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PubMed2004

Forced selection of a human immunodeficiency virus type 1 variant that uses a non-self tRNA primer for reverse transcription: involvement of viral RNA sequences and the reverse transcriptase enzyme.

Human immunodeficiency virus type 1 uses the tRNA(3)(Lys) molecule as a selective primer for reverse transcription. This primer specificity is imposed by sequence complementarity between the tRNA primer and two motifs in the viral RNA genome: the primer-binding site (PBS) and the primer activation signal (PAS). In addition, there may be specific interactions between the tRNA primer and viral proteins, such as the reverse transcriptase (RT) enzyme. We constructed viruses with mutations in the PAS and PBS that were designed to employ the nonself primer tRNA(Pro) or tRNA(1,2)(Lys). These mutants exhibited a severe replication defect, indicating that additional adaptation of the mutant virus is required to accommodate the new tRNA primer. Multiple independent virus evolution experiments were performed to select for fast-replicating variants. Reversion to the wild-type PBS-lys3 sequence was the most frequent escape route. However, we identified one culture in which the virus gained replication capacity without reversion of the PBS. This revertant virus eventually optimized the PAS motif for interaction with the nonself primer. Interestingly, earlier evolution samples revealed a single amino acid change of an otherwise well-conserved residue in the RNase H domain of the RT enzyme, implicating this domain in selective primer usage. We demonstrate that both the PAS and RT mutations improve the replication capacity of the tRNA(1,2)(Lys)-using virus.

Amino Acid Substitution↗
PubMed1992

Transgenic animal studies on the evolution of genetic regulatory circuitries.

The ability to transfer genes from one species to another provides a powerful method to study genetic regulatory differences between species in a homogeneous genetic background. A survey of several transgenic animal experiments indicates that the vast majority of regulatory differences observed between species are due to differences in the cis-acting elements associated with the genes under study. A corollary is that in almost all cases the host species provides the necessary regulatory proteins for expression of the transgenes in specific tissues in which the endogenous homolog is not expressed. Although the details of the cis-acting differences are unknown for most cases, it appears that these differences may consist of the acquisition or loss of unique elements or subtle variation of conserved elements. It is unknown whether much of this variation is directly related to adaptive evolution. The identification of the promoter/enhancer elements responsible for these differences is an important first step in examining the functional significance of this variation.

Animals↗
PubMed2006

Experimental evolution of resistance to an antimicrobial peptide.

A novel class of antibiotics based on the antimicrobial properties of immune peptides of multicellular organisms is attracting increasing interest as a major weapon against resistant microbes. It has been claimed that cationic antimicrobial peptides exploit fundamental features of the bacterial cell so that resistance is much less likely to evolve than in the case of conventional antibiotics. Population models of the evolutionary genetics of resistance have cast doubt on this claim. We document the experimental evolution of resistance to a cationic antimicrobial peptide through continued selection in the laboratory. In this selection experiment, 22/24 lineages of Escherichia coli and Pseudomonas fluorescens independently evolved heritable mechanisms of resistance to pexiganan, an analogue of magainin, when propagated in medium supplemented with this antimicrobial peptide for 600-700 generations.

Animals↗
PubMed2000

The molecular evolution of signal peptides.

Signal peptides direct mature peptides to their appropriate cellular location, after which they are cleaved off. Very many random alternatives can serve the same function. Of all coding sequences, therefore, signal peptides might come closest to being neutrally evolving. Here we consider this issue by examining the molecular evolution of 76 mouse-rat orthologues, each with defined signal peptides. Although they do evolve rapidly, they evolve about half as fast as neutral sequences. This indicates that a substantial proportion of mutations must be under stabilizing selection. A few putative signal sequences lack a hydrophobic core and these tend to be more slowly evolving than others, indicating even stronger stabilizing selection. However, closer scrutiny suggests that some of these represent mis-annotations in GenBank. It is also likely that some of the substitutions are not neutral. We find, for example, that the rate of protein evolution correlates with that of the mature peptide. This may be a result of compensatory evolution. We also find that signal peptides of immune genes tend to be faster evolving than the average, which suggests an association with antagonistic co-evolution. Previous reports also indicated that the signal peptide of the imprinted gene, Igf2r, is also unusually fast evolving. This, it was hypothesized, might also be indicative of antagonistic co-evolution. Comparison of Igf2r's signal peptide evolution shows that, although it is not an outlier, its rate of evolution is comparable to that of many of the faster evolving immune system signal sequences and 5/6 of the amino acid changes do not conserve hydrophobicity. This is at least suggestive that there is something unusual about Igf2r's signal sequence.

