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PubMed2004

Conserved elements in Pax6 intron 7 involved in (auto)regulation and alternative transcription.

Pax6 is a transcription factor with an essential role in eye, central nervous system, and pancreas development. Its expression pattern is restricted to these specific domains within the developing embryo. Here four conserved elements are identified in Pax6 intron 7, showing a high level of sequence conservation between human, mouse, pufferfish, and zebrafish. Three of these are shown to act as cis-regulatory elements, directing expression of a reporter gene to distinct subsets of the Pax6 expression domain. CE1 regulates gene expression in late eye development, CE2 drives expression in the diencephalon and in the developing heart tube where Pax6 is not normally expressed, while CE3 directs expression in rhombencephalon. CE2 is shown to be autoregulated in the diencephalon, responding to absence of Pax6. We identify a highly conserved Pax6 recognition site and demonstrate its ability to bind Pax6 specifically. CE1 is embedded in a CpG island, and we identify a novel Pax6 transcript which initiates from this region. Functional analysis of evolutionary conserved sequences pinpoints novel cis-acting elements that govern the regulation of the complex spatio-temporal and quantitative expression of Pax6.

Alternative Splicing↗
PubMed2000

The role of complement in B cell activation and tolerance.

It is becoming well accepted that innate immunity serves as a natural adjuvant in enhancing and directing the adaptive immune response. In this review, I have discussed how the complement system, a major mediator of innate immunity, links the two systems. The recent availability of knockout mice bearing selective deficiencies in the critical complement proteins and receptors has allowed formal demonstration of the importance of complement in enhancement of humoral immunity. Characterization of the mice has also uncovered mechanisms for maintaining survival of activated B cells within the lymphoid compartment. For example, co-ligation of the CD21/CD19/Tapa-1 receptor with the BCR not only reduces the threshold for B cell follicular survival but provides a unique signal for survival in the germinal centers. In addition complement receptors are critical for localization of antigen and C3d ligand to FDCs for maintenance of long-term B cell memory. A surprise that has come from analysis of the deficient mice is that complement is also important in negative selection of B lymphocytes. This observation provides new insight to a long-standing enigma that the major predisposing factor in lupus is deficiency in complement C1q or C4. The seeming contradiction of dual role for complement in both B cell activation and tolerance is reconciled by the hypothesis that natural IgM provides a mechanism to selectively identify self-antigens that are highly conserved and cross-react with microbial ones such as DNA and nuclear proteins. Thus, the importance of complement in tolerance to self-antigens is restricted to those self-antigens that are evolutionary conserved, and they are identified by natural antibody. The future should hold further surprises as to the intricate interactions between the complement system and acquired immunity.

Adjuvants, Immunologic↗
PubMed2005

An evolutionary proteomics approach identifies substrates of the cAMP-dependent protein kinase.

Protein kinases are important mediators of much of the signal transduction that occurs in eukaryotic cells. Unfortunately, the identification of protein kinase substrates has proven to be a difficult task, and we generally know few, if any, of the physiologically relevant targets of any particular kinase. Here, we describe a sequence-based approach that simplified this substrate identification process for the cAMP-dependent protein kinase (PKA) in Saccharomyces cerevisiae. In this method, the evolutionary conservation of all PKA consensus sites in the S. cerevisiae proteome was systematically assessed within a group of related yeasts. The basic premise was that a higher degree of conservation would identify those sites that are functional in vivo. This method identified 44 candidate PKA substrates, 5 of which had been described. A phosphorylation analysis showed that all of the identified candidates were phosphorylated by PKA and that the likelihood of phosphorylation was strongly correlated with the degree of target site conservation. Finally, as proof of principle, the activity of one particular target, Atg1, a key regulator of autophagy, was shown to be controlled by PKA phosphorylation in vivo. These data therefore suggest that this evolutionary proteomics approach identified a number of PKA substrates that had not been uncovered by other methods. Moreover, these data show how this approach could be generally used to identify the physiologically relevant occurrences of any protein motif identified in a eukaryotic proteome.

