On the origin of the genetic code

It is argued that three chemical criteria determined the evolution of the genetic code: codon-anticodon pairing; codon-amino acid pairing; amino acid pairing. The first criterium determined the set of interactive nucleotides; the second, the set of nucleotides interactive with amino acids; the third, the set of mutually interactive amino acids. The code resulted from the intersection of these sets. This hypothesis explains the specificity and universality of the code as well as the “choice” of the standard amino acids and nucleotides from among those available in nature. The specific mechanism for codon-amino acid pairing assumed here is the “backwards” (Crick, 1967) Pelc-Welton (1966) models. Three types of evidence support “backwards” pairing: parallel genetic coding of amino acid pairs (Root-Bernstein, 1982); results of binding experiments by Saxinger and Ponnamperuma (1974); reinterpretation of Jungck's (1978) correlations between the properties of amino acids and their respective anticodon nucleotides. The inversion of the code to its present state occurred as a result of the evolution of tRNA molecules which supplanted parallel codon-amino acid interactions with antiparallel codon-anticodon ones. The paper concludes with suggestions for testing the hypothesis.     

Is canavanine a natural component of mammalian metabolism?

A group of non-protein amino acids of higher plants, namely l-canavanine, l-canaline, 0-ureido-l-homoserine, and l-canavaninosuccinic acid, have been implicated in mammalian intermediary metabolism. The clinical observations and biochemical basis for this hypothesis as well as conflicting experimental evidence are presented. A possible explanation for the apparent role of these non-protein amino acids in mammalian metabolism is offered.     

A quantitative investigation of the chemical space surrounding amino acid alphabet formation

To date, explanations for the origin and emergence of the alphabet of amino acids encoded by the standard genetic code have been largely qualitative and speculative. Here, with the help of computational chemistry, we present the first quantitative exploration of nature's “choices” set against various models for plausible alternatives. Specifically, we consider the chemical space defined by three fundamental biophysical properties (size, charge, and hydrophobicity) to ask whether the amino acids that entered the genetic code exhibit a higher diversity than random samples of similar size drawn from several different definitions of amino acid possibility space.We found that in terms of the properties studied, the full, standard set of 20 biologically encoded amino acids is indeed significantly more diverse than an equivalently sized group drawn at random from the set of plausible, prebiotic alternatives (using the Murchison meteorite as a model for pre-biotic plausibility). However, when the set of possible amino acids is enlarged to include those that are produced by standard biosynthetic pathways (reflecting the widespread idea that many members of the standard alphabet were recruited in this way), then the genetically encoded amino acids can no longer be distinguished as more diverse than a random sample. Finally, if we turn to consider the overlap between biologically encoded amino acids and those that are prebiotically plausible, then we find that the biologically encoded subset are no more diverse as a group than would be expected from a random sample, unless the definition of “random sample” is adjusted to reflect possible prebiotic abundance (again, using the contents of the Murchison meteorite as our estimator). This final result is contingent on the accuracy of our computational estimates for amino acid properties, and prebiotic abundances, and an exploration of the likely effect of errors in our estimation reveals that our results should be treated with caution. We thus present this work as a first step in quantifying and thus testing various origin-of-life hypotheses regarding the origin and evolution of life's amino acid alphabet, and advocate the progress that would add valuable information in the future.     

Amino Acid Analyses of Desert Varnish from the Sonoran and Mojave Deserts

There has long been a debate as to whether desert varnish deposits are microbially mediated or are deposited by inorganic processes. Several researchers have cultured bacteria from the surface of desert varnish suggesting that bacteria are intimately associated with varnish coatings and may play a role in their formation. To test this hypothesis, we have collected scrapings of desert varnish from the Sonoran Desert in Arizona and the Mojave Desert in California and analyzed them for amino acids. Thirteen amino acids were found in desert varnish indicating a biogenic component of these varnishes. Two protein amino acids that were not detected in any of the varnishes are cysteine and tryptophan. Two nonprotein amino acids,β-alanine andγ-amino butyric acid, were found. These are known to be formed by enzymatic decarboxylation, thereby indicating possible organismal activity in varnish. Some D -enantiomers of the amino acids were also found. In addition to small amounts of the D -enantiomer of aspartic acid, which is rapidly formed by racemization and was present in most samples, D -alanine and D -glutamic acid were found. These latter two amino acids are components of the peptidoglycan cell wall material of bacteria. L -lysine was also detected, but not diaminopimelic acid. The combination of L -lysine, D -alanine, and D -glutamic acid is characteristic of the peptidoglycan from Gram-positive bacteria. Although the presence of these biomarkers does not prove that Gram-positive bacteria produce the coatings, finding them is consistent with the hypothesis that they may play a role in desert varnish formation.     