Amino Acid Substitution↗
PubMed2002

A high-throughput optical screening method for the optimization of colloidal water oxidation catalysts.

A high-throughput method has been developed for screening and optimization of colloidal water oxidation catalysts. The catalysts are irradiated in parallel by visible light from an overhead projector in solutions containing tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)) and persulfate. The array of reaction solutions is held in a 96-well plate, and absorbance readings are taken intermittently using a bioassay plate reader. The absorbance at 430 nm is indicative of the amount of Ru(bpy)(3)(2+) remaining in solution. The best catalysts give the most persistent absorbance, because the oxygen evolution reaction is kinetically competitive with decomposition of Ru(bpy)(3)(3+). Reagent concentrations were varied using a factorial design-of-experiment approach in order to optimize reaction conditions for a IrO(2).xH(2)O colloidal catalyst. A higher colloid concentration, a lower Ru(bpy)(3)(2+) concentration, and a higher pH buffer doubled the number of turnovers relative to the original conditions. Metal oxide colloids consisting of IrO(2).xH(2)O doped with varying amounts of Pt, Ru, and Os were made using a parallel microwave synthesis technique and were tested both by the parallel screening method and by direct measurement of oxygen evolution. The correlation between the two methods was good, with Ir-Pt-Os oxide compositions showing the highest activity. The effect of adding small amounts of Pt and Os to IrO(2).xH(2)O appears to be predominantly to reduce the particle size of the colloids.

Journal Article↗
PubMed2007

The evolution of infidelity in socially monogamous passerines: neglected components of direct and indirect selection.

A recent study by Goran Arnqvist and Mark Kirkpatrick in the American Naturalist (165:S26-S37) suggested that female polyandry in birds is not driven by females because quantitative genetic approximations of selection demonstrated that indirect selection for female infidelity is weaker than natural selection against it. Instead, it was argued that extrapair copulations are the result of antagonistic selection on male behavior driving female coercion. While the approach and framework of the study were very good, the conclusions of the study were premature because a number of potential adaptive components of polyandry were unaccounted for, and several critical assumptions are unsupported by the current empirical data. Our understanding of extrapair paternity in birds, and perhaps polyandry in general, will be improved by a better empirical understanding of the direct benefits of fertility assurance and postcopulatory cryptic female choice and the relationship between polyandry and male investment. In addition, we need to develop a greater awareness of the limitations of trying to study behavior by proxy in the molecular laboratory. Together, these challenges and the framework recently presented should improve our understanding of the true function of extrapair paternity in birds.

Agonistic Behavior↗
PubMed1995

Evolution of the fibropellin gene family and patterns of fibropellin gene expression in sea urchin phylogeny.

This study documents evolutionary modifications in the expression patterns of the sea urchin EGF I and EGF III genes, which encode a family of extracellular matrix proteins, the fibropellins. We show that the sea urchin apical lamina, a macromolecular extracellular matrix that surrounds the sea urchin embryo and is made up of the fibropellins, has been conserved through at least 250 million years of echinoid evolution. The contribution of different fibropellin family members to this structure has, however, changed over the course of sea urchin phylogeny, and between two congeneric species that exhibit different developmental modes. Mapping the evolutionary history of the EGF genes on a cladogram of relationships among sea urchins reveals that EGF I is present in all echinoids examined, while EGF III appears to have arisen by duplication and divergence from EGF I during the radiation of a suborder of the camarodont sea urchins some 35-45 million years ago. Alterations in the temporal expression patterns of these genes as well as the loss of one of the two EGF I transcripts and encoded protein are coincident with the evolution of a direct-developing larval form in Heliocidaris erythrogramma. H. erythrogramma and its congener Heliocidaris tuberculata, which develops via a typical echinopluteus larva, shared a common ancestor about 10 million years ago. The differences in fibropellin representation within the apical lamina of the various taxa indicate that a homologous embryonic structure can undergo substantial changes in composition during its evolutionary history.

