Ancient origin of lactalbumin from lysozyme: Analysis of DNA and amino acid sequences

Summary Parsimony trees relating DNA sequences coding for lysozymesc and -lactalbumins suggest that the gene duplication that allowed lactalbumin to evolve from lysozyme preceded the divergence of mammals and birds. Comparisons of the amino acid sequences of additional lysozymes and lactalbumins are consistent with this view. When all base positions are considered, the probability that the duplication leading to the lactalbumin gene occurred after the start to mammalian evolution is estimated to be 0.05–0.10. Elimination of the phylogenetic noise generated by fast evolution and compositional bias at third positions of codons reduced this probability to 0.002–0.03. Thus the gene duplication may have long preceded the acquisition of lactalbumin function.     

Evolution of DNA structure: Direction,mechanism, rate

Summary On the basis of the results of an analysis of frequencies of pyrimidine oligonucleotides, the degree of pyrimidine clustering of DNA in species from different taxa has been determined. A tendency for an increase in the index of clustering of DNA was revealed in the sequence: invertebrates, fishes, amphibians, reptiles, birds, mammals. A mechanism is postulated, according to which the increase in the degree of clustering of DNA during the evolution may be associated with the accumulation of mutations, Purine Pyrimidine transversions, resulting in a selective enrichment of one of the chains of DNA with pyrimidines and the otherwith purines, i.e. in an increase in the degree of purine-pyrimidine imbalance (asymmetry) of DNA complementary chains. This mechanism of DNA evolution is supported by the presence of positive correlation between the degree of clustering and the degree of the chain asymmetry of natural DNAs, as well as the character of the amino acid substitutions in cytochromes c in different species. The progressive evolution of different groups of organisms on the whole may have been accompanied by an acceleration of the rates of evolution of the DNA structure.On the basis of the amino acid sequence of cytochromes c in different species the degree of clustering and the degree of the chain asymmetry of the corresponding structural genes of DNA was found to have a general tendency towards an increase in the following order: invertebrates, fishes, amphibians, reptiles, birds, mammals. Thus, evolution of cytochrome c cistron is a vector process based on a selection of mutations which, on the one hand, are neutral to protein, and, on the other hand, result in the sense chain of DNA being enriched with pyrimidines and the nonsense one (and the corresponding mRNA) - with purines. Hence, it is the polynucleotide template rather than protein, that must have been the object of selection. The frequency of substitutions in cytochromes c cistron for vertebrates is 1.56×10^–9 per nucleotide per year. It is believed that the evolutionary modification of the DNA structure may be associated with an increase in the interference resistance of the translation, i.e. with selection for codons of highest readout stability.Abbreviations Used Py pyrimidine - Pu purine - H heavy, i.e. the pyrimidine rich strand of DNA - L light, i.e. the purine rich strand of DNA     

The regularities of the changes of amino acid physico-chemical properties within the genetic code

Summary All the codons of the genetic code can be arranged into the closed one-step mutation ring, containing three periods of the same sequence of mutations (2,3,3,3,1,3,3,3,1,3,3,3,1,3,3,3,2,3,3,3). The codons of Gly play a role of the connecting element between the end of the third, and the beginning of the first period of the genetic code. The reactivity of amino acids, expressed by the reaction rates of aminolysis reaction of N-hydroxysuccinimide esters of protected amino acids with p-anisidine, changes periodically with the respect to the mutation periods of the genetic code. Chou-Fasman P as well as P conformational parameters of amino acids, and also the compositional frequencies of amino acids in proteins, demonstrate the pseudosymmetry pattern with respect to the center of one-step mutation ring, which is situated between Thr ACY and ACR codons.     

A cybernetic approach to the origin of the genetic coding mechanism

The sequential fulfillment of theprinciple of succession necessarily guides the main steps of the genetic code evolution to be reflected in its structure. The general scheme of the code series formation is proposed basing on the idea of group coding (Woese, 1970). The genetic code supposedly evolved by means of successive divergence of pra-ARS's loci, accompanied by increasing specification of recognition capacity of amino acids and triplets.The sense of codons had not been changed on any step of stochastic code evolution. The formulated rules for code series formation produce a code version, similar to the contemporary one. Based on these rules the scheme of pra-ARS's divergence is proposed resulting in the grouping of amino acids by their polarity and size. Later steps in the evolution of the genetic code were probably based on more detailed features of the amino acids (for example, on theirfunctional similarities like their interchangeabilities in isofunctional proteins).     

