Origin of the genetic code: A physical-chemical model of primitive codon assignments

Selective compartmentalization of amino acids and nucleotides according to their polarities is proposed as a physical-chemical model for the origin of the genetic code. Assumptions made in this hypothesis are: (1) an oil-slick covered the surface of the primitive ocean, constituents of which formed association colloids or micelles at the water-oil-air interfaces; (2) depending on the polarity of the media, these aggregates possessed hydrophilic and hydrophobic interiors where selective uptake of amino acids and nucleic acid constituents could take place; and 93) condensation and polymerization in the micellar phase were enhanced. According to the chromatographically observed polarities, for example, lysine and uridylate fall into the hydrophilic compartment, and phenylalanine and adenylate are enriched in the hydrophobic environment. These components could eventually be condensed to form a charged adaptor loop with an anticodon which is complementary to the presently valid codon. Only two groups of amino acids, hydrophilic and hydrophobic, were recognized by the primitive translation mechanism. Implications of this hypothesis for the further development of the genetic code is discussed. The catalytic power of micelles have been substantiated by successful synthesis of nucleotides under relatively mild conditions using thiophosphates as high energy phosphates.     

Stacking specificity and polarization. Comparative synopsis of affinity data

An explanation is given of the specificity of stacking associations based on suggestions of the contribution of dipole-induced dipole interactions, which emphasize bond moments as the origin of the polarizing power and polarizability of the π-electron ring system concerned. This explanation is consistent with our own experimental data and with those of other authors on nucleoside self-association, the mixed interaction of different nucleic acid constituents, and the interaction of aromatic amino acids with nucleosides, nucleotides, and nucleic acids.     

Synthesis of alanyl nucleobase amino acids and their incorporation into proteins

Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail.     

Relative character-state space, amount of potential phylogenetic information, and heterogeneity of nucleotide and amino acid characters

We examined a broad selection of protein-coding loci from a diverse array of clades and genomes to quantify three factors that determine whether nucleotide or amino acid characters should be preferred for phylogenetic inference. First, we quantified the difference in observed character-state space between nucleotides and amino acids. Second, we quantified the loss of potential phylogenetic signal from silent substitutions when amino acids are used. Third, we used the disparity index to quantify the relative compositional heterogeneity of nucleotides and amino acids and then determined how commonly convergent (rather than unique) shifts in nucleotide and amino acid composition occur in a phylogenetic context. The greater potential phylogenetic signal for nucleotide characters was found to be enormous (on average 440% that of amino acids), whereas the greater observed character-state space for amino acids was less impressive (on average 150.4% that of nucleotides). While matrices of amino acid sequences had less compositional heterogeneity than their corresponding nucleotide sequences, heterogeneity in amino acid composition may be more homoplasious than heterogeneity in nucleotide composition. Given the ability of increased taxon sampling to better utilize the greater potential phylogenetic signal of nucleotide characters and decrease the potential for artifacts caused by heterogeneous nucleotide composition among taxa, we suggest that increased taxon sampling be performed whenever possible instead of restricting analyses to amino acid characters.     

Sequential GC/MS analysis of sialic acids,monosaccharides, and amino acids of glycoproteins on a single sample as heptafluorobutyrate derivatives

A GC/MS procedure was developed for the analysis of all major constituents of glycoproteins. The rationale for this approach is that by using GC/MS analysis of the constituents as heptafluorobutyrate derivatives, it was possible to quantitatively determine the sialic acid, monosaccharide, fatty acids (when present), and the amino acid composition with the sample remaining in the same reaction vessel during the entire procedure. A mild acid hydrolysis was used to liberate sialic acids and was followed by formation of methyl-esters of heptafluorobutyrate (HFB) derivatives. After GC/MS analysis of sialic acids, the remaining material was submitted to acid-catalyzed methanolysis followed by the formation of HFB derivatives. After GC/MS analysis of the monosaccharides, the sample was supplemented with norleucine (as internal standard) and hydrolyzed with 6 M HCl followed by the formation of isoamyl-esters of HFB derivatives and GC/MS analysis. His and Trp residues were modified during the step of acid-catalyzed methanolysis, but the resulting derivatives were stable during acid hydrolysis and quantitatively recovered by GC/MS analysis. As a result, all constituents of glycoproteins (sialic acids, monosaccharides (or di- and trisaccharides) and amino acids) are identified in the electron impact mode of ionization and quantified using three GC/MS analysis in the same chromatographic conditions and using a limited number of reagents, a considerable advantage over previous techniques. This method is very sensitive, all data (qualitative and quantitative) being obtained at the sub-nanomolar level of initial material.     

