Contribution of temperature gradient to aggregation of thermal heterocopolymers of amino acids in aqueous milieu.

We prepared thermal heterocopolymers of amino acids, aspartic acid and proline, and solubilized them in distilled water at boiling temperature. The resulting suspension was then cooled down while controlling the cooling rate. The maximum growth of phase-separated microspheres was found to occur at a finite, nonzero cooling rate. Temperature-gradient controlled growth of these microspheres indicates that aggregation of thermal heterocopolymers of amino acids in their aqueous milieu is irreversible. In view of the fact that evolutionary process is an indication of lasting physical irreversibility, thermal heterocopolymers of amino acids were shown to have an evolutionary significance in maintaining the capacity of irreversibility lasting over at least several hours.     

Amino acids are general growth inhibitors of Nicotiana silvestris in tissue culture

The growth of Nicotiana silvestris in suspension culture is inhibited by all of the common protein amino acids at the millimolar level, except for L-glutamine. A defined experimental system for growth/inhibition studies has been established, and growth studies were carried out with cells that had been maintained in the exponential growth phase for at least 10 generations (EE cells). The following results were obtained after particularly detailed studies with aromatic amino acids. The onset of inhibition was preceded by a duration of normal growth rate which varied within a range of 12 to 48 h. The degree of inhibition was directly proportional to amino acid concentration and inversely related to the initial cell density of the inoculum. A slowed, but still exponential rate of growth persisted during an early phase of inhibition. Under sufficiently severe conditions, this was followed by progressive diminution of growth rate and eventual lysis. The most drastic inhibitory effects caused by aromatic amino acids were in the order: phenylalanine, tryptophan and tyrosine. When EE cells cultivated under conditions of growth inhibition were diluted into fresh medium, immediate resumption of growth at the uninhibited rate occurred and persisted. On the other hand, when growth-inhibited EE cells were diluted into medium containing the same concentration of amino acid used in the first round of growth, an initial burst of uninhibited growth lasting about 24 h was followed by a drastic, progressively declining growth rate which deteriorated to cell death and lysis. When cells in stationary phase were used as an inoculum, as is done in typical growth characterizations with suspension cultures, the sensitivity to inhibition during the subsequent exponential growth phase was several-fold greater than was the case with EE cells. Hypotheses that growth inhibition might be caused by ammonia toxicity, keto-acid toxicity, or by inhibition of nitrate utilization were ruled out. Observations that provide new insight are: (i)growth-inhibited cells undergo drastic plasmolysis, (ii) L-glutamine is an effective antagonist of amino-acid inhibitors, and (iii) growth-inhibited cells exhibit a transient restoration of normal growth rate upon dilution into fresh growth medium. These results implicate a linkage of amino acids with osmotic regulation and nitrogen metabolism.     

Natural self-organization of polynucleotides and polypeptides in protobiogenesis: Appearance of a protohypercycle

Basic thermal polyamino acids or proteinoids have been reported to be catalytic for both self-instructing polymerization of amino acids and internucleotide synthesis. We show theoretically that a complex suspension of thermal proteinoids, free amino acids, nucleotides and ATP as an energy source can exhibit an evolutionary character. The suspension can produce a prototype of Eigen's hypercycle, or protohypercycle, for which translation proceeds from amino acid to nucleotide. The protohypercycle is suggested to be an evolutionary precursor of the hypercycle, in which translation is from nucleotide to amino acid. The possibility that the Fox-Nakashima microsphere containing both lysine-rich and acidic proteinoids may work as a model of a protohypercycle is considered.     