The transmembrane domain of influenza hemagglutinin exhibits a stringent length requirement to support the hemifusion to fusion transition

Glycosylphosphatidylinositol-anchored influenza hemagglutinin (GPI-HA) mediates hemifusion, whereas chimeras with foreign transmembrane (TM) domains mediate full fusion. A possible explanation for these observations is that the TM domain must be a critical length in order for HA to promote full fusion. To test this hypothesis, we analyzed biochemical properties and fusion phenotypes of HA with alterations in its 27-amino acid TM domain. Our mutants included sequential 2-amino acid (Delta2-Delta14) and an 11-amino acid deletion from the COOH-terminal end, deletions of 6 or 8 amino acids from the NH(2)-terminal and middle regions, and a deletion of 12 amino acids from the NH(2)-terminal end of the TM domain. We also made several point mutations in the TM domain. All of the mutants except Delta14 were expressed at the cell surface and displayed biochemical properties virtually identical to wild-type HA. All the mutants that were expressed at the cell surface promoted full fusion, with the notable exception of deletions of >10 amino acids. A mutant in which 11 amino acids were deleted was severely impaired in promoting full fusion. Mutants in which 12 amino acids were deleted (from either end) mediated only hemifusion. Hence, a TM domain of 17 amino acids is needed to efficiently promote full fusion. Addition of either the hydrophilic HA cytoplasmic tail sequence or a single arginine to Delta12 HA, the hemifusion mutant that terminates with 15 (hydrophobic) amino acids of the HA TM domain, restored full fusion activity. Our data support a model in which the TM domain must span the bilayer to promote full fusion.     

Death ofLactobacillus acidophilus caused by incorporated3H-thymine during incubation without amino acids

Of the cells ofLactobacillus acidophilus R-26 incorporated³H-thymine (specific radioactivity 1.57 Ci/mmol or 3.15 Ci/mmol), their transfer to a medium without essential amino acids resulted in their death. This death may be interpreted in such a way that cell damage caused by disintegration of tritium cannot be effectively repaired under conditions of amino acid deprivation. The experimental conditions make it possible to explain this death either as a result of inhibition of protein or RNA synthesis or as a result of the absence of amino acids. These possibilities were tested in experiments, in which the synthesis of proteins and RNA was inhibited by specific inhibitors in the presence of amino acids. Under these conditions no death of cells was detected, thus indicating that free amino acids play a role in the repair of radiation damage.     

Effect of dietary amino acid on the amino acid pool of Aedes aegypti

Ion-exchange chromatography analysis of whole body extracts of Aedes aegypti mosquitoes which had received amino acids in their diet revealed that generally there were changes in the titre of two or more amino acids. Cysteine produced the greatest number of changes and was toxic to the insect. Of the ten amino acids provided, none resulted in the significant change in the concentration of tyrosine following a blood meal as was observed in previous studies. Evidence is presented for the conversion of arginine to ornithine and for the synthesis of arginine from glutamic acid. The data presented tend to support the hypothesis of lysine synthesis from α-ketoglutarate and for the use of proline as an energy reserve in the insect.     

Excssive DNA synthesis inLactobacillus acidophilus during amino acid starvation

Replication of the bacterial chromosome was studied in two substrains ofLactobacillus acidophilus R-26 during amino acid starvation. According to the hypothesis of Maaløe and Hanawalt (1961), already initiated DNA replication cycles are completed under such conditions, with a corresponding 40% increase in the DNA content; new cycles cannot be initiated in the absence of proteosynthesis. Our findings are considerably at variance with this hypothesis. It was found that the course of DNA synthesis and the size of DNA increments during amino acid starvation were influenced by some low molecular weight substances, in particular by deoxyadenylate and spermidine. In the presence of these substances in media without the essential amino acids, prolonged DNA synthesis accompanied by large DNA increments was observed, suggesting that new DNA replication cycles were initiated. The possibility that deoxyadenylate and spermidine influence the regulation of synthesis of the bacterial chromosome is discussed.     

