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PubMed1993

The RNA-world and co-evolution hypotheses and the origin of life: implications, research strategies and perspectives.

The applicability of the RNA-world and co-evolution hypotheses to the study of the very first stages of the origin of life is discussed. The discussion focuses on the basic differences between the two hypotheses and their implications, with regard to the reconstruction methodology, ribosome emergence, balance between ribozymes and protein enzymes, and their major difficulties. Additional complexities of the two hypotheses, such as membranes and the energy source of the first reactions, are not treated in the present work. A central element in the proposed experimental strategies is the study of the catalytic activities of very small peptides and RNA-like oligomers, according to existing, as well as to yet-to-be-invented scenarios of the two hypotheses under consideration. It is suggested that the novel directed molecular evolution technology, and molecular computational modelling, can be applied to this research. This strategy is assumed to be essential for the suggested goal of future studies of the origin of life, namely, the establishment of a 'Primordial Darwinian entity'.

Biological Evolution↗

PubMed2002

Molecular evolution within the L-malate and L-lactate dehydrogenase super-family.

The NAD(P)-dependent malate (L-MalDH) and NAD-dependent lactate (L-LDH) form a large super-family that has been characterized in organisms belonging to the three domains of life. In the first part of this study, the group of [LDH-like] L-MalDH, which are malate dehydrogenases resembling lactate dehydrogenase, were analyzed and clearly defined with respect to the other enzymes. In the second part, the phylogenetic relationships of the whole super-family were presented by taking into account the [LDH-like] L-MalDH. The inferred tree unambiguously shows that two ancestral genes duplications, and not one as generally thought, are needed to explain both the distribution into two enzymatic functions and the observation of three main groups within the super-family: L-LDH, [LDH-like] L-MalDH, and dimeric L-MalDH. In addition, various cases of functional changes within each group were observed and analyzed. The direction of evolution was found to always be polarized: from enzymes with a high stringency of substrate recognition to enzymes with a broad substrate specificity. A specific phyletic distribution of the L-LDH, [LDH-like] L-MalDH, and dimeric L-MalDH over the Archaeal, Bacterial, and Eukaryal domains was observed. This was analyzed in the light of biochemical, structural, and genomic data available for the L-LDH, [LDH-like] L-MalDH, and dimeric L-MalDH. This analysis led to the elaboration of a refined evolutionary scenario of the super-family, in which the selection of L-LDH and the fate of L-MalDH during mitochrondrial genesis are presented.

Animals↗

PubMed2004

Butterfly selected lines explore the hormonal basis of interactions between life histories and morphology.

Hormonal mechanisms underlie many life-history traits and their interactions. We studied the role of ecdysteroids with regard to wing pattern and development time of the polyphenic butterfly Bicyclus anynana. Ecdysteroid titers and sensitivity to ecdysone injection were assayed for two-trait selected lines (ventral eyespot size and development time concurrently). These two traits are genetically and phenotypically coupled, having a common endocrinal basis. Two-trait selection had been applied both antagonistically (opposite the correlation) and synergistically (in the same direction as the correlation). Although selected lines had diverged most in eyespot size, the widest differences in timing of ecdysteroid titers were observed between the development time selection regimes; fast selected lines had an earlier hormonal increase after pupation than slow selected lines (even when corrected for differential pupal times). This endocrine peak was also earlier for females than for males. Furthermore, sensitivity to ecdysone injection as measured by a subsequent decrease in pupal time was significantly lower for slow selected lines than for fast or unselected lines. We conclude that the observed response in eyespot size to artificial selection must have been achieved via alteration of, or selection on, other developmental mechanisms, because the dynamics of the alternative, hormonal, pathway were dictated by development time selection. The developmental system is flexible enough to allow evolution in directions opposing the correlation between wing pattern and developmental time, and responses to selection are not constrained by a shared hormonal system.

Animals↗

PubMed1996

Patterns of distal-less gene expression and inductive interactions in the head of the direct developing frog Eleutherodactylus coqui.