Protocell-like Microspheres from Thermal Polyaspartic Acid

One of the most prominent amino acids to appear in monomer-generating origin-of-life experiments is aspartic acid. Hugo Schiff found in 1897 that aspartic acid polymerizes when heated to form polyaspartylimide which hydrolyzes in basic aqueous solution to form thermal polyaspartic acid which is a branched polypeptide. We recently reported at the ISSOL 2005 Conference that commercially made thermal polyaspartic acid forms microspheres when heated in boiling water and allowed to cool. In a new experiment we heated aspartic acid at 180°C for up to 100 h to form thermal polyaspartylimide which when heated in boiling water without addition of base hydrolyzed to form thermal polyaspartic acid which upon cooling formed microspheres. Thermal polyaspartic acid microspheres appear protocell-like in the sense of being prebiotically plausible lattices or containers that could eventually have been filled with just the right additions of primordial proteins, nucleic acids, lipids, and metabolites so as to constitute protocells capable of undergoing further chemical and biological evolution. Thermal polyaspartic acid microspheres are extremely simple models of protocells that are more amenable to precise quantitative experimental investigation than the proteinoid microspheres of Sidney W. Fox. We present here scanning electron microscope images of such thermal polyaspartic acid microspheres. Figure 1 shows thermal polyaspartic acid microspheres from l-aspartic acid heated at 180°C for 50 h, at a magnification of 3,500×. Figure 2 shows thermal polyaspartic acid microspheres from the same sample at a magnification of 7,000×. The thermal polyaspartic acid microspheres have a diameter of approximately 1 μm These images were viewed with a Hitachi S2460N scanning electron microscope at 20 kV acceleration voltage. Figure 1 Thermal polyaspartic acid microspheres from l-aspartic acid heated at 180°C for 50 h, at a magnification of 3,500×.

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Figure 2 Thermal polyaspartic acid microspheres from l-aspartic acid heated at 180°C for 50 h, at a magnification of 7,000×.

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Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005.     

Aldocyanoin microspheres: Partial amino acid analysis of the microparticulates formed from simple reactants under various conditions

Summary The work of Kenyon and Nissenbaum on aldocyanoin microspheres was repeated and extended. It was determined that the microspheres contained amino acids and that specific amino acids could be incorporated into the microspheres by adding the requisite aldehyde or ketone precursor to the model mixture. Microsphere formation was found to be dependent on the availability of oxygen. Under anaerobic conditions of synthesis, no micro-spheres formed in the time allotted and the amino acid composition of the macromolecular material was simple. Microparticulate material synthesized by C. Folsome using a quenched spark technique was analyzed and found to contain amino acids that had a qualitative composition similar to both a Miller-Urey discharge and the Kenyon-Nissenbaum microspheres.     

Replaceability of amino acids and the self-facilitation of evolution

The self-facilitating aspect of evolution is formulated in terms of measures of amino acid replaceability. The incorporation of highly replaceable amino acids into a protein increases the rate at which it can incorporate highly irreplaceable amino acids. The reality of self-facilitation is supported by sequence data which imply that silent mutations which change the replaceability of codons are subject to selection. There are a number of interesting implications for the dependence of evolutionary rate on protein diversity and variability. In general, the incorporation of low information value amino acids into a protein increases the maximum rate at which the value of the information in the protein can increase.     

Amino acid limited growth of starter cultures in milk

The specific growth rates of several Streptococcus cremoris strains were 10–40% lower in milk than in other growth in media. The growth rates in milk increased when an amino acid mixture or casein was added, whereas, when milk was diluted, the specific growth rate of the streptococci decreased. This decrease could be overcome by bringing the casein concentration in the diluted milk back to the normal value (3%). This indicates that casein-hydrolysis proceeded at a rate too low for the streptococci to reach their potential maximum specific growth rates in milk so that growth in milk is essentially amino acid-limited. This was subsequently demonstrated for S. cremoris by continuous cultivation in media with low casein concentrations. At a low dilution rate casein hydrolysis was fast enough to supply the cells with enough amino acids and lactose was growth-limiting, whereas at higher dilution rates amino acids became growth-limiting. In cultures exponentially growing in milk the concentration of free amino acids was measured to determine which amino acid(s) was(were) absent and could possibly limit growth. A number of essential amino acids (leucine, methionine, glutamate and in some cases phenylalanine) were not detected and addition of these, together, stimulated the growth of S. cremoris in milk. The amino acids leucine and phenylalanine appeared to play a particularly important role in this stimulation. These two are, supposedly, the first amino acids that become limiting during growth in milk. The effect of competition for casein and amino acids by different organisms was studied in continuous cultures. At different dilution rates different strains became dominant in these mixed cultures, suggesting that differences in apparent affinity constants (K_S) for casein, leucine and glutamate existed between the strains.     

Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein.

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.     

Some analogies between prebiotic thermal conversions of glycine and metabolic pathways

The reaction schemes suggested earlier for thermal transformation of glycine into amino acids and carboxylic acids are considered in detail. Close analogy with some wide-spread biochemical reactions of amino acids is observed. The pathway suggested has some common stages with the tricarboxylic acid cycle and other metabolic processes. The possible role of alpha-imino or alpha-keto acids as prebiological analogs of pyridoxal-phosphate-containing enzymes is discussed. The thermal transformations of glycine under primitive Earth conditions could be considered as evolutionary precursors of some present-day metabolic pathways.     

Inclusion of nonproteinous amino acids in thermally prepared models for prebiotic protein

Sixteen nonproteinous amino acids (those not coded for in contemporary protein biosynthesis) were incorporated during the thermal formation of polyamino acids under postulated prebiotic conditions, although not all into a single polyamino acid. The copresence of proteinous or even α-amino acids was not required. (Norleucine color equivalents and elution times on a Beckman model 120C amino acid analyzer were determined for these nonproteinous amino acids). The results suggest that prebiotically available nonproteinous amino acids would have been constituents of prebiotic protein if the latter were formed thermally. Some differences in properties of the polyamino acids could be attributed to particular nonproteinous amino acid residues; however, the tested properties did not suggest a means for evolutionary selection against nonproteinous amino acids as a group. Selection against this class of amino acids in toto was likely a later, biotic, event.     

Adaptation of a Cyanobacterium to a Biochemically Rich Environment in Experimental Evolution as an Initial Step toward a Chloroplast-Like State

Chloroplasts originated from cyanobacteria through endosymbiosis. The original cyanobacterial endosymbiont evolved to adapt to the biochemically rich intracellular environment of the host cell while maintaining its photosynthetic function; however, no such process has been experimentally demonstrated. Here, we show the adaptation of a model cyanobacterium, Synechocystis sp. PCC 6803, to a biochemically rich environment by experimental evolution. Synechocystis sp. PCC 6803 does not grow in a biochemically rich, chemically defined medium because several amino acids are toxic to the cells at approximately 1 mM. We cultured the cyanobacteria in media with the toxic amino acids at 0.1 mM, then serially transferred the culture, gradually increasing the concentration of the toxic amino acids. The cells evolved to show approximately the same specific growth rate in media with 0 and 1 mM of the toxic amino acid in approximately 84 generations and evolved to grow faster in the media with 1 mM than in the media with 0 mM in approximately 181 generations. We did not detect a statistically significant decrease in the autotrophic growth of the evolved strain in an inorganic medium, indicating the maintenance of the photosynthetic function. Whole-genome resequencing revealed changes in the genes related to the cell membrane and the carboxysome. Moreover, we quantitatively analyzed the evolutionary changes by using simple mathematical models, which evaluated the evolution as an increase in the half-maximal inhibitory concentration (IC₅₀) and estimated quantitative characteristics of the evolutionary process. Our results clearly demonstrate not only the potential of a model cyanobacterium to adapt to a biochemically rich environment without a significant decrease in photosynthetic function but also the properties of its evolutionary process, which sheds light of the evolution of chloroplasts at the initial stage.     

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.     

