Evolutionary Sequence Conservation Research Articles

Tyrosine–heme ligation in heme–peptide complex: design based on conserved motif of catalase

On the basis of evolutionary conservation of sequence in catalases, we have designed a heme-binding peptide (Ac-RLKSYTDTQISR12-(GGGG)-CRIVHC22-NH2) for the 'redox activity modulation' of heme. Heme-binding studies showed a blue-shifted Soret (369 nm) in the presence of TFE and a red-shifted Soret (418 nm) in the absence of TFE. These blue- and red-shifted Sorets suggest ligation through tyrosinate and histidine, respectively. This is the first designed peptide ligating to heme through tyrosine. NMR studies have confirmed that tyrosine ligation to heme in this heme-peptide complex occurs only in the presence of TFE. We suggest that TFE induces helicity in the peptide and brings the arginine and tyrosine in proximity, resulting in ionization of the phenolic side chain of tyrosine. In the absence of TFE, the unstructured peptide lacks the intra-molecular Arg(+)Tyr(-) ion pair, allowing heme binding to histidine. This peptide has significant peroxidase activity though it does not have catalase activity.

Genomix: a method for combining gene-finders' predictions, which uses evolutionary conservation of sequence and intron–exon structure

Correct gene predictions are crucial for most analyses of genomes. However, in the absence of transcript data, gene prediction is still challenging. One way to improve gene-finding accuracy in such genomes is to combine the exons predicted by several gene-finders, so that gene-finders that make uncorrelated errors can correct each other. We present a method for combining gene-finders called Genomix. Genomix selects the predicted exons that are best conserved within and/or between species in terms of sequence and intron-exon structure, and combines them into a gene structure. Genomix was used to combine predictions from four gene-finders for Caenorhabditis elegans, by selecting the predicted exons that are best conserved with C.briggsae and C.remanei. On a set of approximately 1500 confirmed C.elegans genes, Genomix increased the exon-level specificity by 10.1% and sensitivity by 2.7% compared to the best input gene-finder. Scripts and Supplementary Material can be found at http://www.sanger.ac.uk/Software/analysis/genomix

Bio-Informatic Trends for the Determination of miRNA–Target Interactions in Mammals

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate mRNAs through a sequence-specific mechanism. By virtue of their structure and mechanism of action, computational methods have been devised to investigate the encoding of miRNA genes and the targets of miRNA action. A variety of assumptions have predicated the implementation of these various computational solutions. Evolutionary sequence conservation, secondary structure, and folding energetics are some of the assumptions that have been used. The success of these different computational solutions has been evaluated for both elucidation of new miRNAs and deducing targets of miRNA action. While the focus is on search techniques for new miRNAs, we have compared the programs miRseeker, miRScan, PalGrade, ProMiR, and miRAlign as examples of implementation of these techniques. For these programs, a benchmark comparison between theoretical estimation and actual identification is possible. We have also compared the target prediction programs TargetScanS, PicTar, DIANA-microT, miRanda, and RNAhybrid. However, it is difficult to rigorously assess the benchmark performance of these programs due to the difficulty in confirming their theoretical predictions.

Evolutionary conservation of a unique amino acid sequence in human DICER protein essential for binding to Argonaute family proteins

The Argonaute family and DICER proteins are major key proteins involved in the RNA-mediated gene silencing mechanism of various species. In this mechanism, cleavage of messenger RNAs (mRNA) or suppression of mRNA translation takes place via small RNAs that are uniquely processed by DICER. Previously, we demonstrated that human Argonaute family proteins bind to DICER. In this study, we identified a unique amino acid sequence of 127 amino acids in the RIBOc-A domain of human DICER protein as a “binding site” to Argonaute proteins. Comparative genomics analysis revealed that this unique amino acid sequence is highly conserved in the vertebrates, but not found in the non-vertebrate species. Significant difference in the RIBOc-A domain of DICER protein between vertebrate and non-vertebrate species may help exploring the functional complexity in the RNA-mediated gene silencing mechanism.