Autophagy↗
PubMed2003

Evolution of learning in three aplysiid species: differences in heterosynaptic plasticity contrast with conservation in serotonergic pathways.

We investigated the neurobiological basis of variation in sensitization between three aplysiid species: Aplysia californica, Phyllaplysia taylori and Dolabrifera dolabrifera. We tested two different forms of sensitization induced by a noxious tail shock: local sensitization, expressed near the site of shock, and general sensitization, tested at remote sites. Aplysia showed both local and general sensitization, whereas Phyllaplysia demonstrated only local sensitization, and Dolabrifera lacked both forms of learning. We then investigated a neurobiological correlate of sensitization, heterosynaptic modulation of sensory neuron excitability by tail-nerve stimulation. We found (1) an increase in sensory neuron (SN) excitability after both ipsilateral and contralateral nerve stimulation in Aplysia, (2) a smaller and shorter-lasting increase in Phyllaplysia, and (3) no effect in Dolabrifera. Because sensitization in Aplysia is strongly correlated with serotonergic (5-HT) neuromodulation, we hypothesized that the observed interspecific variation in sensitization and SN neuromodulation might be correlated with variation in the anatomy and/or functional response of the serotonergic system. However, using immunohistochemistry, we found that all three species showed a similar pattern of 5-HT innervation. Furthermore, they also showed comparable 5-HT release evoked by tail-nerve shock, as measured with chronoamperometry. These observations indicate that interspecific variation in learning is correlated with differences in SN heterosynaptic plasticity within a background of evolutionary conservation in the 5-HT neuromodulatory pathway. We thus hypothesize that evolutionary changes in learning phenotype do not involve modifications of the 5-HT pathway per se, but rather, changes in the response of SNs to the activation of this or other neuromodulatory pathways upon noxious stimulation.

Animals↗
PubMed2025

Conserved HSFA1-dependent chromatin dynamics drive heat stress responses in plants.

Eukaryotic organisms remodel chromatin landscapes to regulate gene expression in response to environmental stress. In plants, heat stress (HS) induces widespread chromatin changes, yet the role of heat shock transcription factors (HSFs) in chromatin remodeling and their evolutionary conservation remains unclear. Using Marchantia polymorpha Mphsf mutants and Arabidopsis thaliana Athsfa1s mutants, we identify HSFA1 as a key regulator of HS-induced cis-regulatory element (CRE) accessibility, a mechanism conserved across land plants, mice, and humans. Gene regulatory network modeling reveals parallel transcription factor subnetworks, with MpWRKY10 and MpABI5B acting as indirect and negative HS regulators. We further showed that ABA modulates gene expression in an HSFA1-dependent manner without inducing chromatin remodeling. Finally, we develop a machine learning framework integrating chromatin accessibility and CRE information to predict gene expression across species, revealing stress-responsive regulatory logic at the transcriptional level. These findings provide insights into how TFs coordinate chromatin architecture to drive stress adaptation.

Heat-Shock Response↗
PubMed1989

Conserved epitopes on plant H1 histones recognized by monoclonal antibodies.

A series of monoclonal antibodies specific for distinct regions of H1 histone from the plant Nicotiana tabacum were obtained from fusion experiments with spleen cells of mice immunized with tobacco nuclear extracts. These monoclonal antibodies were characterized and the evolutionary conservation of the epitopes in higher plants and animals studied by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Whereas some epitopes appear restricted to the Solanaceae plant family, others are common to all higher eukaryotes tested and even detectable on nuclear proteins of yeast. ELISA experiments performed with isolated tobacco chromatin give some indications of the differential accessibility of the epitopes after interaction of H1 histone with the nucleosome.

Antibodies, Monoclonal↗
PubMed1995

Conserved high-affinity NF-kappa B binding site in the interferon regulatory factor-1 promoter is not occupied by NF-kappa B in vivo and is transcriptionally inactive.