The direct developing frog Eleutherodactylus coqui exhibits radical changes in its embryogenesis. A frog-like head forms directly with no appearance of a cement gland or several jaw cartilages characteristic of tadpoles, and limbs appear early in development. The numerous differences in the embryogenesis of E. coqui provide an opportunity to examine developmental causes for the evolutionary shift from biphasic to direct development. We have cloned DNA fragments corresponding to four E. coqui genes related to the Drosophila distal-less gene Dll. While the expression patterns of the distal-less genes are generally conserved, there are some spatiotemporal differences when embryos of E. coqui are compared to those of Xenopus laevis. The changes in gene expression are correlated with the embryonic changes in head structures including craniofacial cartilages and in particular, the cement gland. We have then examined inductive interactions involved in cement gland formation by interspecific transplants and recombinants. E. coqui embryos can generate signaling that culminates in cement gland formation, but E. coqui ectoderm appears to be incapable of a cement gland response. These results show here that inductive interactions in the anterior region of the E. coqui embryo have been modified during the evolution of direct development, and that changes in the competence of the E. coqui ectoderm may be responsible for the loss of certain tadpole-specific structures, such as cement gland.

Amino Acid Sequence↗

PubMed2005

A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities.

Yeast surface display and sorting by flow cytometry are now widely used to direct the evolution of protein binding such as single-chain antibodies or scFvs. The available commercial yeast display vector pYD1 (Invitrogen) displays the protein of interest flanked on the N-terminus by Aga2, the disulfide of which binds the myristylated surface membrane protein Aga1. We have noted that two anti-CD3epsilon scFvs expressed as fusion proteins suffer a 30- to 100-fold loss of affinity when placed NH(2) terminal to either truncated toxins or human serum albumin. In the course of affinity maturing one of these scFv (FN18) using pYD1 we noted that the affinity towards the ectodomain of monkey CD3epsilongamma was too low to measure. Consequently we rebuilt pYD1 tethering the scFv off the NH(2) terminus of Aga2. This display vector, pYD5, now gave a positive signal displaying FN18 scFv with its ligand, monkey CD3epsilongamma. The apparent equilibrium association constant of the higher affinity scFv directed at human CD3epsilongamma increased approximately 3-fold when displayed on pYD5 compared with pYD1. These data show that for certain yeast-displayed scFvs a carboxy-tethered scFv can result in increased ligand-scFv equilibrium association constants and thereby extend the low range of affinity maturation measurements.

Amino Acid Sequence↗

PubMed2005

A simple and rapid approach for screening of SARS-coronavirus genotypes: an evaluation study.

BACKGROUND: The Severe Acute Respiratory Syndrome (SARS) was a newly emerged infectious disease which caused a global epidemic in 2002-2003. Sequence analysis of SARS-coronavirus isolates revealed that specific genotypes predominated at different periods of the epidemic. This information can be used as a footprint for tracing the epidemiology of infections and monitor viral evolution. However, direct sequencing analysis of a large number of clinical samples is cumbersome and time consuming. We present here a simple and rapid assay for the screening of SARS-coronavirus genotypes based on the use of fluorogenic oligonucleotide probes for allelic discrimination. METHODS: Thirty SARS patients were recruited. Allelic discrimination assays were developed based on the use of fluorogenic oligonucleotide probes (TaqMan). Genotyping of the SARS-coronavirus isolates obtained from these patients were carried out by the allelic discrimination assays and confirmed by direct sequencing. RESULTS: Genotyping based on the allelic discrimination assays were fully concordant with direct sequencing. All of the 30 SARS-coronavirus genotypes studied were characteristic of genotypes previously documented to be associated with the latter part of the epidemic. Seven of the isolates contained a previously reported major deletion but in patients not epidemiologically related to the previously studied cohort. CONCLUSION: We have developed a simple and accurate method for the characterization and screening of SARS-coronavirus genotypes. It is a promising tool for the study of epidemiological relationships between documented cases during an outbreak.

