expressed it in muscles. We found that it negatively regulates endogenous Pum, producing gene dosage-dependent pum loss-of-function NMJ phenotypes. NQ1 coexpression also suppresses lethality and NMJ phenotypes caused by overexpression of Pum in muscles. The Q/N block of NQ1 is required for these phenotypic effects. Negative regulation of Pum by NQ1 might be explained by formation of inactive aggregates, but we have been unable to demonstrate that NQ1 aggregates in Drosophila. NQ1 could also regulate Pum by a "dominant-negative" effect, in which it would block Q/N-mediated interactions of Pum with itself or with cofactors required for translational repression.

Predicting RNA-binding residues from evolutionary information and sequence conservation

BackgroundRNA-binding proteins (RBPs) play crucial roles in post-transcriptional control of RNA. RBPs are designed to efficiently recognize specific RNA sequences after it is derived from the DNA sequence. To satisfy diverse functional requirements, RNA binding proteins are composed of multiple blocks of RNA-binding domains (RBDs) presented in various structural arrangements to provide versatile functions. The ability to computationally predict RNA-binding residues in a RNA-binding protein can help biologists reveal important site-directed mutagenesis in wet-lab experiments.ResultsThe proposed prediction framework named “ProteRNA” combines a SVM-based classifier with conserved residue discovery by WildSpan to identify the residues that interact with RNA in a RNA-binding protein. Although these conserved residues can be either functionally conserved residues or structurally conserved residues, they provide clues on the important residues in a protein sequence. In the independent testing dataset, ProteRNA has been able to deliver overall accuracy of 89.78%, MCC of 0.2628, F-score of 0.3075, and F0.5-score of 0.3546.ConclusionsThis article presents the design of a sequence-based predictor aiming to identify the RNA-binding residues in a RNA-binding protein by combining machine learning and pattern mining approaches. RNA-binding proteins have diverse functions while interacting with different categories of RNAs because these proteins are composed of multiple copies of RNA-binding domains presented in various structural arrangements to expand the functional repertoire of RNA-binding proteins. Furthermore, predicting RNA-binding residues in a RNA-binding protein can help biologists reveal important site-directed mutagenesis in wet-lab experiments.

Predicting Protein Ligand Binding Sites by Combining Evolutionary Sequence Conservation and 3D Structure

Identifying a protein's functional sites is an important step towards characterizing its molecular function. Numerous structure- and sequence-based methods have been developed for this problem. Here we introduce ConCavity, a small molecule binding site prediction algorithm that integrates evolutionary sequence conservation estimates with structure-based methods for identifying protein surface cavities. In large-scale testing on a diverse set of single- and multi-chain protein structures, we show that ConCavity substantially outperforms existing methods for identifying both 3D ligand binding pockets and individual ligand binding residues. As part of our testing, we perform one of the first direct comparisons of conservation-based and structure-based methods. We find that the two approaches provide largely complementary information, which can be combined to improve upon either approach alone. We also demonstrate that ConCavity has state-of-the-art performance in predicting catalytic sites and drug binding pockets. Overall, the algorithms and analysis presented here significantly improve our ability to identify ligand binding sites and further advance our understanding of the relationship between evolutionary sequence conservation and structural and functional attributes of proteins. Data, source code, and prediction visualizations are available on the ConCavity web site (http://compbio.cs.princeton.edu/concavity/).

Small RNA analysis in Petunia hybrida identifies unusual tissue-specific expression patterns of conserved miRNAs and of a 24mer RNA

Two pools of small RNAs were cloned from inflorescences of Petunia hybrida using a 5'-ligation dependent and a 5'-ligation independent approach. The two libraries were integrated into a public website that allows the screening of individual sequences against 359,769 unique clones. The library contains 15 clones with 100% identity and 53 clones with one mismatch to miRNAs described for other plant species. For two conserved miRNAs, miR159 and miR390, we find clear differences in tissue-specific distribution, compared with other species. This shows that evolutionary conservation of miRNA sequences does not necessarily include a conservation of the miRNA expression profile. Almost 60% of all clones in the database are 24-nucleotide clones. In accordance with the role of 24mers in marking repetitive regions, we find them distributed across retroviral and transposable element sequences but other 24mers map to promoter regions and to different transcript regions. For one target region we observe tissue-specific variation of matching 24mers, which demonstrates that, as for 21mers, 24mer concentrations are not necessarily identical in different tissues. Asymmetric distribution of a putative novel miRNA in the two libraries suggests that the cloning method can be selective for the representation of certain small RNAs in a collection.