The promoter of the interferon regulatory factor-1 (IRF-1) gene contains at position -47 to -38 an evolutionary conserved binding sequence for the inducible transcription factor NF-kappa B. This site is highly homologous to a transcriptionally active site from the MHC class I enhancer. In this study, we show by in vitro assays using purified NF-kappa B that the kappa B motif in the IRF-1 promoter binds the factor specifically and with high affinity, comparable to various other cis-acting kappa B elements. Two copies of the IRF-1 kappa B site fused to the heterologous c-fos promoter conferred induction of a chloramphenicol acetyl transferase (CAT) reported gene in response to stimulation of L929 fibroblasts with various NF-kappa B inducers, such as tumor necrosis factor alpha (TNF alpha) or phorbol 12-myristate 13-acetate (PMA). Mutation of the binding site completely abolished transcriptional inducibility of the heterologous promoter. Surprisingly, the same IRF-1 kappa B motif in context of the homologous IRF-1 promoter was transcriptionally inactive in CAT assays. The very weak induction of the IRF-1 promoter in response to TNF treatment or infection of fibroblasts with Newcastle disease virus (NDV) was barely affected by point mutation of the kappa B site or loss of the site by truncation of the promoter. Analysis of the occupational state of the chromosomal IRF-1 kappa B site by in vivo foot-printing revealed that no footprint was induced over the kappa B motif in the IRF-1 promoter after PMA treatment of L929 fibroblast cells, despite the simultaneous induction of IRF-1 mRNA and NF-kappa B binding activity. Constitutive footprints were detected at a CCAAT and GC-rich region in the promoter. This is the first example of a high-affinity NF-kappa B binding site within a promoter which may not participate in transcriptional regulation under conditions activating NF-kappa B DNA binding and gene expression.

Animals↗
PubMed2004

Upregulation of rat P23 (a member of the YjgF protein family) by fasting, glucose diet and fatty acid feeding.

In a previous study, we identified and purified a 99-amino-acid rat liver-kidney perchloric-acid-soluble 23-kDa protein (P23) which displays 30% identity with a highly conserved domain of heat shock proteins (HSPs), as well as an AT-rich 3' untranslated region, which has also been described to play a role in H70 mRNA life span and protein expression. An identical perchloric-acid-soluble protein inhibiting protein synthesis in a rabbit reticulocyte lysate system was also found 2 years later by another group. More recently, the novel, the YjgF, protein family has been described, comprising, 24 full-length homologues, including P23, highly conserved through evolution, and consisting of approximately 130 residues each and sharing a common ternary structure. Independent studies from different laboratories have provided various hypothetical functions for each of these proteins. The high degree of evolutionary conservation may suggest that these proteins play an important role in cellular regulation. Although the function of none of these proteins is known precisely, we present experimental evidence which, combined with the relationship to glucose-regulating protein revealed here, and the relationship to fatty-acid-binding protein revealed by others, allow us to propose a role for P23. In rat liver, P23 expression is developmentally regulated and modulated by dietary glucose, and its mRNA is induced by starvation, in the presence of fatty-acids and in 3-MeDAB-induced hepatomas. The mRNA encoding mouse liver P23 is also hormonally modulated in a mouse line AT1F8. These data indicate that P23 protein might be a key controller of intermediary metabolism during fasting.

Animals↗
PubMed2005

How did the platypus get its sex chromosome chain? A comparison of meiotic multiples and sex chromosomes in plants and animals.

The duck-billed platypus is an extraordinary mammal. Its chromosome complement is no less extraordinary, for it includes a system in which ten sex chromosomes form an extensive meiotic chain in males. Such meiotic multiples are unprecedented in vertebrates but occur sporadically in plant and invertebrate species. In this paper, we review the evolution and formation of meiotic multiples in plants and invertebrates to try to gain insights into the origin of the platypus meiotic multiple. We describe the meiotic hurdles that translocated mammalian chromosomes face, which make longer chains disadvantageous in mammals, and we discuss how sex chromosomes and dosage compensation might have affected the evolution of sex-linked meiotic multiples. We conclude that the evolutionary conservation of the chain in monotremes, the structural properties of the translocated chromosomes and the highly accurate segregation at meiosis make the platypus system remarkably different from meiotic multiples in other species. We discuss alternative evolutionary models, which fall broadly into two categories: either the chain is the result of a sequence of translocation events from an ancestral pair of sex chromosomes (Model I) or the entire chain came into being at once by hybridization of two populations with different chromosomal rearrangements sharing monobrachial homology (Model II).