Characterization of the extracellular ribonuclease of Tetrahymena pyriformis W

Several investigations have indicated that Tetrahymena pyriformis secretes ribonuclease activity into culture media. The extracellular ribonuclease from strain W has been purified and partially characterized. The molecular weight was determined by gel filtration to be 26,500. The amino acid composition of the enzyme was compared with those of the three intracellular ribonucleases characterized by Trangas, and substantial differences were demonstrated. The extracellular enzyme hydrolyzed both polyadenylic and polyuridylic acids, indicating lack of absolute base specificity. The hydrolysis of polyadenylic acid followed normal Michaelis-Menten kinetics, but substrate inhibition occurred at high concentrations of polyuridylic acid. The hydrolysis of polyuridylic acid was competitively inhibited by 2'- and 3'-cytidine, guanine, and uridine nucleotides, and by 2'AMP. No inhibition of the hydrolysis of Torula yeast RNA was detected. The kinetic properties of the extracellular ribonuclease are compared with those of the intracellular enzymes.     

Characterization of the Extracellular Ribonuclease of Tetrahymena pyriformis W

Several investigations have indicated that Tetrahymena pyriformis secretes ribonuclease activity into culture media. The extracellular ribonuclease from strain W has been purified and partially characterized. The molecular weight was determined by gel filtration to be 26,500. The amino acid composition of the enzyme was compared with those of the three intracellular ribonucleases characterized by Trangas, and substantial differences were demonstrated. The extracellular enzyme hydrolyzed both polyadenylic and polyuridylic acids, indicating lack of absolute base specificity. The hydrolysis of polyadenylic acid followed normal Michaelis-Menten kinetics, but substrate inhibition occurred at high concentrations of polyuridylic acid. The hydrolysis of polyuridylic acid was competitively inhibited by 2'- and 3'-cytidine, guanine, and uridine nucleotides, and by 2'AMP. No inhibition of the hydrolysis of Torula yeast RNA was detected. The kinetic properties of the extracellular ribonuclease are compared with those of the intracellular enzymes.     

Nucleotide,nucleoside and base nutritional requirements of the mosquito Culex pipiens

Nucleic acid subcomponents needed to satisfy the dietary nucleic acid requirement of Culex pipiens were studied in growth experiments using synthetic media in which nucleosides, bases and alternative nucleotides were variously substituted in mixtures of 3 nucleotides (adenylic acid, thymidylic acid, and either cytidylic or uridylic acid) previously shown to be adequate replacements for whole nucleic acid. Any or all 3 nucleotides could be replaced by corresponding nucleosides without adverse effect, except that adenosine substitution moderately delayed pupation. All base substitutions were unsatisfactory: substitution of thymine for thymidylic acid allowed development to the adult stage but at a greatly reduced rate; single substitution of adenine, cytosine or uracil for the corresponding nucleotides allowed scarcely more development than in the total absence of nucleic acid derivatives. Inosinic acid or inosine were adequate substitutes for adenylic acid, but orotic acid or orotidine were ineffective in place of the pyrimidine ribonucleotides, cytidylic or uridylic acids. Deoxyadenylic acid could take the place of adenylic acid, though inefficiently, but deoxycytidylic and deoxyuridylic acids were very poor replacements for the corresponding ribonucleotides. The minimal required nucleic acid derivatives thus appear to be a purine ribonucleotide (adenylic or inosinic acids), a pyrimidine ribonucleoside (either uridine or cytidine), and the pyrimidine deoxyribonucleoside, thymidine.     

Phagostimulation of larval Culexpipiens L. by nucleic acid nucleotides, nucleosides and bases

ABSTRACT. The influence of nucleic acid constituents on the rate of ingestion of charcoal powder filtered from ambient water by larvae of Culex pipiens L. was examined. All nucleotides tested stimulated ingestion to some extent. Various mono-, di- and tri-phosphates of adenosine were most effective and at concentrations of 1 mM stimulated ingestion nearly as well as yeast extract, a powerful phagostimu-lant. Guanylic, thymidylic, cytidylic and uridylic acids were less stimulatory, the latter two even at 10 mM. Cyclic AMP and deoxyadenylic acid were less effective than other adenine nucleotides. The nucleosides adenosine, guanosine and uridine were almost as effective as their corresponding nucleotides (adenylic, guanylic and uridylic acids); thymidine was less effective than thymidylic acids, whereas cytidine was non-stimulatory. Adenine, guanine, uracil and cytosine, the bases of the ribose nucleotides, were non-phagostimulatory, whereas thymine, base of the deoxynucleotide, thymidylic acid, caused low but significantly increased ingestion. These findings are compared with the reported phagostimulation by nucleic acid constituents of certain plant feeding insects and with the stimulation of engorgement of the blood meal by many blood feeding insects.     