Structure and Function of the c-myc DNA-unwinding Element-binding Protein DUE-B

Local zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNA-unwinding element-binding protein DUE-B in vivo. We present here the 2.0A crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both D-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA.

Interpreting missense variants: comparing computational methods in human disease genesCDKN2A,MLH1,MSH2,MECP2, and tyrosinase (TYR)

The human genome contains frequent single-basepair variants that may or may not cause genetic disease. To characterize benign vs. pathogenic missense variants, numerous computational algorithms have been developed based on comparative sequence and/or protein structure analysis. We compared computational methods that use evolutionary conservation alone, amino acid (AA) change alone, and a combination of conservation and AA change in predicting the consequences of 254 missense variants in the CDKN2A (n = 92), MLH1 (n = 28), MSH2 (n = 14), MECP2 (n = 30), and tyrosinase (TYR) (n = 90) genes. Variants were validated as either neutral or deleterious by curated locus-specific mutation databases and published functional data. All methods that use evolutionary sequence analysis have comparable overall prediction accuracy (72.9-82.0%). Mutations at codons where the AA is absolutely conserved over a sufficient evolutionary distance (about one-third of variants) had a 91.6 to 96.8% likelihood of being deleterious. Three algorithms (SIFT, PolyPhen, and A-GVGD) that differentiate one variant from another at a given codon did not significantly improve predictive value over conservation score alone using the BLOSUM62 matrix. However, when all four methods were in agreement (62.7% of variants), predictive value improved to 88.1%. These results confirm a high predictive value for methods that use evolutionary sequence conservation, with or without considering protein structural change, to predict the clinical consequences of missense variants. The methods can be generalized across genes that cause different types of genetic disease. The results support the clinical use of computational methods as one tool to help interpret missense variants in genes associated with human genetic disease.

DP-Bind: a web server for sequence-based prediction of DNA-binding residues in DNA-binding proteins

This article describes DP-Bind, a web server for predicting DNA-binding sites in a DNA-binding protein from its amino acid sequence. The web server implements three machine learning methods: support vector machine, kernel logistic regression and penalized logistic regression. Prediction can be performed using either the input sequence alone or an automatically generated profile of evolutionary conservation of the input sequence in the form of PSI-BLAST position-specific scoring matrix (PSSM). PSSM-based kernel logistic regression achieves the accuracy of 77.2%, sensitivity of 76.4% and specificity of 76.6%. The outputs of all three individual methods are combined into a consensus prediction to help identify positions predicted with high level of confidence. Freely available at http://lcg.rit.albany.edu/dp-bind. http://lcg.rit.albany.edu/dp-bind/dpbind_supplement.html.

Evolutionary conservation of sequence and secondary structures in CRISPR repeats

The categorisation and structural analysis of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) sequences from 195 microbial genomes show that repeats from diverse organisms can be grouped based on sequence similarity, and that some groups have pronounced secondary structures with compensatory base changes.

ECRbase: database of evolutionary conserved regions, promoters, and transcription factor binding sites in vertebrate genomes

Evolutionary conservation of DNA sequences provides a tool for the identification of functional elements in genomes. We have created a database of evolutionary conserved regions (ECRs) in vertebrate genomes, entitled ECRbase, which is constructed from a collection of whole-genome alignments produced by the ECR Browser. ECRbase features a database of syntenic blocks that recapitulate the evolution of rearrangements in vertebrates and a comprehensive collection of promoters in all vertebrate genomes generated using multiple sources of gene annotation. The database also contains a collection of annotated transcription factor binding sites (TFBSs) in evolutionary conserved and promoter elements. ECRbase currently includes human, rhesus macaque, dog, opossum, rat, mouse, chicken, frog, zebrafish and fugu genomes. It is freely accessible at http://ecrbase.dcode.org.

