Non-enzymatic transfer of sequence information under plausible prebiotic conditions

We simulated in our laboratory a prebiotic environment where dry and wet periods were cycled. Under anhydrous conditions, lipid molecules present in the medium could form fluid lamellar matrices and work as organizing agents for the condensation of nucleic acid monomers into polymers. We exposed a mixture of 2′-deoxyribonucleoside 5′-monophosphates and a ssDNA oligomer template to this dry environment at 90 °C under a continuous gentle stream of CO₂ and we followed it with rehydration periods. After five dry/wet cycles we were able to detect the presence of a product that was complementary to the template. The reaction had a 0.5% yield with respect to the template, as measured by staining with the Pico Green^® fluorescent probe. Absent initial template, the product of the reaction remained below the detection limit. In order to characterize the fidelity of replication, the synthesized strand was ligated to adapters, amplified by PCR, and sequenced. The alignment of the sequenced DNA to the expected complementary sequence revealed that the misincorporation rate was 9.9%. We present these results as a proof of concept for the possibility of having non-enzymatic transfer of sequence information in a prebiotically plausible environment.     

Encapsulation of macromolecules by lipid vesicles under simulated prebiotic conditions

Summary Phospholipid vesicles (liposomes) were subjected to dehydration-hydration cycles in the presence of 6-carboxyfluorescein or salmon sperm DNA. We found that the vesicles fused into multilamellar structures during dehydration with solutes trapped between the lamellae. Upon rehydration the lamellae swelled and formed large vesicular structures containing solute. This model can be used to study encapsulation of macromolecules by lipid membranes to form protocellular structures under prebiotic conditions.     

Substrate-Directed formation of small biocatalysts under prebiotic conditions

One of the most debated issues concerning the origin of life, is how enzymes which are essential for existence of any living organism, evolved. It is clear that, regardless of the exact mechanism, the process should have been specific and reproducible, involving interactions between different molecules. We propose that substrate templating played a crucial role in maintaining reproducible and specific formation of prebiotic catalysts. This work demonstrates experimentally, for the first time, substrate-directed formation of an oligopeptide that possesses a specific catalytic activity toward the substrate on which it was formed. In our experiments we used the substrate o-nitrophenol-β-d-galactopyranoside (ONPG) as a molecular template for the synthesis of a specific catalyst that is capable of cleaving the same substrate. This was achieved by incubation of the substrate with free amino acids and a condensing agent (dicyandiamide) at elevated temperatures. A linear increase with time of the reaction rate (d[product]/d²t), pointed to an acceleration regime, where the substrate generates the formation of the catalyst. The purified catalyst, produced by a substrate-directed mechanism, was analyzed, and identified as Cys₂-Fe⁺². The mechanism of substrate-directed formation of prebiotic catalysts provides a solution to both the specificity and the reproducibility requirements from any prebiotic system which should evolve into the biological world. Received: 26 January 1996 / Accepted: 22 April 1997     

Formation of the imidazolides of dinucleotides under potentially prebiotic conditions

Summary Imidazolides of dinucleotides such as ImpApA can be formed from the corresponding dinucleotides in a two-stage process, which gives up to 15% yields under potentially prebiotic conditions. First a solution of the dinucleotide and sodium trimetaphosphate is dried out at constant temperature and humidity. This produces polyphosphates such as pnApA in excellent yield (80%). The products are dissolved in water, imidazole is added, and the solution is dried out again. This yields the 5-phosphorimidazolides.Abbreviations P₃! trimetaphosphate - A adenosine - U uridine - EDTA ethylenediaminetetraacetic acid - Ap adenosine 2(3)-phosphate - Ap! adenosine cyclic 2:3-phosphate - pA adenosine 5-phosphate - pA²p adenosine 2, 5-diphosphate - pA³p adenosine 3, 5-diphosphate - pAp! 5-phospho-adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - ImpA adenosine 5-phosphorimidazolide - A²pA adenylyl-[25]-adenosine - A³pA adenylyl-[35]-adenosine - A²pU adenylyl-[25]-uridine - A³pU adenylyl-[35]-uridine - pA²pA 5-phosphoadenylyl-[25]-adenosine - pA³pA 5-phospho-adenylyl-[35]-adenosine - pA²pU 5-phospho-adenylyl-[25]-uridine - pA³pU 5-phospho-adenylyl-[35]-uridine - pApN (N= A, U) 5-phosphate of a dinucleoside phosphate - p_nApN (N = A, U; n = 2, 3, 4.) 5-polyphosphate of a dinucleoside phosphate - ImpA²pA imidazolide of pA²pA - ImpA³pA imidazolide of pA³pA - ImpA²pU imidazolide of pA²pU - ImpA³pU imidazolide of pA³pU - ImpApN imidazolide of pApN     