Mercury Exposure: Protein Biomarkers of Mercury Exposure in Jaraqui Fish from the Amazon Region

Poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132) is a water-soluble organogermanium compound that exerts various physiological effects, including anti-inflammatory activity and pain relief. In water, Ge-132 is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP), which in turn is capable of interacting with cis-diol compounds through its trihydroxy group, indicating that this compound could also interact with diol-containing nucleic acid constituents. In this study, we evaluated the ability of THGP to interact with nucleosides or nucleotides via nuclear magnetic resonance (NMR) analysis. In addition, we evaluated the effect of added THGP on the enzymatic activity of adenosine deaminase (ADA) when using adenosine or 2′-deoxyadenosine as a substrate. In solution, THGP indeed formed complexes with nucleotides or nucleosides through their cis-diol group. Moreover, the ability of THGP to form complexes with nucleotides was influenced by the number of phosphate groups present on the ribose moiety. Notably, THGP also inhibited the catalysis of adenosine by ADA in a concentration-dependent manner. Thus, interactions between THGP and important biological nucleic acid constituents might be implicated in the physiological effects of Ge-132.     

Synthesis of proteins and nucleic acids by the pea aphid Acyrthosiphon pisum (aphididae) in the absence of a full complement of dietary amino acids

Protein, nucleic acids, and nucleotide syntheses were studied in pea aphids, Acyrthosiphon pisum (Harris), by feeding them labeled ¹⁴C-amino acids and [5-³H]-orotic acid in sucrose. It was demonstrated that in the absence of dietary essential amino acids, aphids were capable of synthesizing nucleic acids, nucleotides, and proteins when provided with a single dietary amino acid in sucrose. It is suggested that other required amino acids were possibly supplied by the symbionts present in the pea aphid and/or were obtained from the amino acid pool in the hemolymph or glucose, one of the end products of sucrose digestion. Of the various amino acids tested, synthesis of measurable amounts of protein or other compounds occurred when alanine, aspartic acid, glutamic acid, glycine, proline, or serine were provided, but no synthesis occurred with cysteine.     

Molecular Cloning and Expression of Mn2+-Dependent Sphingomyelinase/Hemolysin of an Aquatic Bacterium, Pseudomonas sp. Strain TK4

We report here the molecular cloning and expression of a hemolytic sphingomyelinase from an aquatic bacterium, Pseudomonas sp. strain TK4. The sphingomyelinase gene was found to consist of 1,548 nucleotides encoding 516 amino acid residues. The recombinant 57.7-kDa enzyme hydrolyzed sphingomyelin but not phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine, indicating that the enzyme is a sphingomyelin-specific sphingomyelinase C. The hydrolysis of sphingomyelin by the enzyme was found to be most efficient at pH 8.0 and activated by Mn(2+). The enzyme shows quite a broad specificity, i.e., it hydrolyzed 4-nitrobenz-2-oxa-1,3-diazole (NBD)-sphingomyelin with short-chain fatty acids and NBD-sphingosylphosphorylcholine, the latter being completely resistant to hydrolysis by any sphingomyelinase reported so far. Significant sequence similarities were found in sphingomyelinases from Bacillus cereus, Staphylococcus aureus, Listeria ivanovii, and Leptospira interrogans, as well as a hypothetical protein encoded in Chromobacterium violaceum, although the first three lacked one-third of the sequence corresponding to that from the C terminus of the TK4 enzyme. Interestingly, the deletion mutant of strain TK4 lacking 186 amino acids at the C-terminal end hydrolyzed sphingomyelin, whereas it lost all hemolytic activity, indicating that the C-terminal region of the TK4 enzyme is indispensable for the hemolytic activity.     