In vivo enhancer analysis of human conserved non-coding sequences

Identifying the sequences that direct the spatial and temporal expression of genes and defining their function in vivo remains a significant challenge in the annotation of vertebrate genomes. One major obstacle is the lack of experimentally validated training sets. In this study, we made use of extreme evolutionary sequence conservation as a filter to identify putative gene regulatory elements, and characterized the in vivo enhancer activity of a large group of non-coding elements in the human genome that are conserved in human-pufferfish, Takifugu (Fugu) rubripes, or ultraconserved in human-mouse-rat. We tested 167 of these extremely conserved sequences in a transgenic mouse enhancer assay. Here we report that 45% of these sequences functioned reproducibly as tissue-specific enhancers of gene expression at embryonic day 11.5. While directing expression in a broad range of anatomical structures in the embryo, the majority of the 75 enhancers directed expression to various regions of the developing nervous system. We identified sequence signatures enriched in a subset of these elements that targeted forebrain expression, and used these features to rank all approximately 3,100 non-coding elements in the human genome that are conserved between human and Fugu. The testing of the top predictions in transgenic mice resulted in a threefold enrichment for sequences with forebrain enhancer activity. These data dramatically expand the catalogue of human gene enhancers that have been characterized in vivo, and illustrate the utility of such training sets for a variety of biological applications, including decoding the regulatory vocabulary of the human genome.

Polypeptide binding proteins: what remains to be discovered?

Many proteins have developed the potential to sequester a client polypeptide chain in its various folding states as a specific intermolecular ligand and, thus, exhibit the properties of a holding chaperone. The resulting complexes can be of a diverse nature in terms of structure and reaction dynamics and are characterized on the basis of various microscopic properties including formation and decay of encounter and Michaelis complexes as well as reactant and product stability. Interpretation of the functional consequences of complex formation in the cell generally tends to be rather complicated, with notable exceptions including complexes formed during the reaction pathways of proteases, protein kinases and protein phosphatases. Peptide bond cis/trans isomerases take up an intermediate position among the poly(oligo)peptide binding proteins because, although the relationship between chain sequestration and catalysis of isomerization can easily be delineated in vitro, it is sometimes difficult to resolve in the cell. Time-resolved studies on interactions involving peptide bond cis/trans isomerases have led to the establishment of generally applicable methods for studying protein-poly(oligo)peptide interactions that are capable of identifying new types of biocatalysis.

Toward a Molecular Understanding of Pleiotropy

Pleiotropy refers to the observation of a single gene influencing multiple phenotypic traits. Although pleiotropy is a common phenomenon with broad implications, its molecular basis is unclear. Using functional genomic data of the yeast Saccharomyces cerevisiae, here we show that, compared with genes of low pleiotropy, highly pleiotropic genes participate in more biological processes through distribution of the protein products in more cellular components and involvement in more protein-protein interactions. However, the two groups of genes do not differ in the number of molecular functions or the number of protein domains per gene. Thus, pleiotropy is generally caused by a single molecular function involved in multiple biological processes. We also provide genomewide evidence that the evolutionary conservation of genes and gene sequences positively correlates with the level of gene pleiotropy.

Structural study of elements of Tetrahymena telomerase RNA stem–loop IV domain important for function

Tetrahymena telomerase RNA (TER) contains several regions in addition to the template that are important for function. Central among these is the stem-loop IV domain, which is involved in both catalysis and RNP assembly, and includes binding sites for both the holoenzyme assembly protein p65 and telomerase reverse transcriptase (TERT). Stem-loop IV contains two regions with high evolutionary sequence conservation: a central GA bulge between helices, and a terminal loop. We solved the solution structure of loop IV and modeled the structure of the helical region containing the GA bulge, using NMR and residual dipolar couplings. The central GA bulge with flanking C-G base pairs induces a approximately 50 degrees semi-rigid bend in the helix. Loop IV is highly structured, and contains a conserved C-U base pair at the top of the helical stem. Analysis of new and previous biochemical data in light of the structure provides a rationale for some of the sequence conservation in this region of TER. The results suggest that during holoenzyme assembly the protein p65 recognizes a bend in stem IV, and this binding to central stem IV helps to position the structured loop IV for interaction with TERT and other region(s) of TER.