A possible role of fluctuating clay-water systems in the production of ordered prebiotic oligomers

Summary A model is proposed for the intermediate stages of prebiotic evolution, based on the characteristics of the adsorption and condensation of amino acids and nucleotides on the surface area of clay minerals in a fluctuating environment. Template replication and translation of adsorbed oligonucleotides and catalytic effects by peptide products on further condensation are proposed, due to specific properties of hypohydrous clay surfaces as well as the biomolecules themselves. Experimental evidence supports some of the proposed interactions, and all of them can be tested experimentally.on leave from the Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel, 1975–76     

Condensation of amino acids to form peptides in aqueous solution induced by the oxidation of sulfur(iv): An oxidative model for prebiotic peptide formation

Condensation of amino acids to peptides is an important step during the origin of life. However, up to now, successful explanations for plausible prebiotic peptide formation pathways have been limited. Here we report that the oxidation of sulfur (IV) can induce the condensation reaction of carboxylic acids and amines to form amides, and the condensation reaction of amino acids to form peptides. This might be a general reaction contributing to prebiotic peptide formation.     

A physical model for the condensation and decondensation of eukaryotic chromosomes

During the eukaryotic cell cycle, chromatin undergoes several conformational changes, which are believed to play key roles in gene expression regulation during interphase, and in genome replication and division during mitosis. In this paper, we propose a scenario for chromatin structural reorganization during mitosis, which bridges all the different scales involved in chromatin architecture, from nucleosomes to chromatin loops. We build a model for chromatin, based on available data, taking into account both physical and topological constraints DNA has to deal with. Our results suggest that the mitotic chromosome condensation/decondensation process is induced by a structural change at the level of the nucleosome itself.     

A simple,chisel-assisted mechanical microdissection system for harvesting homogenous plant tissue suitable for the analysis of nucleic acids and proteins

Many physiological processes are limited to specific tissues or even specific cell types. Analysing entire plants or organs results in averaged data of all cell types contained in the sample; thus, specific metabolic functions cannot be assigned to individual cell types. A higher spatial resolution is required. By microdissecting plant organs, homogeneous material can be obtained. If a suitable amount of material is collected, standard analytical methods can be applied to elucidate cell type-specific processes. The collection of sufficient quantities of homogeneous material can be done by means of mechanical microdissection. This technique is a low-cost alternative to laser-coupled microdissection techniques. Here we describe a protocol for chisel-assisted mechanical microdissection of embedded plant material and demonstrate that the collected material is suitable to obtain nucleic acids and proteins.     

A possible route for the formation of protoprotein and its microspheres from primitive gases under possible prebiotic conditions

In this study, a possible reaction pathway for the formation of protein-like copolyamino acids from gaseous starting materials is presented. The sequence of the reaction pathway is composed of four steps. These are a) formation of fumaronitrile, b) hydrolysis of fumaronitrile to thermal precursors of aspartic acid such as ammonium fumarate or malate, c) thermal copolycondensation of the precursors of aspartic acids with various amino acids (s), and d) formation of microspheres from the copolyamino acids. The steps b), c) and d) were demonstrated experimentally in this study. The materials and the conditions used in this study could be regarded as prebiological. The thermal copolycondensation is also a convenient synthetic method of aspartic acid containing copolyamino acids.     