Animals↗
PubMed1994

Conserved domain structure of beta-neurexins. Unusual cleaved signal sequences in receptor-like neuronal cell-surface proteins.

Neurexins, a family of neuronal cell-surface proteins, consist of the longer alpha-neurexins (I alpha, II alpha, and III alpha) and the shorter beta-neurexins (I beta and II beta) with identical C termini but distinct N termini. alpha-Neurexins have the structure of cell surface receptors, but the membrane topology and conservation of beta-neurexins is unknown. We have now characterized cDNA clones encoding bovine neurexins I beta and III beta, thereby demonstrating the presence of a beta-form for neurexin III and the evolutionary conservation of beta-neurexins in mammals. Similar to alpha-neurexins, beta-neurexins were found to be highly O-glycosylated after expression by transfection in COS cells, suggesting that alpha- and beta-neurexins utilize the same O-glycosylation cassette and have similar transmembrane orientations. To determine if beta-neurexins contain a cleaved or uncleaved signal sequence for membrane translocation, beta-neurexin-IgG fusion proteins were expressed in COS cells, and their N termini were directly sequenced. This revealed that the N terminus of all three beta-neurexins contains an unusual cleaved signal sequence. Together our data show that all known neurexin genes generate alpha and beta forms with similar transmembrane organizations and receptor-like structures. Due to the presence of a long atypical cleaved signal peptide, beta-neurexins contain only a short unique sequence before splicing into the alpha-neurexin sequence. Thus, beta-neurexins are essentially N terminally truncated alpha-neurexins.

Amino Acid Sequence↗
PubMed2002

Transcription factors in cardiogenesis: the combinations that unlock the mysteries of the heart.

Heart formation is one of the first signs of organogenesis within the developing embryo and this process is conserved from flies to man. Completing the genetic roadmap of the molecular mechanisms that control the cell specification and differentiation of cells that form the developing heart has been an exciting and fast-moving area of research in the fields of molecular and developmental biology. At the core of these studies is an interest in the transcription factors that are responsible for initiation of a pluripotent cell to become programmed to the cardiac lineage and the subsequent transcription factors that implement the instructions set up by the cells commitment decision. To gain a better understanding of these pathways, cardiac-expressed transcription factors have been identified, cloned, overexpressed, and mutated to try to determine function. Although results vary depending on the gene in question, it is clear that there is a striking evolutionary conservation of the cardiogenic program among species. As we move up the evolutionary ladder toward man, we encounter cases of functional redundancy and combinatorial interactions that reflect the complex networks of gene expression that orchestrate heart development. This review focuses on what is known about the transcription factors implicated in heart formation and the role they play in this intricate genetic program.

Alternative Splicing↗
PubMed2002

Monte Carlo simulations of the peptide recognition at the consensus binding site of the constant fragment of human immunoglobulin G: the energy landscape analysis of a hot spot at the intermolecular interface.

Monte Carlo simulations of molecular recognition at the consensus binding site of the constant fragment (Fc) of human immunoglobulin G (Ig) protein have been performed to analyze structural and thermodynamic aspects of binding for the 13-residue cyclic peptide DCAWHLGELVWCT. The energy landscape analysis of a hot spot at the intermolecular interface using alanine scanning and equilibrium-simulated tempering dynamics with the simplified, knowledge-based energy function has enabled the role of the protein hot spot residues in providing the thermodynamic stability of the native structure to be determined. We have found that hydrophobic interactions between the peptide and the Met-252, Ile-253, His-433, and His-435 protein residues are critical to guarantee the thermodynamic stability of the crystallographic binding mode of the complex. Binding free energy calculations, using a molecular mechanics force field and a solvation energy model, combined with alanine scanning have been conducted to determine the energetic contribution of the protein hot spot residues in binding affinity. The conserved Asn-434, Ser-254, and Tyr-436 protein residues contribute significantly to the binding affinity of the peptide-protein complex, serving as an energetic hot spot at the intermolecular interface. The results suggest that evolutionary conserved hot spot protein residues at the intermolecular interface may be partitioned in fulfilling thermodynamic stability of the native binding mode and contributing to the binding affinity of the complex.