In Vitro Release of Endogenous Excitatory Sulfur-Containing Amino Acids from Various Rat Brain Regions

Efflux of various amino acids from rat brain slices was determined under resting or depolarizing conditions. Slices of neocortex, hippocampus, striatum, cerebellum, mesodiencephalon, pons-medulla, and spinal cord were depolarized by K+ (50 mM) or veratrine (33 micrograms/ml). The 4-N,N-dimethylamino-azobenzene-4'-isothiocyanate (DABITC) derivatization method of Chang [Biochem. J. 199, 537-545 (1981)] for HPLC was adapted for analysis of amino acids and peptides in superfusion solutions. It allowed the separation and simultaneous detection of the sulfur-containing amino acids cysteine sulfinic acid (CSA), cysteic acid (CA), homocysteine sulfinic acid (HCSA), and homocysteic acid (HCA) at the picomole level. All four were shown to be released on depolarization in a Ca2+-dependent manner from brain slices. CSA and HCSA were released from cortex, hippocampus, mesodiencephalon, and, for HCSA only, striatum. HCA release, observed in all regions, was most prominent in cortex and hippocampus. CA was slightly increased by depolarization in hippocampus and mesodiencephalon. These sulfur-containing amino acids have been shown to exert an excitatory action on CNS neurons. The fact that these sulfur-containing amino acids are released as endogenous substances from nervous tissue supports the hypothesis that they play a role in CNS neurotransmission.     

In vivo engineering of proteins with nitrogen-containing tryptophan analogs

Recently, it has become possible to reprogram the protein synthesis machinery such that numerous noncanonical amino acids can be translated into target sequences yielding tailor-made proteins. The canonical amino acid tryptophan (Trp) encoded by a single nucleotide triplet (UGG) is a particularly interesting target for protein engineering and design. Trp-residues can be substituted with a variety of analogs and surrogates generated biosynthetically or by organic chemistry. Among them, nitrogen-containing tryptophan analogs occupy a central position, as they have distinct chemical properties in comparison with aliphatic amines and imines. They resemble purine bases of DNA and share their capacity for pH-sensitive intramolecular charge transfer. These special properties of the analogs can be directly transmitted into related protein structures via in vivo ribosome-mediated translation. Proteins expressed in this way are further endowed with unique properties like new spectral, altered redox and titration features or might serve as useful biomaterials. We present and discuss current works and future developments in protein engineering with nitrogen-containing tryptophan analogs and related compounds as well as their relevance for academic and applicative research.The term noncanonical amino acid refers to an amino acid that does not belong, in contrast to a canonical amino acid, to the genetically encoded, proteinogenic amino acids. The term analog defines a strict isosteric exchange of a canonical/noncanonical amino acid (e.g., tryptophan/azatryptophan), while the term surrogate defines a nonisosteric change (e.g., tryptophan/azulene). Mutant denotes a protein in which the wild-type sequence was changed by site-directed mutagenesis (codon manipulation on the DNA level) within the repertoire of the standard amino acids. Variant denotes a protein in which one or more canonical amino acids derived from a wild-type or a mutant sequence were replaced by a noncanonical one (expanded amino acid repertoire, codon reassignment on the protein translation level).     

Rabbit lutropin: preparation, characterization of the hormone, its subunits and radioimmunoassay.

The purification of rabbit lutropin is described. A product with a potency of 1.53 X NIH-LH-Sl was obtained as assayed by the ovarian ascorbic acid depletion assay. In a homologous radioimmunoassay, which is described, rabbit lutropin has a potency 4.83 X NIH-LH-Sl. In a radioligand assay, utilizing labeled ovine lutropin as the trace, the relative potency was 0.47 X NIH-LH-Sl measured by 50% inhibition comparison since rabbit lutropin response in this system did not parallel ovine lutropin. A counter-current distribution procedure for separation of rabbit lutropin subunits is described. Amino acid composition of the isolated subunits and intact rabbit lutropin was determined. The carbohydrate composition of the latter is presented; only amino sugar determinations are available for the subunits. The NH2-terminal amino acids are phenylalanine (alpha subunit) and alanine (beta subunit). Preliminary data on COOH-terminal amino acids are provided.     