Molecular characterization and expression of a novel homolog of uncoupling protein 5 (UCP5) from the eastern oyster Crassostrea virginica (Bivalvia: Ostreidae)

Uncoupling proteins (UCPs) belong to the mitochondrial anion carrier gene family which has been implicated in diverse physiological functions ranging from thermoregulation to antioxidant defense. In mammals, the UCP family is well characterized and contains five members (UCP1-5). In contrast, invertebrate homologues of uncoupling proteins are much less studied both from the viewpoints of structure and function. In this study we report nucleotide and predicted protein structure of an important member of UCP family, UCP5 from eastern oysters Crassostrea virginica. UCP5 from oysters appears to be a close homolog of the mammalian brain mitochondrial carrier protein (BMCP1, or UCP5) and is the first full-length UCP described from a Lophotrochozoan invertebrate. Evolutionary analysis of UCP sequences indicates at least three monophyletic UCP branches (UCP1-3, UCP4 and UCP5) that have diverged early in the evolution, prior to the divergence of vertebrates and invertebrates. In oysters, two forms of UCP5 transcript are found (UCP5S and UCP5L) that differ by 152 bp in length due to the presence of an intron in UCP5L. UCP5 was expressed in all studied oyster tissues, unlike mammals, where UCP5 is predominantly expressed in brains and male gonads. Hypoxia-reoxygenation stress, sublethal Cd exposure (50 μg L − 1 Cd for 56 days) and acclimation to different temperatures (12 and 20 °C) had no significant effect on UCP5 mRNA expression in oysters indicative of its relative unimportance in antioxidant defense and temperature adaptation of oyster mitochondria. These data suggest that despite the relatively high degree of evolutionary conservation of the UCP5 amino acid sequence, its functional significance in mitochondria changed in the course of evolution of mollusks and vertebrates.

Reciprocal regulation of glycine-rich RNA-binding proteins via an interlocked feedback loop coupling alternative splicing to nonsense-mediated decay in Arabidopsis

The Arabidopsis RNA-binding protein AtGRP8 undergoes negative autoregulation at the post-transcriptional level. An elevated AtGRP8 protein level promotes the use of a cryptic 5′ splice site to generate an alternatively spliced transcript, as_AtGRP8, retaining the 5′ half of the intron with a premature termination codon. In mutants defective in nonsense-mediated decay (NMD) abundance of as_AtGRP8 but not its pre-mRNA is elevated, indicating that as_AtGRP8 is a direct NMD target, thus limiting the production of functional AtGRP8 protein. In addition to its own pre-mRNA, AtGRP8 negatively regulates the AtGRP7 transcript through promoting the formation of the equivalent alternatively spliced as_AtGRP7 transcript, leading to a decrease in AtGRP7 abundance. Recombinant AtGRP8 binds to its own and the AtGRP7 pre-mRNA, suggesting that this interaction is relevant for the splicing decision in vivo. AtGRP7 itself is part of a negative autoregulatory circuit that influences circadian oscillations of its own and the AtGRP8 transcript through alternative splicing linked to NMD. Thus, we identify an interlocked feedback loop through which two RNA-binding proteins autoregulate and reciprocally crossregulate by coupling unproductive splicing to NMD. A high degree of evolutionary sequence conservation in the introns retained in as_AtGRP8 or as_AtGRP7 points to an important function of these sequences.

S12.18 A genetic system to study mitochondrial DNA mutations and their propagation in mice

The number of mutations in mitochondrial DNA (mtDNA) thought to causemetabolic disorders in patients is growing. At present, we lack the technology to introduce these mutations into the mtDNA of model organisms for detailed study. In this study, random mtDNA mutations were generated using the transgenic mtDNA mutator mouse, which expresses an engineered mitochondrial polymerase defective in its proofreading function. Female lines were then established to transmit and segregate the resulting mutations. We observed the selective loss of nonsense mutations in the protein coding genes, consistent with strong purifying selection by the female germline. The selection was evident two generations after the founder mtDNA mutator mouse, eliminating these nonsense mutations before they were detectable by Sanger sequencing methods. Curiously, the tRNA and rRNA genes do not show the same rapid selection against mutations despite strong evolutionary sequence conservation. Despite this purifying selection, putative deleterious mutations were identified and propagated in the mouse lines. Also, mutations analogous to human pathogenic mtDNA mutations were identified. These results will aid in the generation of more accurate models of inheritance of disease-causing mtDNA mutations and allow for the generation of laboratory mouse models of mtDNA mutations known in human disease.