Functional Correlation in Amino Acid Residue Mutations of Yeast Iso-2-Cytochrome c that Is Consistent with the Prediction of the Concomitantly Variable Codon Theory in Cytochrome c Evolution

Fitch and Markowitz' theory of concomitantly variable codons (covarions) in evolution predicted the existence of functional correlation in amino acid residue mutations among present-day cytochromes c. Mutational analysis was carried out on yeast iso-2-cytochrome c, where hydrophobic core residues I20, M64, L85, and M98 and surface residue L9 were mutated, in selected combinations, to those found in mammalian and bird cytochromes c. The functionality assay is based upon the ability of yeast cells to grow in YPGE medium. Furthermore, experiments on the single M64L and M98L mutations as well as the double M64L/M98L mutation using NMR showed that the effects of these mutations are to perturb the structural integrity of the protein. We identified functional correlation in two cases of a pair of residue mutations, the I20 V and M98 L pair and the L9 I and L85 I pair. In both cases, only one of the two alternative, putative evolutionary pathways leads to a functional protein and the corresponding pairs of residue mutations are among those found in present-day cytochromes c. Since valine is predicted to be at position 20 in the ancestral form of cytochrome c, the present data provide an explanation for the ancient requirement of leucine rather than methionine in position 98. The present data provide further evidence for the role of those specific atom–atom interactions in directing a pathway in the evolutionary changes of the amino acid sequence that have taken place in cytochrome c, in accordance with Fitch and Markowitz.     

Photosynthesis and respiration of Griffithsia monilis (Rhodophyceae): Effect of light,salinity, and oxygen

The giant-celled alga Griffithsia monilis has a low light compensation point and saturates photosynthesis at 60–90 mol photons m^-2s^-1 (oxygen evolution and CO₂ fixation). Under dark and low light intensities ¹⁴C is preferentially incorporated into amino acids (mainly aspartate and alanine). With increasing light a gradual change was observed and, under light saturation, compounds of the anionic fraction (digeneaside and hexosephosphates) were the most strongly labeled compounds, together with the amino acids glycine and serine. To a large extent (30–40% of the total) ¹⁴C was fixed into EtOH-insoluble products, the hydrolysates of which consisted mainly of glucose and mannose. In the steady state the rates of photosynthesis and respiration decreased with increasing salinity. Changes in the rates after hyperosmotic shocks were less severe in cells adapted to high salinities. Photorespiration exists in Griffithsia: Glycine and serine are the major labeled compounds in O₂-saturated media.     

Active transcription of the pseudogene for subunit 7 of the NADH dehydrogenase in Marchantia polymorpha mitochondria

A pseudogene, nad7, which has significant sequence similarity (66.7% amino acid identity) with the bovine nuclear gene for a 49 kDa subunit of the NADH dehydrogenase (NADH:ubiquinone oxidoreductase, EC 1.6.99.3), has been identified on the mitochondrial genome of the liverwort Marchantia polymorpha. The predicted coding region, which includes six termination codons, is actively transcribed into RNA molecules of 16 and 9.6 kb in length, but RNA splicing products were not detected in the liverwort mitochondria. Genomic DNA blot analysis and RNA blot analysis using poly(A)⁺ RNA suggest that a structurally related nuclear gene encodes the mitochondrial ND7 polypeptide. These results imply that this nad7 is a relic of a gene transfer event from the mitochondrial genome into the nuclear genome during mitochondrial evolution in M. polymorpha.Communicated by R. G. Herrmann     

A working hypothesis on the interdependent genesis of nucleotide bases,protein amino acids,and primitive genetic code

In the course of experimental approach to the chemical evolution in the primeval sea, we have found that the main products from formaldehyde and hydroxylamine are glycine, alanine, serine, aspartic acid etc., and the products from glycine and formaldehyde are serine and aspartic acid. Guanine is found in the two-letter genetic codons of all these amino acids.Based upon the finding and taking into consideration the probable synthetic pathways of nucleotide bases and protein amino acids in the course of chemical evolution and a correlation between the two-letter codons and the number of carbon atoms in the carbon skeleton of amino acids, 1 have been led to a working hypothesis on the interdependent genesis of nucleotide bases, protein amino acids, and primitive genetic code as shown in Table I.Protein amino acids can be classified into two groups: Purine Group amino acids and Pyrimidine Group amino acids. Purine bases and Pyrimidine bases are predominant in two-letter codons of amino acids belonging to the former and the latter group respectively.Guanine, adenine, and amino acids of the Purine Group may be regarded as synthesized from C₁ and C₂ compounds and N₁ compounds (including C₁N₁ compunds such as HCN), probably through glycine, in the early stage of chemical evolution.Uracil, cytosine, and amino acids of the Pyrimidine Group may be regarded as synthesized directly or indirectly from three-carbon chain compounds. This synthesis became possible after the accumulation of three-carbon chain compounds and their derivatives in the primeval sea.The Purine Group can be further classified into a Guanine or (Gly+nC₁) Subgroup and an Adenine or (Gly+nC₂) Subgroup or simply nC₂ Subgroup. The Pyrimidine Group can be further classified into a Uracil or C₃C₆C₉ Subgroup and a Cytosine or C₅-chain Subgroup (Table I).It is suggested that the primitive genetic code was established by a specific interaction between amino acids and their respective nucleotide bases. The interaction was dependent upon their concentration in the primeval environments and the binding constants between amino acids and their respective bases.Presented at the International Symposium (Lipmann Symposium) on The Concepts of Chemical Recognition in Biology held in Grignon near Versailles (France) on July 18–20, 1979.     