Alleles↗

PubMed2002

Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

Binding Sites↗

PubMed1999

Apextrin, a novel extracellular protein associated with larval ectoderm evolution in Heliocidaris erythrogramma.

During the evolution of direct development in the sea urchin Heliocidaris erythrogramma major modifications occurred, which allowed the precocious formation of adult-specific structures and led to a novel larval body that surrounds these structures. The HeET-1 gene was isolated in a differential screen for transcripts enriched in the early embryos of H. erythrogramma relative to those of its indirect-developing congener, H. tuberculata. HeET-1 was unique among the three genes found in that no homologous transcript was detected in H. tuberculata total embryonic RNA blots. To verify this apparently extreme differential expression of the HeET-1 genes in Heliocidaris, we isolated the HeET-1 homologue from H. tuberculata genomic DNA and used it to probe blots of poly(A)+ RNA prepared from H. tuberculata embryos. It is expressed in H. tuberculata embryos at levels undetectable by this technique. The predicted amino acid sequence of HeET-1 suggested that it encodes a novel secreted protein. To assess the function of HeET-1, we raised polyclonal antisera to the HeET-1-encoded protein. We find that it is present in eggs in a type of secretory vesicle and that this maternal pool is gradually secreted after fertilization. As cells acquire apical-basal polarity in the blastula the protein becomes localized to the apical extracellular matrix, leading us to name the protein apextrin. The apical extracellular localization of apextrin is maintained in the columnar cells of the larval ectoderm until their internalization at metamorphosis. Ingressing mesenchyme cells rapidly endocytose apextrin upon leaving the vegetal plate. Comparison with fibropellin III, an apical lamina component, suggests that apextrin is an extracellular protein that is in tighter association with the plasma membrane than is the hyalin layer or apical lamina. We propose that apextrin is involved in apical cell adhesion and that its high level of expression may represent an adaptive cooption necessary for strengthening the large H. erythrogramma embryo.

Amino Acid Sequence↗

PubMed2003

Global incorporation of norleucine in place of methionine in cytochrome P450 BM-3 heme domain increases peroxygenase activity.

In this study we have replaced all 13 methionine residues in the cytochrome P450 BM-3 heme domain (463 amino acids) with the isosteric methionine analog norleucine. This experiment has provided a means of testing the functional limits of globally incorporating into an enzyme an unnatural amino acid in place of its natural analog, and also an efficient way to test whether inactivation during peroxide-driven P450 catalysis involves methionine oxidation. Although there was no increase in the stability of the P450 under standard reaction conditions (in 10 mM hydrogen peroxide), complete substitution with norleucine resulted in nearly two-fold-increased peroxygenase activity. Thermostability was significantly reduced. The fact that the enzyme can tolerate such extensive amino acid replacement suggests that we can engineer enzymes with unique chemical properties via incorporation of unnatural amino acids while retaining or improving catalytic properties. This system also provides a platform for directing enzyme evolution using an extended set of protein building blocks.

Culture Media↗

PubMed2005

Yeast surface display of a noncovalent MHC class II heterodimer complexed with antigenic peptide.

Microbial protein display technologies have enabled directed molecular evolution of binding and stability properties in numerous protein systems. In particular, dramatic improvements to antibody binding affinity and kinetics have been accomplished using these tools in recent years. Examples of successful application of display technologies to other immunological proteins have been limited to date. Herein, we describe the expression of human class II major histocompatibility complex allele (MHCII) HLA-DR4 on the surface of Saccharomyces cerevisiae as a noncovalently associated heterodimer. The yeast-displayed MHCII is fully native as assessed by binding of conformationally specific monoclonal antibodies; failure of antibodies specific for empty HLA-DR4 to bind yeast-displayed protein indicates antigenic peptide is bound. This report represents the first example of a noncovalent protein dimer displayed on yeast and of successful display of wild-type MHCII. Results further point to the potential for using yeast surface display for engineering and analyzing the antigen binding properties of MHCII.