Alanine↗
PubMed2000

Secondary structure of vertebrate telomerase RNA.

Telomerase is a ribonucleoprotein enzyme that maintains telomere length by adding telomeric sequence repeats onto chromosome ends. The essential RNA component of telomerase provides the template for telomeric repeat synthesis. To determine the secondary structure of vertebrate telomerase RNA, 32 new telomerase RNA genes were cloned and sequenced from a variety of vertebrate species including 18 mammals, 2 birds, 1 reptile, 7 amphibians, and 4 fishes. Using phylogenetic comparative analysis, we propose a secondary structure that contains four structural domains conserved in all vertebrates. Ten helical regions of the RNA are universally conserved while other regions vary significantly in length and sequence between different classes of vertebrates. The proposed vertebrate telomerase RNA structure displays a strikingly similar topology to the previously determined ciliate telomerase RNA structure, implying an evolutionary conservation of the global architecture of telomerase RNA.

Animals↗
PubMed1998

Crystal structure of Y34F mutant human mitochondrial manganese superoxide dismutase and the functional role of tyrosine 34.

Tyrosine 34 is a prominent and conserved residue in the active site of the manganese superoxide dismutases in organisms from bacteria to man. We have prepared the mutant containing the replacement Tyr 34 --> Phe (Y34F) in human manganese superoxide dismutase (hMnSOD) and crystallized it in two different crystal forms, orthorhombic and hexagonal. Crystal structures of hMnSOD Y34F have been solved to 1.9 A resolution in a hexagonal crystal form, denoted as Y34Fhex, and to 2.2 A resolution in an orthorhombic crystal form, denoted as Y34Fortho. Both crystal forms give structures that are closely superimposable with that of wild-type hMnSOD, with the phenyl rings of Tyr 34 in the wild type and Phe 34 in the mutant very similar in orientation. Therefore, in Y34F, a hydrogen-bonded relay that links the metal-bound hydroxyl to ordered solvent (Mn-OH to Gln 143 to Tyr 34 to H2O to His 30) is broken. Surprisingly, the loss of the Tyr 34 hydrogen bonds resulted in large increases in stability (measured by Tm), suggesting that the Tyr 34 hydroxyl does not play a role in stabilizing active-site architecture. The functional role of the side chain hydroxyl of Tyr 34 can be evaluated by comparison of the Y34F mutant with the wild-type hMnSOD. Both wild-type and Y34F had kcat/Km near 10(9) M-1 s-1, close to diffusion-controlled; however, Y34F showed kcat for maximal catalysis smaller by 10-fold than the wild type. In addition, the mutant Y34F was more susceptible to product inhibition by peroxide than the wild-type enzyme. This activity profile and the breaking of the hydrogen-bonding chain at the active site caused by the replacement Tyr 34 --> Phe suggest that Tyr 34 is a proton donor for O2* - reduction to H2O2 or is involved indirectly by orienting solvent or other residues for proton transfer. Up to 100 mM buffers in solution failed to enhance catalysis by either Y34F or the wild-type hMnSOD, suggesting that protonation from solution cannot enhance the release of the inhibiting bound peroxide ion, likely reflecting the enclosure of the active site by conserved residues as shown by the X-ray structures. The increased thermostability of the mutant Y34F and equal diffusion-controlled activity of Y34F and wild-type enzymes with normal superoxide levels suggest that evolutionary conservation of active-site residues in metalloenzymes reflects constraints from extreme rather than average cellular conditions. This new hypothesis that extreme rather than normal substrate concentrations are a powerful constraint on residue conservation may apply most strongly to enzyme defenses where the ability to meet extreme conditions directly affects cell survival.