Fast comparison of DNA sequences by oligonucleotide profiling

BackgroundThe comparison of DNA sequences is a traditional problem in genomics and bioinformatics. Many new opportunities emerge due to the improvement of personal computers, allowing the implementation of novel strategies of analysis.FindingsWe describe a new program, called UVWORD, which determines the number of times that each DNA word present in a sequence (target) is found in a second sequence (source), a procedure that we have called oligonucleotide profiling. On a standard computer, the user may search for words of a size ranging from k = 1 to k = 14 nucleotides. Average counts for groups of contiguous words may also be established. The rate of analysis on standard computers is from 3.4 (k = 14) to 16 millions of words per second (1 ≤ k ≤ 8). This makes feasible the fast screening of even the longest known DNA molecules.DiscussionWe show that the combination of the ability of analyzing words of relatively long size, which occur very rarely by chance, and the fast speed of the program allows to perform novel types of screenings, complementary to those provided by standard programs such as BLAST. This method can be used to determine oligonucleotide content, to characterize the distribution of repetitive sequences in chromosomes, to determine the evolutionary conservation of sequences in different species, to establish regions of similar DNA among chromosomes or genomes, etc.

Amelogenin proteins of developing dental enamel.

The amelogenins of developing dental enamel are tissue-specific proteins, rich in proline, leucine, histidine and glutamyl residues, and synthesized by the ameloblast cells of the inner enamel epithelium. These proteins comprise the bulk of the extracellular matrix that becomes mineralized with a hydroxyapatite phase to become the mature enamel. Examination of the amino acid sequences of amelogenins from a range of mammals shows a high degree of evolutionary sequence conservation, suggestive of specialized function. Recently it has been shown that multiple amelogenin components, observed in the matrix, arise both by a sequence of post-secretory proteolytic processing and by the expression of alternatively spliced mRNAs generated from the amelogenin gene(s) that are located on the sex chromosomes. Although the function of these amelogenins in enamel biomineralization is unknown, physico-chemical studies of recombinant amelogenins have shown that they undergo a self-assembly process in vitro generating supra-molecular 'nanosphere' structures, and recent observations in vivo point to a functional role for the nanospheres in the ultrastructural organization of the secretory enamel matrix, conducive to the organized development of the earliest mineral crystallites.

Comparative genomics of transcription factors driving expression of the immunoglobulin heavy chain locus in teleost fish

The immunoglobulin heavy chain locus of teleost fish is driven by an enhancer (Eμ3′) that contains, in all species examined, octamer and E‐box motifs (typically μE5). Thus, the transcription factors known (through experiments in fish) or predicted (from knowledge of their mammalian homologues) to be involved in the function of the enhancer are the octamer‐binding (Oct) transcription factors Oct1 and Oct2, their coactivator BOB.1 and the μE5‐binding E‐proteins (E2A, HEB and E2‐2). Comparative genomic analysis of the genes encoding these transcription factors in teleost fish permits some conclusions to be drawn concerning the fate of these genes following the genome‐wide duplication event that occurred in this lineage. Although problems in the assembly of some regions of the zebrafish genome may complicate the analysis, the pattern that emerges from these studies is that Oct1, Oct2, BOB.1 and E2‐2 are all single‐copy genes in teleosts, whereas in the case of E2A and HEB, two copies of each gene are present. The evolutionary conservation of sequences between related genes is variable, but with the highest similarities always being observed in the domains with ligand‐binding function, whether the ligand is a DNA motif or, as in the case of BOB.1, another protein domain. A major challenge that must be faced is the assignment of precise functions to these diverse transcription factors in teleosts; assumptions drawn by inference from their mammalian homologues will likely be misleading.

Identifying Structural Noncoding RNAs Using RNAz

The functions of many noncoding RNAs and cis-acting regulatory elements of mRNAs depend on a defined RNA secondary structure. RNAz predicts such functional RNA structures on the basis of thermodynamic stability and evolutionary conservation of homologous sequences. It can be used to efficiently filter multiple alignments for noncoding RNA candidates in genomic screens.