Animals↗
PubMed2004

Protein evolution by codon-based random deletions.

A method to delete in-phase codons throughout a defined target region of a gene has been developed. This approach, named the codon-based random deletion (COBARDE) method, is able to delete complete codons in a random and combinatorial mode. Robustness, automation and fine-tuning of the mutagenesis rate are essential characteristics of the method, which is based on the assembly of oligonucleotides and on the use of two transient orthogonal protecting groups during the chemical synthesis. The performance of the method for protein function evolution was demonstrated by changing the substrate specificity of TEM-1 beta-lactamase. Functional ceftazidime-resistant beta-lactamase variants containing several deleted residues inside the catalytically important omega-loop region were found. The results show that the COBARDE method is a useful new molecular tool to access previously unexplorable sequence space.

Amino Acids↗
PubMed1996

An enzyme-linked oligonucleotide assay.

The recent development of in vitro methods to select high-affinity ligands by combinatorial chemistry methodologies promises unique and theoretically unlimited supplies of novel therapeutic and diagnostic reagents. One such combinatorial chemistry process, systematic evolution of ligands by exponential enrichment (SELEX), allows rapid identification, from large random sequence pools, of the few oligonucleotide sequences that bind to a desired target molecule with high affinity and specificity. We describe an enzyme-linked sandwich assay that uses a SELEX-derived oligonucleotide. This assay demonstrates that these oligonucleotides can be effective and useful analytical reagents.

Animals↗
PubMed2005

Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A.

Botulism is caused by botulinum neurotoxin (BoNT), the most poisonous substance known. Potential use of BoNT as a biothreat agent has made development of sensitive assays for toxin detection and potent antitoxin for treatment of intoxication a high priority. To improve detection and treatment of botulism, molecular evolution and yeast display were used to increase the affinity of two neutralizing single chain Fv (scFv) antibodies binding BoNT serotype A (BoNT/A). Selection of yeast displayed scFv libraries was performed using methods to select for both increased association rate constant (k(on)) and decreased dissociation rate constants (k(off)). A single cycle of error prone mutagenesis increased the affinity of the 3D12 scFv 45-fold from a K(D) of 9.43x10(-10)M to a K(D) of 2.1x10(-11)M. Affinity of the HuC25 scFv was increased 37-fold from 8.44x10(-10)M to 2.26x10(-11)M using libraries constructed by both random and site directed mutagenesis. scFv variable region genes were used to construct IgG for use in detection assays and in vivo neutralization studies. While IgG had the same relative increases in affinity as scFv, (35-fold and 81-fold, respectively, for 3D12 and HuC25) higher solution equilibrium binding constants were observed for the IgG, with the 3D12 K(D) increasing from 6.07x10(-11)M to 1.71x10(-12)M and the HuC25 K(D) increasing from 4.51x10(-11)M to 5.54x10(-13)M. Affinity increased due to both an increase in k(on), as well as slowing of k(off). Higher affinity antibodies had increased sensitivity, allowing detection of BoNT/A at concentrations as low as 1x10(-13)M. The antibodies will also allow testing of the role of affinity in in vivo toxin neutralization and could lead to the generation of more potent antitoxin.

Antibody Affinity↗
PubMed2006

Frame shuffling: a novel method for in vitro protein evolution.

We describe 'frame shuffling', a novel method for preparing artificial protein libraries. With this method, a Y-family DNA polymerase known to introduce frame shift mutations at high rates is utilized to scramble the reading frames of a parental gene. The resultant progeny produce mutant proteins having segmental sequence changes. Such frame-shuffled mutant proteins exhibit physicochemical properties that differ from those of proteins obtained using conventional mutagenesis.

Amino Acid Sequence↗
PubMed2006

RNA aptamers: from basic science towards therapy.