On the trends in protein molecular evolution: Amino acid composition

Data on the amino acid composition of proteins having various functions from organisms representing different evolutionary levels (83 superfamilies) are used in order to elucidate the trends in protein molecular evolution. The interconnections evolutionary rate (rate of mutation acceptance) — amino acid composition, and evolutionary level of the organism — amino acid composition (in case of proteins of the same or very similar function) are studied. The amino acid compositions of proteins performing jointly an evolutionarily old functions are also juxtaposed. The mean contemporary protein composition is used as a basis for comparison. The obtained results are evidence in favour of the existence of a trend for an increase of the special amino acids (Met, Ile, Gln, His, Lys, Asn, Phe, Tyr, Trp, Cys) at the expense of the usual ones (Thr, Pro, Ala, Ser, Arg, Gly, Leu, Val, Glu, Asp). The tests of statistical significance of the obtained results (comparison of the mean compositions of proteins from low evolutionary level organisms with that of all sequenced proteins; comparison of the mean contemporary protein composition with that obtained after simulation of the evolutionary process) confirm and universalize the observed trend. The above results direct the attention to the concept of a smaller number of amino acids in the ancient proteins and respectively simpler genetic code. A fluctuation around the initial primitive level is suggested to explain the conservatism of proteins of the same function in evolutionarily low level organisms. The observed trend could be applied for designing new proteins.     

Self-sequencing of amino acids and origins of polyfunctional protocells

The primal role of the origins of proteins in molecular evolution is discussed. On the basis of this premise, the significance of the experimentally established self-sequencing of amino acids under simulated geological conditions is explained as due to the fact that the products are highly nonrandom and accordingly contain many kinds of information. When such thermal proteins are aggregated into laboratory protocells, an action that occurs readily, the resultant protocells also contain many kinds of information. Residue-by-residue order, enzymic activities, and lipid quality accordingly occur within each preparation of proteinoid (thermal protein).In this paper are reviewed briefly the phenomenon of self-sequencing of amino acids, its relationship to evolutionary processes, other significance of such self-ordering, and the experimental evidence for original polyfunctional protocells.     

The rate of molecular evolution of alpha-fetoprotein approaches that of pseudogenes

We conducted the present study in an attempt to correlate function with the rate of molecular evolution for serum albumin and alpha-fetoprotein. We found a high rate of silent substitution (between 5 X 10(-9) and 7 X 10(-9)/site/year) for both the albumin and alpha-fetoprotein genes, perhaps the highest so far reported for an expressed nuclear gene. The rates of effective substitution and amino acid changes were also very high, but in contrast to silent substitutions, they are higher for alpha-fetoprotein than for albumin by approximately 70%. For alpha-fetoprotein, the rate of effective substitution (1.5 X 10(-9)/site/year) may be approaching that for nonfunctional pseudogenes (about 3 X 10(-9)/site/year). Evolutionary divergence was also estimated at the amino acid level. It was found that the rate of change of alpha-fetoprotein (55% amino acids replaced in 100 Myr) approaches that of the fastest-evolving fibrinopeptides (92% amino acids replaced in 100 Myr). This high rate may indicate that alpha-fetoprotein can tolerate a great deal of molecular variation without its function being impaired in the process. Albumin evolves at a slower rate (39% amino acids replaced in 100 Myr), although still faster than either hemoglobin (17% amino acids replaced in 100 Myr) or cytochrome c (5% amino acids replaced in 100 Myr). The slower evolutionary rate may indicate that albumin has more refined functional specifications and hence can tolerate fewer mutational changes. The latter conclusion remains, however, to be reconciled with the condition of inherited analbuminemia, where a virtually complete absence of albumin produces surprisingly few symptoms.     

Parallel acceleration of evolutionary rates in symbiont genes underlying host nutrition