KINETICS OF COMPETITIVE INHIBITION OF NEUTRAL AMINO ACID TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER

The transport of tryptophan across the blood-brain barrier is used as a specific example of a general approach by which rates of amino acid influx into brain may be predicted from existing concentrations of amino acids in plasma. The kinetics of inhibition of [¹⁴C]tryptophan transport by four natural neutral amino acids (phenylalanine, leucine, methionine, and valine) and one synthetic amino acid (α-methyl tyrosine) is studied with a tissue-sampling, single injection technique in the barbiturate-anesthetized rat. The equality of the K₁ (determined from cross-inhibition studies) and the K_m (determined from auto-inhibition data) for neutral amino acid transport indicate that these amino acids compete for a single transport site in accordance with the kinetics of competitive inhibition. Based on equations derived for competitive inhibition, apparent K_m values are computed for the essential neutral amino acids from known data on amino acid transport K_m and plasma concentrations. The apparent K_m values make possible predictions of the in vivo rates of amino acid influx into brain based on given plasma amino acid concentrations. Finally, a method is presented for determining transport constants from saturation data obtained with single injection techniques.     

AN EXAMINATION OF THE CATALYTIC PROPERTIES OF SEVERAL AMINOTRANSFERASES IN BRAIN AND LIVER BY MEANS OF AN IMPROVED RADIOMETRIC ASSAY WITH DINITROPHENYLHYDRAZINE

Abstract— The assay of aminotransferases, performed by solvent extraction of keto acids formed from labelled amino acids, has been modified to enhance the recovery of both aliphatic and aromatic keto acid products. The keto acids are first converted to their respective dinitrophenylhydrazones which are more completely extracted into less polar organic solvents. By this manoeuvre, both keto acid extraction is increased and the extraction of the precursor amino acid is reduced. Employing this technique, the kinetics of brain-stem γ-aminobutyric acid (GABA), tryptophan, 3,4-dihydroxyphenylalanine (DOPA) aminotransferases and brain-stem and liver tyrosine aminotransferases were examined. Brain-stem aminotransferases, particularly the aromatic amino acid transferases, have a higher affinity for both the amino acid and the keto acid when the aromatic keto acid, phenylpyruvate (0·8 mM), is employed as amino group acceptor, whereas maximal velocities for aminotransferase reactions are much greater when α-ketoglutarate (0·8 m m ) is the amino group acceptor. Brain-stem tyrosine aminotransferase exhibits a much lower affinity for tyrosine in the presence of either 0·8m m -α-ketoglutarate or 0·8 m m -phenylpyruvate than does liver tyrosine aminotransferase. p -Chlorophenylpyruvate and phenylpyruvate exhibit similar properties as amino group acceptors for brain-stem tryptophan aminotransferase. Cysteine inhibits tryptophan aminotransferase when phenylpyruvate is the amino group acceptor, in a manner which is competitive with the amino acid. Benzoylformate inhibits both tryptophan and DOPA aminotransferases when phenylpyruvate is the amino group acceptor, but this inhibition does not appear to be competitive with phenylpyruvate.     

Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization

A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC–ESI–MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performed using l-norvaline as standard. A limit of detection as low as 1 fmol/µl with a linear range of up to 125 pmol/µl could be obtained. Intraday and interday precisions were lower than 10 % relative standard deviations for most of the amino acids. Quantification using l-norvaline as internal standard gave very similar results compared to the quantification using deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm).     

Synthesis of sugar-amino acid-nucleosides as potential glycosyltransferase inhibitors

Sugar-amino acid-nucleosides (SAAN) were synthesized to mimic glycosyl nucleotide donors based on the hypothesis that a basic amino acid may interact with carboxylate groups of the enzyme in a manner similar to the diphosphate metal ion complex. C-Glycoside analogues of the d-galactopyranose or l-arabinofuranose ring systems, and four amino acids (lysine, glutamine, tryptophan, and histidine), were chosen for this study. The targets were synthesized and tested against GlfT2, a galactofuranosyltransferase essential for cell wall galactan biosynthesis in Mycobacterium tuberculosis. The inhibition assay showed that analogues containing histidine and tryptophan are moderate inhibitors of GlfT2.     