Antibodies, Monoclonal↗

PubMed2004

Commentary: The evolution of evidence-based psychotherapy.

What has been identified as a revolution actually represents an evolution, stemming directly from the basic behavioral tenets of the early to mid 20th century. Cognitive-behavior approaches within behavior therapy are not really new. As early as the 1950s a substantial number of behaviorists saw a clear role for cognition and relied on hypothetical constructs to explain behavior. There are at least two major challenges for cognitive-behavior approaches. The first is the assurance of the validity and reliability of the measurement of both outcome and the cognitive factors that are presumed to serve as mediators of the observed effects. As the cognitive factors being studied become more complex, the measurement of the variables of interest becomes more difficult. The second challenge is to be open to new paradigms and approaches that may bring into question well-established concepts. The preeminence of the cognitive-behavioral approach is directly related to the fact that its concepts and methods can be subjected to empirical scrutiny.

Evidence-Based Medicine↗

PubMed2002

Hydrogen/deuterium exchange for higher specificity of protein identification by peptide mass fingerprinting.

Genome sequencing projects produce large amounts of information that could be translated into potential protein sequences. Such amounts of material continuously increase protein database sizes. At present, 22 times more protein sequences are available in the SWISS-PROT and TrEMBL databases than 8 years ago in SWISS-PROT. One of the methods of choice for protein identification makes use of specific endoproteolytic cleavage followed by matrix-assisted laser desorption/ionisation mass spectrometric (MALDI-MS) analysis of the digested product. Since 1993, when this technique was first demonstrated, the conditions required for a correct identification have changed dramatically. Whilst 4-5 peptides with an uncertainty of 2-3 Da were sufficient for a correct identification in 1993, 10-13 peptides with less than 60 ppm mass error are now required for human and E. coli proteins. This evolution is directly related to the continuous increase in protein database sizes, which causes an increase in the number of false positive matches in identification results. Use of an information complement deduced from the primary protein sequence, in the process of identification by peptide mass fingerprints, can help to increase confidence in the identification results. In this article, we propose the exchange of labile hydrogen atoms with deuterium atoms to provide an alternative information complement. The exchange reaction with optimised techniques has shown an average 95% of hydrogen/deuterium (H/D) exchange on tryptic peptides. This level of exchange was sufficient to single out one or more peptides from a list of potential candidate proteins due to the dependence of H/D exchange on the peptide primary structure. This technique also has clear advantages in the identification of small proteins where direct protein identification is impaired by the limited number of endoproteolytic peptides. Then, information related to primary sequence obtained with this technique could help to identify proteins with high confidence without any expensive tandem mass spectrometry instruments.

Amino Acid Sequence↗

PubMed1998

Display-induced antigenic variation in recombinant peptides.

Peptide display on solvent-exposed surfaces of carrier proteins is a promising approach pursuing the identification and improvement of reactive amino acid sequences. However, the contribution of the molecular environment where the peptide is inserted on its interactive properties remains essentially unexplored. By an exhaustive antigenic analysis of the same peptide displayed on 20 structurally distinct frameworks, we show that peptide accommodation into the acceptor site has dramatic effects on its immunoreactivity. Conformational constraints can modulate the molecular recognition properties of the insert within a surprisingly wide range, probably by affecting the positioning of critical contact residues. The observed display-induced antigenic variation prompts a careful consideration of the molecular context when evaluating output amino acid sequences from screening of peptide libraries or application of directed molecular evolution technologies.

Amino Acid Sequence↗

PubMed2025

Experimental Evolution of Poxviruses.

Experimental evolution is the process of exposing virus populations to defined selective pressures in a laboratory setting to identify adaptive changes. Coupled with deep sequencing, this experimental approach allows for nucleotide-level resolution of poxvirus adaptive strategies over time. Here, we present a general method of poxvirus experimental evolution, Illumina-based deep sequencing, and bioinformatic analyses to identify structural changes (e.g., gene duplication) as well as local adaptive changes (e.g., small indels and single nucleotide polymorphisms).