The overproduction of essential amino acids by Buchnera aphidicola, the primary bacterial mutualist of aphids, is considered an adaptation for increased production of nutrients that are lacking in aphids' diet of plant sap. Given their shared role in host nutrition, amino acid biosynthetic genes of Buchnera are expected to experience parallel changes in selection that depend on host diet quality, growth rate, and population structure. This study evaluates the hypothesis of parallel selection across biosynthetic pathways by testing for correlated changes in evolutionary rates at biosynthetic genes of Buchnera. Previous studies show fast evolutionary rates at tryptophan biosynthetic genes among Buchnera associated with the aphid genus Uroleucon and suggest reduced purifying selection on symbiont nutritional functions in this aphid group. Here, we test for parallel rate acceleration at other amino acid biosynthetic genes of Buchnera-Uroleucon, including those for leucine (leuABC) and isoleucine/valine biosynthesis (ilvC). Ratios of nonsynonymous to synonymous substitutions (d(N)/d(S)) were estimated using codon-based maximum-likelihood methods that account for the extreme AT compositional bias of Buchnera sequences. A significant elevation in d(N)/d(S) at biosynthetic loci but not at two housekeeping genes sampled (dnaN and tuf) suggests reduced host-level selection on biosynthetic capabilities of Buchnera-Uroleucon. In addition, the discovery of trpEG pseudogenes in Buchnera-U. obscurum further supports reduced selection on amino acid biosynthesis.     

Nitrogen Metabolism of Lemna minor. I. Growth, Nitrogen Sources and Amino Acid Inhibition

Lemna minor grown in sterile culture on a minerals-sucrose medium can utilize as nitrogen source, in order of increasing growth rate: ammonia, nitrate, a mixture of glutamic and aspartic acids plus arginine, or a balanced mixture of amino acids (hydrolyzed casein). Maximum growth is found with nitrate plus hydrolyzed casein.Many synthetic mixtures of amino acids are unable to support growth. Many single amino acids are inhibitory, and when added (at 2 mm or less) to cultures, growing in the presence of nitrate, cause a decrease in growth rate or even death of the plants (e.g. with alanine, valine, methionine or leucine). Some of these inhibitory effects are also found when the amino acid is added to cultures growing on ammonia or hydrolyzed casein. Arginine was the only amino acid of those tested which gave a marked stimulation of growth when added to cultures growing with inorganic nitrogen.The rapid rate of growth, sterile nature of tissue, decreased biological variation of samples containing many plants and ability to utilize different culture media make this an attractive organism for studies on higher plant metabolism.     

Influence of Polyamine Limitation on the Chain Growth Rates of β-Galactosidase and of Its Messenger Ribonucleic Acid

The rates of elongation of beta-galactosidase and its messenger ribonucleic acid (RNA) were estimated in a polyamine-deficient mutant of Escherichia coli through an analysis of the kinetics of enzyme induction. The chain growth of beta-galactosidase was calculated from the time required after the appearance of an amino terminal fragment of 60 amino acids (auto-alpha) until completed enzyme began to accumulate. The elongation rate of beta-galactosidase messenger RNA was estimated from the time after induction at which streptolydigen-resistant, enzyme-forming capacity first appeared. Upon polyamine starvation, the rate of polypeptide elongation slowed from 17 to 10 amino acids per s and the messenger RNA elongation rate decreased from 47 to 30 nucleotides per s. These reductions in polymerization rates were proportional to the decrease in cellular growth rate produced by polyamine starvation. It was concluded that, although it is quite unlikely that polyamine levels are involved in regulation of cell growth, they may be acting as cofactors in the synthesis of RNA or protein, or both.     

The capability of the algaeChlorella vulgaris andScenedesmus obliquus of utilizing amino acids as the sole nitrogen source

The capability of utilizing 20 amino acids and 2 amides as the sole nitrogen source for growth was studied in two green algae (Chlorophyceae). A comparison was made of the growth rate of algae in a mineral nutrient solution containing nitrate as the nitrogen source, with that in the same solution in which nitrogen in the form of nitrate was substituted by an equivalent nitrogen amount in the form of various amino acids. In addition to this, another series of experiments was carried out in whioh both culture media were supplied with glucose. The results show that both algae utilize a series of amino acids in dependence of their structure (mostly 3-carbon amino acids). The growth rate ofChlorella in the presence of these sources is the same as in nitrate, that ofScenedesmus even much higher. In the cultures containing glucose both algal species exhibit a higher growth rate in the media with the nitrate nitrogen source than in those with amino acids (with the exception of glycine inScenedesmus).