Dipeptide frequencies in proteins and the CpG deficiency in vertebrate DNA

Summary Analysis of vertebrate protein sequences totalling 4040 residues shows that amino acids with a high proportion of codons ending in C occur with significantly reduced frequency before amino acids whose codons start with G. This effect is not shown by control bacterial protein sequences. The consequent implication of shortage of XXC. GXX codon pairs in vertebrate messenger RNA is discussed in relation to the extreme rarity of the base doublet CpG in vertebrate DNA.     

Nonrandom Intragenic Variations in Patterns of Codon Bias Implicate a Sequential Interplay Between Transitional Genetic Drift and Functional Amino Acid Selection

Although most codon third bases appear to be functionless, the synonymous codons so defined exhibit a strikingly nonrandom distribution (codon bias) within human and other genes. To examine this phenomenon further, we generated a database of DNA sequences encoding human transmembrane cell-surface receptor proteins. Using this database we show here that the guanine and cytosine content of codon third bases (GC3) varies intragenically with the nature of the specified receptor domains (transmembrane > extracellular > intracellular domains; p < 0.001), the phenotype of the encoded amino acids (hydrophobic > hydrophilic > neutral amino acids; p < 0.001), and the receptor affiliation of the transmembrane domain superfamily (G-protein- coupled receptors > receptor tyrosine kinases; p < 0.001). Within gene regions specifying transmembrane domains, GC3 declines as domain functionality becomes redundant with increasing hydrophobicity (p < 0.001). Codons containing the second-base cytosine (XCZ, which encodes neutral amino acids) are selectively depleted of third-base adenine content (A3: XCA codons) when encoding transmembrane domain residues, consistent with positive selection for transitional mutation of XCG to XTG (which encodes hydrophobic amino acids) rather than to the synonymous XCA. Supporting this XCG XTG mechanism of codon bias, the G3:A3 ratio of codons specifying the transmembrane amino acid glycine (GGZ) is intermediate between that of its functional homolog alanine (GCZ) and that of hydrophobic valine (GTZ), even though the C3:T3 ratios are similar. Conversely, nearest-neighbor analysis of third bases 5 to codons specifying valine and leucine (CTZ) confirms a significant difference in C3:T3 but not G3:A3 ratios (i.e., C3/G1 T3/G1 > C3/A1; p < 0.001), consistent with the functionally advantageous retention of hydrophobic residues. These data raise the possibility that patterns of intragenic codon bias reflect a balance between negative and positive selection, suggesting in turn that analysis of codon third-base usage may help to predict the functional significance of encoded products. Supplementary information: Current address: (K. Lin) College of Life Sciences, Beijing Normal University, Beijing 100875, China     

Use of Chou-Fasman amino acid conformational parameters to analyze the organization of the genetic code and to construct protein genealogies