Poxviridae↗

PubMed1987

The social ecology of alcohol treatment in the United States.

This chapter reviews the literature on the social ecology of alcohol-related treatment in the United States. It begins with an examination of differences in the population characteristics of alcoholics and problem drinkers within and without alcohol treatment institutions. Recent trends in treatment populations are described. Also considered are a variety of factors that influence both entry into treatment and the distribution of clients across the treatment system, including gender, ethnicity, problem severity, social networks, client wants, gatekeeping practices among treatment providers, and referral patterns. The general applicability of the "health belief model" to treatment entry is assessed as well as the place of a number of well-known barriers to alcohol-related treatment in accounting for nonentry. The distribution of the society's alcohol-related caseload--both across alcohol-specific and other health and social service institutions--is considered. Also examined are recent trends in the social handling of alcohol-related problems, with special reference to the growing reliance on coercion in case recruitment. Finally, the review offers a number of suggestions regarding implications for further research and broad directions the evolution of American alcohol-related treatment institutions may take.

Alcoholism↗

PubMed1991

Tissue-specific expression of the HLA-DRA gene in transgenic mice.

Transgenic mice were produced containing a 33 kilobase (kb) DNA fragment encompassing the five exons and all the known regulatory regions of the class II HLA-DRA gene. The transgene displayed regulated expression [constitutive and interferon-gamma (IFN)-gamma induced] of the human products in most mouse tissues. The tissue distribution of the DRA transgene products more closely resembled that of their mouse homologues, the endogenous H-2 Ea products, than the wider distribution of DRA products in humans. This was evident in several tissues (endothelia of small vessels, especially those of glomerular capillaries, Kupffer cells, and epithelial cells lining the gastrointestinal tract), known to differentially express class II molecules in the two species. Thus, the wider human specific pattern of expression requires an exact cis/trans complementation which is incompletely reconstituted in transgenic mice, suggesting that human-specific cis-acting elements may have arisen during evolution to direct the expression of class II genes to those anatomical regions which usually lack them in the mouse. The only example of aberrant expression of the DRA gene in the present series of transgenic mice was in the dendritic and/or epithelial cells of the thymic cortex, which displayed greatly reduced DR alpha levels in spite of a normal expression of the endogenous E alpha molecules.

Animals↗

PubMedDate not supplied

Peliosis hepatis with pseudotumoral and hemorrhagic evolution: CT and MR findings.

Peliosis hepatis is an uncommon benign disorder characterized by blood-filled cavities within the liver. We describe the sonographic, computed tomographic, and magnetic resonance appearance of a patient affected by peliosis hepatis with pseudotumoral and hemorrhagic evolution and direct demonstration of the ectatic vascular components.

Adult↗

PubMed2005

Resolution enhanced homonuclear carbon decoupled triple resonance experiments for unambiguous RNA structural characterization.

Large RNAs (>30 nucleotides) suffer from extensive resonance overlap that can seriously hamper unambiguous structural characterization. Here we present a set of 3D multinuclear NMR experiments with improved and optimized resolution and sensitivity for aiding with the assignment of RNA molecules. In all these experiments strong base and ribose carbon-carbon couplings are eliminated by homonuclear band-selective decoupling, leading to improved signal to noise and resolution of the C5, C6, and C1' carbon resonances. This decoupling scheme is applied to base-type selective 13C-edited NOESY, 13C-edited TOCSY (HCCH, CCH), HCCNH, and ribose H1C1C2 experiments. The 3D implementation of the HCCNH experiment with both carbon and nitrogen evolution enables direct correlation of 13C and 15N resonances at different proton resonant frequencies. The advantages of the new experiments are demonstrated on a 36 nucleotides hairpin RNA from domain 5 (D5) of the group II intron Pylaiella littoralis using an abbreviated assignment strategy. These four experiments provided additional separation for regions of the RNA that have overlapped chemical shift resonances, and enabled the assignment of critical D5 bulge nucleotides that could not be assigned using current experimental schemes.

Carbon Isotopes↗