The SELEX technique (systematic evolution of ligands by exponential enrichment) provides a powerful tool for the in vitro selection of nucleic acid ligands (aptamers) from combinatorial oligonucleotide libraries against a target molecule. In the beginning of the technique's use, RNA molecules were identified that bind to proteins that naturally interact with nucleic acids or to small organic molecules. In the following years, the use of the SELEX technique was extended to isolate oligonucleotide ligands (aptamers) for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. These oligonucleotides bind their targets with similar affinities and specificities as antibodies do. The in vitro selection of oligonucleotides with enzymatic activity, denominated aptazymes, allows the direct transduction of molecular recognition to catalysis. Recently, the use of in vitro selection methods to isolate protein inhibitors has been extended to complex targets, such as membrane-bound receptors, and even entire cells. RNA aptamers have also been expressed in living cells. These aptamers, also called intramers, can be used to dissect intracellular signal transduction pathways. The utility of RNA aptamers for in vivo experiments, as well as for diagnostic and therapeutic purposes, is considerably enhanced by chemical modifications, such as substitutions of the 2'-OH groups of the ribose backbone in order to provide resistance against enzymatic degradation in biological fluids. In an alternative approach, Spiegelmers are identified through in vitro selection of an unmodified D-RNA molecule against a mirror-image (i.e. a D-peptide) of a selection target, followed by synthesis of the unnatural nuclease-resistant L-configuration of the RNA aptamer that recognizes the natural configuration of its selection target (i.e. a L-peptide). Recently, nuclease-resistant inhibitory RNA aptamers have been developed against a great variety of targets implicated in disease. Some results have already been obtained in animal models and in clinical trials.

Animals↗
PubMed2006

Small molecules: big players in the evolution of protein synthesis.

The aminoacyl-tRNA synthetases (aaRSs) are responsible for selecting specific amino acids for protein synthesis, and this essential role in translation has garnered them much attention as targets for novel antimicrobials. Understanding how the aaRSs evolved efficient substrate selection offers a potential route to develop useful inhibitors of microbial protein synthesis. Here, we discuss discrimination of small molecules by aaRSs, and how the evolutionary divergence of these mechanisms offers a means to target inhibitors against these essential microbial enzymes.

Amino Acyl-tRNA Synthetases↗
PubMed2005

In-depth analysis of a heterosexually acquired human immunodeficiency virus type 1 superinfection: evolution, temporal fluctuation, and intercompartment dynamics from the seronegative window period through 30 months postinfection.

Human immunodeficiency virus type 1 (HIV-1) superinfection refers to the acquisition of another strain by an already infected individual. Here we report a comprehensive genetic analysis of an HIV-1 superinfection acquired heterosexually. The infected individual was in a high-risk cohort in Tanzania, was exposed to multiple subtypes, and was systematically evaluated every 3 months with a fluorescent multi-region genotyping assay. The subject was identified in the window period and was first infected with a complex ACD recombinant strain, became superinfected 6 to 9 months later with an AC recombinant, and was monitored for >2.5 years. The plasma viral load exceeded 400,000 copies/ml during the first 9 months of infection but resolved to the set point of 67,000 copies/ml by 3 months after superinfection; the CD4 cell count was 377 cells/mul at 30 months. Viral diversity was evaluated with techniques designed to fully sample the quasi-species, permitting direct observation of the evolution, temporal fluctuation, and intercompartment dynamics of the initial and superinfecting strains and recombinants derived from them. Within 3 months of superinfection, seven different molecular forms were detected in gag and six were detected in env. The proportions of forms fluctuated widely over time in plasma and peripheral blood mononuclear cells, illustrating how challenging the detection of dually infected individuals can be. Strain-specific nested PCR confirmed that the superinfecting strain was not present until the 9 month follow-up. This study further defines the parameters and dynamics of superinfection and will foster appropriate studies and approaches to gain a more complete understanding of risk factors for superinfection and its impact on clinical progression, epidemiology, and vaccine design.

Evolution, Molecular↗
PubMed2005

Evolution of a microbial acetyltransferase for modification of glyphosate: a novel tolerance strategy.