Chromatographic separation as selection process for prebiotic evolution and the origin of the genetic code

A model for the evolution of a translation apparatus has been suggested where oligonucleotides in a hairpin conformation act as primordial adapters. Specifically activated amino acids are assumed to be attached to these hairpin molecules. For the specific activation, a chromatographic separation of, e.g. ala and CMP from gly and GMP can be accomplished on silica (e.g. of volcanic origin) with aqueous salt solutions. Other adsorbents like clays (kaolin, bentonite, montmorillonite), different silicates (florisil, magnesium trisilicate, calcium silicate, talc), hydroxyapatite, barium sulfate, calcium carbonate, calcium fluoride and titanoxide have been examined as model systems for the separation of nucleotides, nucleosides and amino acids on mineral surfaces. The possible role of chromatographic separation of amino acids for the formation of proteinoids, composed of selected amino acids, is also considered.     

Genes in diploid triticinae compensating for the low temperature regulating gene Ltp in chromosome 5D of Triticum aestivum

Summary Hybrids of Triticum aestivum (monosomic 5D or ditelosomic 5DL) x T. speltoides (= Ae. speltoides) showed that the genotype of T. speltoides carries gene(s) which can partially compensate for the expected decrease in chromosome association at low temperatures (10°C) in the absence of chromosome 5D. In hybrids of T. aestivum (normal, ditelosomic 3DL or ditelosomic 3DL-monosomic 5D) x T. longissimum (= Ae. sharonensis), this compensation was not observed.In normal F₁ hybrids of T. durum x T. longissimum partial chromosome association occurred at 10°C and this stabilizer effect may be explained by the presence of a Ltp-like gene on chromosome 5A. When a line of T. durum carrying a homozygous translocated 5B-5D chromosome was used in the crosses an even higher chromosome association was observed.These results suggest either the existence of a promoter gene for chromosome association in the 5D translocated segment or the loss of a weak suppressor gene in the removed segment of 5B. It was concluded that the translocated 5D segment did not carry the Ltp stabilizer gene.The work was supported by a fellowship of the Gulbenkian Foundation and partly carried out while the author was at the Department of Genetics, Agricultural University, Wageningen, The Netherlands.     

A rapid coupling protocol for the synthesis of peptide nucleic acids

Summary With the current interest in anti-sense and anti-gene technologies, an efficient, fast and less toxic synthesis protocol would be advantageous for the oligomerisation of Peptide Nucleic Acids (PNA). Most of the methods currently in use for thet-Boc synthesis of PNA's use TFA/m-cresol, pyridine, piperidine and capping reagents. In this work, a rapid synthesis protocol has been adapted from an earlier published peptide synthesis method allowing a reduction in cycle time from around 30 min down to 16 min. By utilising quantitative deprotection with 100% TFA, a coupling time of 10 min and a four-fold excess of monomer, this synthesis protocol has been used to synthesise a number of PNA's incorporating all four nucleotides of varying sequence, up to 17 residues in length.     

A rapid coupling protocol for the synthesis of peptide nucleic acids

With the current interest in anti-sense and anti-gene technologies, an efficient, fast and less toxic synthesis protocol would be advantageous for the oligomerisation of Peptide Nucleic Acids (PNA). Most of the methods currently in use for the t-Boc synthesis of PNA's use TFA/m-cresol, pyridine, piperidine and capping reagents. In this work, a rapid synthesis protocol has been adapted from an earlier published peptide synthesis method allowing a reduction in cycle time from around 30 min down to 16 min. By utilising quantitative deprotection with 100% TFA, a coupling time of 10 min and a four-fold excess of monomer, this synthesis protocol has been used to synthesise a number of PNA's incorporating all four nucleotides of varying sequence, up to 17 residues in length.     

Gene regulation under growth conditions. A model for the regulation of initiation of replication in Escherichia coli

A stochastic model is presented to describe gene regulation during growth conditions (non-steady-state), with an emphasis on the distribution of gene activation times. A non-Poissonian distribution, with a smaller variability than in steady-state, is obtained when gene activation by the regulatory protein(s) occurs before the protein(s) reach their saturation concentration. The model is applied to the regulation of initiation of chromosomal replication in Escherichia coli. The rate of initiation is shown to depend linearly on the concentration of the initiator molecule, DnaA, in good agreement with recently published data. It is suggested that the variability of initiation times could be growth rate dependent, with slow growing cells being more synchronized than fast growing ones.     