Structural organization and expression of human MTUS1, a candidate 8p22 tumor suppressor gene encoding a family of angiotensin II AT2 receptor-interacting proteins, ATIP

The Mitochondrial Tumor suppressor 1 ( MTUS1) gene is a newly identified candidate tumor suppressor gene at chromosomal position 8p22. We report here that MTUS1 encodes a family of proteins whose leader member (ATIP1) was previously isolated in our laboratory as a novel interacting partner of the angiotensin II AT2 receptor involved in growth inhibition (Nouet, JBC 279: 28989–97, 2004). The MTUS1 gene contains 17 coding exons distributed over 112 kb of genomic DNA. Alternative exon usage generates three major transcripts (ATIP1, ATIP3 and ATIP4), each showing different tissue distribution. ATIP polypeptides are identical in their carboxy-terminal region carrying four coiled-coil domains. In their amino-terminal portion, ATIP polypeptides exhibit distinct motifs for localisation in the cytosol, nucleus or cell membrane, suggesting that MTUS1 gene products may be involved in a variety of intracellular functions in an AT2-dependent and independent manner. ATIP1 is ubiquitous and highly expressed in the brain. ATIP3 is the major transcript in tissues (prostate, bladder, breast, ovary, colon) corresponding to cancer types with frequent loss of heterozygosity at 8p22. Interestingly, ATIP4 is a brain-specific transcript highly abundant in the cerebellum and fetal brain. High evolutionary conservation of ATIP amino-acid sequences suggests important biological roles for this new family of proteins in tumor suppression and/or brain function.

The saposin-like proteins 1, 2, and 3 of Fasciola gigantica

The SAP genes of Fasciola encode proteins belonging to the saposin-like protein family. The saposin signature, a compact domain of mainly α-helical character, contains six conserved cysteine residues and has been implicated in membrane-binding, pore formation, and subsequent cell lysis in several family members. Recombinant SAP-2 of F. hepatica has been shown to induce lysis of human erythrocytes and peripheral blood mononuclear cells. This suggests that the SAPs are involved in the nutrition of Fasciola as the released content of lysed host cells is available for further enzymatic processing and uptake by the parasite. In the present study a new SAP-3 cDNA was obtained in an immunoscreen of an adult stage F. gigantica cDNA library with an antiserum against the parasite's excretion/secretion antigens. SAP-1 and SAP-2 cDNAs were isolated from F. gigantica cDNA libraries using oligonucleotide primers specific to the SAP-1 and SAP-2 DNA sequences from F. hepatica. Transcripts of the three SAPs are present from the metacercarial to the adult stage and are located to the gut epithelium. In immatures SAP-1 RNA is the predominant product whereas in adults SAP-2 and -3 are the more abundant products. Polyclonal anti-SAP-1 and SAP-2 antisera confirmed the tissue-specificity and revealed the subcellular localization of SAPs in large granules concentrated in the apical part of the gut epithelial cells of the parasite. Interestingly, evolutionary conservation of the Fasciola SAP sequences among other trematodes is low at 20–30% sequence identity comparable to the Entamoeba amoebapore sequences.

Conservation of RET Regulatory Function from Human to Zebrafish Without Sequence Similarity

Evolutionary sequence conservation is an accepted criterion to identify noncoding regulatory sequences. We have used a transposon-based transgenic assay in zebrafish to evaluate noncoding sequences at the zebrafish ret locus, conserved among teleosts, and at the human RET locus, conserved among mammals. Most teleost sequences directed ret-specific reporter gene expression, with many displaying overlapping regulatory control. The majority of human RET noncoding sequences also directed ret-specific expression in zebrafish. Thus, vast amounts of functional sequence information may exist that would not be detected by sequence similarity approaches.