Summary Chou-Fasman parameters, measuring preferences of each amino acid for different conformational regions in proteins, were used to obtain an amino acid difference index of conformational parameter distance (CPD) values. CPD values were found to be significantly lower for amino acid exchanges representing in the genetic code transitions of purines, GA than for exchanges representing either transitions of pyrimidines, CU, or transversions of purines and pyrimidines. Inasmuch as the distribution of CPD values in these non GA exchanges resembles that obtained for amino acid pairs with double or triple base differences in their underlying codons, we conclude that the genetic code was not particularly designed to minimize effects of mutation on protein conformation. That natural selection minimizes these changes, however, was shown by tabulating results obtained by the maximum parsimony method for eight protein genealogies with a total occurrence of 4574 base substitutions. At the beginning position of the codons GA transitions were in very great excess over other base substitutions, and, conversely, CU transitions were deficient. At the middle position of the codons only fast evolving proteins showed an excess of GA transitions, as though selection mainly preserved conformation in these proteins while weeding out mutations affecting chemical properties of functional sites in slow evolving proteins. In both fast and slow evolving proteins the net direction of transitions and transversions was found to be from G beginning codons to non-G beginning codons resulting in more commonly occurring amino acids, especially alanine with its generalized conformational properties, being replaced at suitable sites by amino acids with more specialized conformational and chemical properties. Historical circumstances pertaining to the origin of the genetic code and the nature of primordial proteins could account for such directional changes leading to increases in the functional density of proteins.In order to further explore the course of protein evolution, a modified parsimony algorithm was developed for constructing protein genealogies on the basis of minimum CPD length. The algorithm's ability to judge with finer discrimination that in protein evolution certain pathways of amino acid substitution should occur more readily than others was considered a potential advantage over strict maximum parsimony. In developing this CPD algorithm, the path of minimum CPD length through intermediate amino acids allowed by the genetic code for each pair of amino acids was determined. It was found that amino acid exchanges representing two base changes have a considerably lower average CPD value per base substitution than the amino acid exchanges representing single base changes. Amino acid exchanges representing three base changes have yet a further marked reduction in CPD per base change. This shows how extreme constraining effects of stabilizing selection can be circumvented, for by way of intermediate amino acids almost any amino acid can ultimately be substituted for another without damage to an evolving protein's conformation during the process.     

Conservation of the secondary structure of protein during evolution and the role of the genetic code

In this communication we demonstrate, in a group of modern proteins, following an algorithm described by Argyle (1980), that the ordination of the amino acids in terms of the most frequent substitutions agrees with the conservation of the-helix,-sheet, and-turn formation tendencies during evolution. The same correspondence has been demonstrated for the conservation of the physico-chemical properties in the amino acid substitutions. Both parameters are similar in showing higher correlation with the most frequent amino acid substitutions than with the feasibility of changes at the level of the respective codons.Some kind of restrictions for the expression of the genomic changes, due to the conservation of the secondary structure of proteins and/or the physicochemical properties of the substituted amino acids, could account for the differences found between the distribution of the amino acid substitutions and the most probable codon changes.This work has been partially supported by Departamento de Investigación y Bibliotecas, Universidad de Chile y Fondo Nacional de Investigación Científica y Tecnológica.     

Botryococcus braunii carbon/nitrogen metabolism as affected by ammonia addition

Carbon metabolism in photosynthesizing and respiring cells of Botryococcus braunii was radically changed by the presence of 1 mM NH₄Cl in the medium, when the so-called resting state previously had been subjected to a nitrogen-deficient medium. Ammonia addition to the algae photosynthesizing with ¹⁴C-labelled HCO ₃ ^- almost completely inhibited the synthesis of ¹⁴C-labelled botryococcenes and other hexane-extractable compounds, and also inhibited the formation of insoluble compounds; however, it resulted in a large increase in the synthesis of alanine, glutamine, other amino acids, and especially of 5-aminolevulinic acid. Total CO₂ fixation decreased about 60% and O₂ evolution decreased more than 50%.CO₂ fixation in the dark with ammonia present led to labelled products derived from phosphoenolpyruvate carboxylation, such as glutamine, glutamate, and malate. Respiratory uptake of O₂ increased by about 70%.The inhibition of terpenoid synthesis and increased synthesis of C₅ amino acids by Botryococcus upon ammonia addition indicates 1) a diversion of acetyl coenzyme A from synthetic pathways leading to terpenoids and 2) increased operation of pathways leading to the synthesis of amino acids, especially 5-aminolevulinic acid, a precursor to chlorophyll biosynthesis.This work was supported in part by the Office of Energy Research, Office of Basic Energy Sciences, Biological Energy Research Division of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098, in part by a grant from SOHIO, and, in part, by a grant from the Japan-U.S. Cooperative Science Program (The Japan Society for the Promotion of Science, National Science Foundation, Division of International Programs)     

Hydropathic anti-complementarity of amino acids based on the genetic code

An interesting pattern in the genetic code has been discovered. Codons for hydrophilic and hydrophobic amino acids on one strand of DNA are complemented by codons for hydrophobic and hydrophilic amino acids on the other DNA strand, respectively. The average tendency of codons for "uncharged" (slightly hydrophilic) amino acids is to be complemented by codons for "uncharged" amino acids.     