A computer program for the calculation of sedimentation coefficients and molecular weights of nucleic acids

A procedure is described for the purification of nuclei and identification of chromatin proteins in transformed epithelial cell lines from mammalian bladder and salivary gland. Nuclear purification was performed by homogenization, in hypotonic buffer containing polyamines to stabilize the nuclear structure, followed by 0.1–0.2% Triton X-100 washing and centrifugation through 2.2 m sucrose. Chromatin was liberated from nuclei by freeze-thawing in hypotonic buffer and the chromatin proteins were extracted with 7 m urea/3 m NaCl. The chromatin proteins were identified using NEPHGE two-dimensional electrophoresis and fluorographic autoradiography. This procedure enabled detection of histones and a range of basic nonhistone chromatin proteins, following cell culture in the presence of low levels of l-(4,5-³H)leucine.     

A rationale for the symmetries by base substitutions of degeneracy in the genetic code

The first symmetry by base substitutions of degeneracy in the genetic code was described by Rumer (1966) and the other symmetries were identified later by Jestin (2006) and Jestin and Soulé (2007). Here, a rationale accounting for these symmetries is reported. The number of non-synonymous substitutions over the replicated coding sequence is written as a function of the substitution matrix, whose elements are the number of substitutions from any codon to any other codon. The p-adic distance used as a similarity measure and applied to this matrix is shown to be biologically relevant. The rationale indicates that symmetries by base substitutions of degeneracy in the genetic code are symmetries of the measures of the number of non-synonymous substitutions for sets of synonymous codons.     

A diffusion model for the evolution of metalloporphyrin

We give a mathematical model of the evolution of enzymes, the molecular structure of which is like metalloporphyrins or chlorophylls. We show, for this model, that even a small amount of these enzymes at the first stage is sufficient to increase and dominate the majority in a cell (like phenomena of gene fixation). For this purpose we use Kimura's equation, which has been explored for the study of evolution of genetics and has been known as a neutral theory of molecular evolution. Our model is a non-linear, non-equilibrium and non-closed (open to the external world) model.     

Non-random pre-transcriptional evolution in HIV-1. A refutation of the foundational conditions for neutral evolution

The complete base sequence of HIV-1 virus and GP120 ENV gene were analyzed to establish their distance to the expected neutral random sequence. An especial methodology was devised to achieve this aim. Analyses included: a) proportion of dinucleotides (signatures); b) homogeneity in the distribution of dinucleotides and bases (isochores) by dividing both segments in ten and three sub-segments, respectively; c) probability of runs of bases and No-bases according to the Bose-Einstein distribution. The analyses showed a huge deviation from the random distribution expected from neutral evolution and neutral-neighbor influence of nucleotide sites. The most significant result is the tremendous lack of CG dinucleotides (p < 10(-50) ), a selective trait of eukaryote and not of single stranded RNA virus genomes. Results not only refute neutral evolution and neutral neighbor influence, but also strongly indicate that any base at any nucleotide site correlates with all the viral genome or sub-segments. These results suggest that evolution of HIV-1 is pan-selective rather than neutral or nearly neutral.     

Mechanistic model for prediction of formate dehydrogenase kinetics under industrially relevant conditions

Formate dehydrogenase (FDH) from Candida boidinii is an important biocatalyst for the regeneration of the cofactor NADH in industrial enzyme‐catalyzed reductions. The mathematical model that is currently applied to predict progress curves during (semi‐)batch reactions has been derived from initial rate studies. Here, it is demonstrated that such extrapolation from initial reaction rates to performance during a complete batch leads to considerable prediction errors. This observation can be attributed to an invalid simplification during the development of the literature model. A novel mechanistic model that describes the course and performance of FDH‐catalyzed NADH regeneration under industrially relevant process conditions is introduced and evaluated. Based on progress curve instead of initial reaction rate measurements, it was discriminated from a comprehensive set of mechanistic model candidates. For the prediction of reaction courses on long time horizons (>1 h), decomposition of NADH has to be considered. The model accurately describes the regeneration reaction under all conditions, even at high concentrations of the substrate formate and thus is clearly superior to the existing model. As a result, for the first time, course and performance of NADH regeneration in industrial enzyme‐catalyzed reductions can be accurately predicted and used to optimize the cost efficiency of the respective processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010