Cloning and developmental expression of WSTF during Xenopus laevis embryogenesis

The gene WSTF is deleted in the autosomal dominant hereditary disorder Williams–Beuren syndrome. This disorder is caused by a 1.3 megabase deletion in human chromosome 7, encompassing at least 17 genes. The WSTF protein contains a bromodomain, found predominantly in chromatin-associated proteins. Reported association of WSTF with chromatin remodeling factors and functional data support a role for WSTF during chromatin remodeling. Here, we report the cloning and developmental expression pattern of Xenopus laevis WSTF. Xenopus laevis WSTF is a protein with a predicted amino acid sequence of 1441 amino acids. Three discrete domains can be identified in the Xenopus laevis WSTF protein, a PHD finger, a DDT domain and a bromodomain. Alignment of Xenopus WSTF with the corresponding orthologues from Homo sapiens, Gallus gallus, Mus musculus and Danio rerio demonstrates an evolutionary conservation of WSTF amino acid sequence and domain organization. In situ hybridization reveals a dynamic expression profile during embryonic development. WSTF is expressed differentially in neural tissue, especially during neurulae stages in the eye, in neural crest cells and the brain.

Evaluation of the RET regulatory landscape reveals the biological relevance of a HSCR-implicated enhancer

Evolutionary sequence conservation is now a relatively common approach for the prediction of functional DNA sequences. However, the fraction of conserved non-coding sequences with regulatory potential is still unknown. In this study, we focus on elucidating the regulatory landscape of RET, a crucial developmental gene within which we have recently identified a regulatory Hirschsprung disease (HSCR) susceptibility variant. We report a systematic examination of conserved non-coding sequences (n=45) identified in a 220 kb interval encompassing RET. We demonstrate that most of these conserved elements are capable of enhancer or suppressor activity in vitro, and the majority of the elements exert cell type-dependent control. We show that discrete sequences within regulatory elements can bind nuclear protein in a cell type-dependent manner that is consistent with their identified in vitro regulatory control. Finally, we focused our attention on the enhancer implicated in HSCR to demonstrate that this element drives reporter expression in cell populations of the excretory system and central nervous system (CNS) and peripheral nervous system (PNS), consistent with expression of the endogenous RET protein. Importantly, this sequence also modulates expression in the enteric nervous system consistent with its proposed role in HSCR.

SVC: structured visualization of evolutionary sequence conservation

We have developed a web application for the detailed analysis and visualization of evolutionary sequence conservation in complex vertebrate genes. Given a pair of orthologous genes, the protein-coding sequences are aligned. When these sequences are mapped back onto their encoding exons in the genomes, a scaffold of the conserved gene structure naturally emerges. Sequence similarity between exons and introns is analysed and embedded into the gene structure scaffold. The visualization on the SVC server provides detailed information about evolutionarily conserved features of these genes. It further allows concise representation of complex splice patterns in the context of evolutionary conservation. A particular application of our tool arises from the fact that around mRNA editing sites both exonic and intronic sequences are highly conserved. This aids in delineation of these sites. SVC is available at .

Evolutionary conservation of nuclear and nucleolar targeting sequences in yeast ribosomal protein S6A

Over 1 billion years ago, the animal kingdom diverged from the fungi. Nevertheless, a high sequence homology of 62% exists between human ribosomal protein S6 and S6A of Saccharomyces cerevisiae. To investigate whether this similarity in primary structure is mirrored in corresponding functional protein domains, the nuclear and nucleolar targeting signals were delineated in yeast S6A and compared to the known human S6 signals. The complete sequence of S6A and cDNA fragments was fused to the 5′-end of the LacZ gene, the constructs were transiently expressed in COS cells, and the subcellular localization of the fusion proteins was detected by indirect immunofluorescence. One bipartite and two monopartite nuclear localization signals as well as two nucleolar binding domains were identified in yeast S6A, which are located at homologous regions in human S6 protein. Remarkably, the number, nature, and position of these targeting signals have been conserved, albeit their amino acid sequences have presumably undergone a process of co-evolution with their corresponding rRNAs.