Tissue distribution and functional analyses of the constitutively active orphan G protein coupled receptors, GPR26 and GPR78

GPR26 and GPR78 are orphan GPCRs (oGPCRs) that share 51% amino acid sequence identity and are widely expressed in selected tissues of the human brain as well as the developing and adult mouse brain. Investigation of the functional activity of GPR26 and GPR78 via expression in HEK293 cells showed that both proteins are constitutively active and coupled to elevated cAMP production. Accordingly, in yeast, GPR26 demonstrated apparent agonist-independent coupling to a chimeric Gpa1 protein in which the 5 C-terminal amino acids were from Galphas. A comparison of the proteins revealed an atypical glutamine residue in GPR78 in place of the conserved arginine residue (R3.50) in the so-called DRY box. Site-directed mutants R3.50 in GPR26 were constructed and retained their constitutive activity suggesting that these 2 receptors activate G proteins in a manner that is distinct from other group 1 GPCRs.     

On the mechanism of interaction between histone II B1 and DNA and histone II B2 and DNA

A mechanism is suggested at the molecular level whereby histone IIB₂ can act as a cross-link between two (or possibly three) adjacent and parallel strands of DNA double helix some 40 Å apart. Application of Prothero's rule and the Lewis probability functions indicate the probable locations of three a-helices and a number of β-turns. This, coupled with the requirement that the tertiary conformation of the histone be complementary to the DNA molecules and for as many basic groups as possible to bind to phosphate oxygens, allows us to suggest, on the basis of model building using accurate space-filling (CPK) models, a complex conformation that achieves this.A similar process applied to histone IIB₁, whose complete amino acid sequence is also known, shows the location of five probable a-helices, a number of β-turns, and a segment of β-pleated sheet. The basic amino acids are gathered in four groupings. Model building experiments suggest that histone IIB₁ forms a complex strut joining four parallel strands of DNA double helix that form a diamond with diameters 100 and 40 Å. In both these models the purpose and function of a fair proportion of the individual amino acids can be specified.This paper is the third and last of a series in this Journal in which models are presented for the tertiary conformation and function of all five histones of known (in whole or in part) amino acid sequence. This suggests that all five are concerned in packing the long DNA double helix, which may be in a “square helix” form, into the confined space of the chromosome. The hypotheses may be tested by a direct investigation of nucleoprotein in situ to see if these 40, 70, and 100 Å interhelical distances can be detected by biophysical methods.     

On the possibility of determining the secondary structure of proteins in solution

A method to determine the contribution of various side chains to the optical rotatory dispersion and circular dichroism or proteins is presented. This method assumes that the side chain of any given amino acid in a similar situation in different proteins contributes the same to the optical rotatory dispersion and circular dichroism of those proteins. The method has a great deal of flexibility in allowing the investigator to postulate any hypothesis about the contributions of such side chains and ultimately to use statistical tests to test that hypothesis. If attained, knowledge of the contribution of side chains will enable investigators to determine the secondary structure of proteins in terms of certain reference conformations. The current use of polyamino acids as standard reference conformations is not entirely satisfactory. Some of the questions raised by their use are discussed and possible solutions proposed.     

Origins of gene, genetic code, protein and life: comprehensive view of life systems from a GNC-SNS primitive genetic code hypothesis

We have investigated the origin of genes, the genetic code, proteins and life using six indices (hydropathy, α-helix, β-sheet and β-turn formabilities, acidic amino acid content and basic amino acid content) necessary for appropriate three-dimensional structure formation of globular proteins. From the analysis of microbial genes, we have concluded that newly-born genes are products of nonstop frames (NSF) on antisense strands of microbial GC-rich genes [GC-NSF(a)] and from SNS repeating sequences [(SNS)_n] similar to the GC-NSF(a) (S and N mean G or C and either of four bases, respectively). We have also proposed that the universal genetic code used by most organisms on the earth presently could be derived from a GNC-SNS primitive genetic code. We have further presented the [GADV]-protein world hypothesis of the origin of life as well as a hypothesis of protein production, suggesting that proteins were originally produced by random peptide formation of amino acids restricted in specific amino acid compositions termed as GNC-, SNS and GC-NSF(a)-0th order structures of proteins. The [GADV]-protein world hypothesis is primarily derived from the GNC-primitive genetic code hypothesis. It is also expected that basic properties of extant genes and proteins could be revealed by considerations based on the scenario with four stages This review is a modified English version of the paper, which was written in Japanese and published inViva Origino 2001 29 66–85.