Binding Sites↗
PubMed2000

Structural basis of the Axin-adenomatous polyposis coli interaction.

Axin and the adenomatous polyposis coli (APC) tumor suppressor protein are components of the Wnt/Wingless growth factor signaling pathway. In the absence of Wnt signal, Axin and APC regulate cytoplasmic levels of the proto-oncogene beta-catenin through the formation of a large complex containing these three proteins, glycogen synthase kinase 3beta (GSK3beta) and several other proteins. Both Axin and APC are known to be critical for beta-catenin regulation, and truncations in APC that eliminate the Axin-binding site result in human cancers. A protease-resistant domain of Axin that contains the APC-binding site is a member of the regulators of G-protein signaling (RGS) superfamily. The crystal structures of this domain alone and in complex with an Axin-binding sequence from APC reveal that the Axin-APC interaction occurs at a conserved groove on a face of the protein that is distinct from the G-protein interface of classical RGS proteins. The molecular interactions observed in the Axin-APC complex provide a rationale for the evolutionary conservation seen in both proteins.

Adenomatous Polyposis Coli Protein↗
PubMed2002

Structural analysis of conserved base pairs in protein-DNA complexes.

Understanding of protein-DNA interactions is crucial for prediction of DNA-binding specificity of transcription factors and design of novel DNA-binding proteins. In this paper we develop a novel approach to analysis of protein-DNA interactions. We bring together two sources of information: (i) structures of protein-DNA complexes (PDB/NDB database) and (ii) experimentally obtained sites recognized by DNA-binding proteins. Sites are used to compute conservation (information content) of each base pair, which indicates relative importance of the base pair in specific recognition. The main result of this study is that conservation of base pairs in a site exhibits significant correlation with the number of contacts the base pairs have with the protein. In particular, base pairs that have more contacts with the protein are more conserved in evolution. As natural as it is, this result has never been reported before. We also observe that for most of the studied proteins, hydrogen bonds and hydrophobic interactions alone cannot explain the pattern of evolutionary conservation in the binding site suggesting cumulative contribution of different types of interactions to specific recognition. Implications for prediction of the DNA-binding specificity are discussed.

Bacterial Proteins↗
PubMedDate not supplied

Genes and chromosomes of Leishmania infantum.

During recent years, several Leishmania infantum genes have been cloned and characterized. Here, we have summarized the available information on the gene organization and expression in this protozoan parasite. From a comparative analysis, the following outstanding features were found to be common to most of the genes characterized: tandemly organized genes with conserved coding regions and divergent untranslated regions, polycistronic transcription and post-transcriptional regulation of gene expression. The analysis of chromosomes of L. infantum by pulsed-field electrophoresis showed the existence of both size and number polymorphisms such that each strain has a distinctive molecular karyotype. Despite this variability, highly conserved physical linkage groups exists among different strains of L. infantum and even among Old World Leishmania species. Gene mapping on the L. infantum molecular karyotype evidenced a bias in chromosomal distribution of, at least, the evolutionary conserved genes.

Animals↗
PubMed2003

Effects of pathological mutations on the stability of a conserved amino acid triad in retinoschisin.

A three-dimensional model has been calculated for the discoidin domain of retinoschisin (RS1), the protein involved in the X-linked juvenile retinoschisis. The model allows for a mapping of the pathological retinoschisis missense mutations and a rationale for the structural effects of an evolutionary conserved surface exposed triad (W122-R200-W163). Molecular dynamics simulations of the triad mutants models, together with ab initio energy calculations of the complexes corresponding to the triad show that the observed pathological mutations sensibly destabilize local interactions and the entire fold. Moreover the presented model reveals evidence of a putative site for membrane association.

Amino Acid Sequence↗