N-Acetylation is a modification of glyphosate that could potentially be used in transgenic crops, given a suitable acetyltransferase. Weak enzymatic activity (k(cat) = 5 min(-1), K(M) = 1 mM) for N-acetylation of glyphosate was discovered in several strains of Bacillus licheniformis (Weigmann) Chester by screening a microbial collection with a mass spectrometric assay. The parental enzyme conferred no tolerance to glyphosate in any host when expressed as a transgene. Eleven iterations of DNA shuffling resulted in a 7000-fold improvement in catalytic efficiency (k(cat)/K(M)), sufficient for conferring robust tolerance to field rates of glyphosate in transgenic tobacco and maize. In terms of k(cat)/K(M), the native enzyme exhibited weak activity (4-450% of that with glyphosate) with seven of the common amino acids. Evolution of the enzyme towards an improved k(cat)/K(M) for glyphosate resulted in increased activity toward aspartate (40-fold improved k(cat)), but activity with serine and phosphoserine almost completely vanished. No activity was observed among a broad sampling of nucleotides and antibiotics. Improved catalysis with glyphosate coincided with increased thermal stability.

Acetyltransferases↗
PubMed2001

The effect of cytidine on the structure and function of an RNA ligase ribozyme.

A cytidine-free ribozyme with RNA ligase activity was obtained by in vitro evolution, starting from a pool of random-sequence RNAs that contained only guanosine, adenosine, and uridine. This ribozyme contains 74 nt and catalyzes formation of a 3',5'-phosphodiester linkage with a catalytic rate of 0.016 min(-1). The RNA adopts a simple secondary structure based on a three-way junction motif, with ligation occurring at the end of a stem region located several nucleotides away from the junction. Cytidine was introduced to the cytidine-free ribozyme in a combinatorial fashion and additional rounds of in vitro evolution were carried out to allow the molecule to adapt to this added component. The resulting cytidine-containing ribozyme formed a 3',5' linkage with a catalytic rate of 0.32 min(-1). The improved rate of the cytidine-containing ribozyme was the result of 12 mutations, including seven added cytidines, that remodeled the internal bulge loops located adjacent to the three-way junction and stabilized the peripheral stem regions.

Base Sequence↗
PubMedDate not supplied

Study of lung antibodies in patients with silicosis.

The presence of lung antibodies was determined in a group of 68 patients with silicosis in various stages of evolution and in a control group of 35 healthy subjects, by the standard indirect immunofluorescent technique with fluorescent conjugate anti human gammaglobulin and monospecific fluorescent conjugates anti human IgG, IgA and IgM. In the group of patients investigated, lung antibodies were detected by means of fluorescent conjugate anti human gammaglobulin, in 47 cases of the 68 examined. As regards the investigations using monospecific fluorescent conjugates it was observed that all the 47 patients presented lung antibodies of the IgG class, 32 presented lung antibodies of the IgA class and 15 presented lung antibodies of the IgM class. The presence of lung antibodies in silicotic patients suggests the participation of acquired humoral immunity in the pathogeny of disease without, however, being directly responsible for the evolution of disease. The association of silicosis with various other autoimmune diseases pleads for the existence of an immune component in the evolution of this pneumoconiosis.

Autoantibodies↗
PubMedDate not supplied

Considerations on the changes of certain immunologic cells in silicosis.

Cell-mediated immunity was estimated in a group of 68 patients divided into three subgroups according to the evolutive stage of disease. It was demonstrated that in silicosis there is a disturbance of immunity. The detection of E rosettes and of activated rosettes was carried out according to the classical method. The mean percentage of total T lymphocytes was found lower in silicosis (62 +/- 2% statistically significant for p < 0.05), compared to the control group (66 +/- 4.6%). The study revealed a decrease of T helper lymphocytes, an increase of T suppressor/cytotoxic lymphocytes and a decrease of the T helper/T suppressor ratio (1.96 +/- 0.13) in silicosis as compared to the control group (2.83 +/- 0.31). The presence of these changes in silicotic patients suggested the participation of cell-mediated immunity in the pathogenesis of disease, the cell mediated immunity being directly responsible for the evolution of silicosis.

Adult↗