Amino acid composition and the evolutionary rates of protein-coding genes

Summary Based on the rates of amino acid substitution for 60 mammalian genes of 50 codons or more, it is shown that the rate of amino acid substitution of a protein is correlated with its amino acid composition. In particular, the content of glycine residues is negatively correlated with the rate of amino acid substitution, and this content alone explains about 38% of the total variation in amino acid substitution rates among different protein families. The propensity of a polypeptide to evolve fast or slowly may be predicted from an index or indices of protein mutability directly derivable from the amino acid composition. The propensity of an amino acid to remain conserved during evolutionary times depends not so much on its being features prominently in active sites, but on its stability index, defined as the mean chemical distance [R. Grantham (1974) Science 185862–864] between the amino acid and its mutational derivatives produced by single-nucleotide substitutions. Functional constraints related to active and binding sites of proteins play only a minor role in determining the overall rate of amino acid substitution. The importance of amino acid composition in determining rates of substitution is illustrated with examples involving cytochrome c, cytochrome b₅,ras-related genes, the calmodulin protein family, and fibrinopeptides.     

Differential effect of amino acid residues on the stability of double helices formed from polyribonucleotides and its possible relation to the evolution of the genetic code

Summary The interaction of amino acid residues with polyribonucleotides was characterized by measurements of melting temperatures (t_m) for poly(A) poly(U) and poly(I)poly(C) as functions of the concentrations of various amino acid amides. The amides of hydrophilic amino acids lead to a continuous increase of tm with increasing concentration, whereas amides of hydrophobic amino acids induce a decrease of t_m at low concentrations (1 mM) followed by an increase at higher concentrations. Analysis of the data by a simple site model provides the affinity of each ligand for the double helix relative to that for the single strands. This parameter decreases in the order Ala>Gly>Ser>Asn>Pro>Met, Val>Ile, Leu for poly(A) poly(U) and Ala, Gly, Ser>Asn>Pro>Val>Ile, Met, Leu for poly(I)poly(C). The special effects of hydrophobic amino acids may be related to the similarity of the codons for these amino acids. A simple model for assignment of codons to amino acids is proposed.     

On origin of genetic code and tRNA before translation

Background  

Synthesis of proteins is based on the genetic code - a nearly universal assignment of codons to amino acids (aas). A major challenge to the understanding of the origins of this assignment is the archetypal "key-lock vs. frozen accident" dilemma. Here we re-examine this dilemma in light of 1) the fundamental veto on "foresight evolution", 2) modular structures of tRNAs and aminoacyl-tRNA synthetases, and 3) the updated library of aa-binding sites in RNA aptamers successfully selected in vitro for eight amino acids.     

Genetic Code-guided Protein Synthesis and Folding in Escherichia coli

Universal genetic codes are degenerated with 61 codons specifying 20 amino acids, thus creating synonymous codons for a single amino acid. Synonymous codons have been shown to affect protein properties in a given organism. To address this issue and explore how Escherichia coli selects its “codon-preferred” DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. As a result, 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence. The soluble expression level varied from completely insoluble aggregates to a soluble yield of ∼2.5 mg/liter, whereas the antigen-binding activity changed from no binding at all to a binding affinity of > 10⁻⁸ m. Not only does our work demonstrate the involvement of genetic codes in regulating protein synthesis and folding but it also provides a novel screening strategy for producing improved proteins without the need to substitute amino acids.     

A rapid method for isolation of purified,physiologically active chloroplasts,used to study the intracellular distribution of amino acids in pea leaves

A procedure is described for the rapid (<5 min) isolation of purified, physiologically active chloroplasts from Pisum sativum L. Mitochondrial and microbody contamination is substantially reduced and broken chloroplasts are excluded by washing through a layer containing a treated silica sol. On average the preparations contain 93% intact chloroplasts and show high rates of ¹⁴CO₂ fixation and CO₂-dependent O₂ evolution (over 100 mol/mg chlorophyll(chl)/h); they are also able to carry out light-driven incorporation of leucine into protein (4 nmol/mg chl/h). The amino-acid contents of chloroplasts prepared from leaves and from leaf protoplasts have been determined. Asparagine is the most abundant amino acid in the pea chloroplast (>240 nmol/mg chl), even thought it is proportionately lower in the chloroplast relative to the rest of the cell. The chloroplasts contain about 20% of many of the amino acids of the cell, but for individual amino acids the percentage in the chloroplast ranges from 8 to 40% of the cell total. Glutamic acid, glutamine and aspartic acid are enriched in the chloroplasts, while asparagine, homoserine and -(isoxazolin-5-one-2-yl)-alanine are relatively lower. Leakage of amino acids from the chloroplast during preparation or repeated washing was ca. 20%. Some differences exist between the amino-acid composition of chloroplasts isolated from intact leaves and from protoplasts. In particular, -aminobutyric acid accumulates to high levels, while homoserine and glutamic acid decrease, during protoplast formation and breakage.