Amino acid composition of aragonitic concholin in the shell of Arctica islandica

Haugen, J. E. & Sejrup, H. P. 1990 04 15: Amino acid composition of aragonitic conchiolin in the shell of Arctica islandica. Lethaia , Vol. 23, pp. 133–141. Oslo. ISSN 0024–1164.
The distribution of amino acids within the two aragonitic shell layers of modern specimens of the mollusc Arctica islandica (Linné) has been studied in detail. The mean total hydrolyzed amino acid content was 19 nmol*mg in the inner layer and 15 nmol/mg in the outer layer. No significant difference in amino acid composition could be found between the two layers. The layers contained minor amounts of free amino acids which made up 0.3–0.7% of the total hydrolyzed amino acid content. The composition of the free amino acid fraction was very similar in each of the two layers, but differed somewhat from the total hydrolyzed fraction. The total hydrolyzed fraction was dominated by aspartic acid, glycine. alanine and glutamic acid, which together made up 62% of the total amount of amino acids, whereas the free fraction was dominated by Asp. Ser, Gly and Tyr. Amino acid content and composition within a single layer showed little variation from umbo to the ventral margin. The amino acid composition is in accordance with previously reported data on similar mineral structures which support the theory of structure specific rather than species specific amino acid composition. * Arctica islandica. amino acids. shell structure .     

Influence of the amino acid moiety on deconjugation of bile acid amidates by cholylglycine hydrolase or human fecal cultures

The influence of the chemical structure of the amino acid (or amino acid analogue) moiety of a number of synthetic cholyl amidates on deconjugation by cholylglycine hydrolase from Clostridium perfringens was studied in vitro at pH 5.4. Conjugates with alkyl homologues of glycine were hydrolyzed more slowly as the number of methylene units increased (cholylglycine greater than cholyl-beta-alanine greater than cholyl-gamma-aminobutyrate). In contrast, for conjugates with the alkyl homologues of taurine, cholylaminopropane sulfonate was hydrolyzed slightly faster than cholyltaurine, whereas cholylaminomethane sulfonate was hydrolyzed much more slowly. When glycine was replaced by other neutral alpha-amino acids, rates of hydrolysis decreased with increasing steric hindrance near the amide bond (cholyl-L-alpha-alanine much much greater than cholyl-L-leucine much greater than cholyl-L-valine greater than cholyl-L-tyrosine much greater than cholyl-D-valine). Conjugation with acidic or basic amino acids also greatly reduced the rates of hydrolysis, as cholyl-L-aspartate, cholyl-L-cysteate, cholyl-L-lysine, and cholyl-L-histidine were all hydrolyzed at a rate less than one-tenth that of cholylglycine. Methyl esterification of the carboxylic group of the amino acid moiety reduced the hydrolysis, but such substrates (cholylglycine methyl ester and cholyl-beta-alanine methyl ester) were completely hydrolyzed after overnight incubation with excess of enzyme. In contrast, cholyl-cholamine was not hydrolyzed at all, suggesting that a negative charge at the end of the side chain is required for optimal hydrolysis. Despite the lack of specificity for the amino acid moiety, a bile salt moiety was required, as the cholylglycine hydrolase did not display general carboxypeptidase activity for other non-bile acid substrates containing a terminal amide bond: hippuryl-L-phenylalanine and hippuryl-L-arginine, as well as oleyltaurine and oleylglycine, were not hydrolyzed. Fecal bacterial cultures from healthy volunteers also hydrolyzed cholyl-L-valine and cholyl-D-valine more slowly than cholylglycine, suggesting that cholylglycine hydrolase from Clostridium perfringens has a substrate specificity similar to that of the deconjugating enzymes of the fecal flora. The results indicate that modification of the position of the amide bond, introduction of steric hindrance near the amide bond, or loss of a negative charge on the terminal group of the amino acid moiety of the bile acid conjugate greatly reduces the rate of bacterial deconjugation in vitro when compared to that of the naturally occurring glycine and taurine conjugates.     

GC constituents and relative codon expressed amino acid composition in cyanobacterial phycobiliproteins

The genomic as well as structural relationship of phycobiliproteins (PBPs) in different cyanobacterial species are determined by nucleotides as well as amino acid composition. The genomic GC constituents influence the amino acid variability and codon usage of particular subunit of PBPs. We have analyzed 11 cyanobacterial species to explore the variation of amino acids and causal relationship between GC constituents and codon usage. The study at the first, second and third levels of GC content showed relatively more amino acid variability on the levels of G3 + C3 position in comparison to the first and second positions. The amino acid encoded GC rich level including G rich and C rich or both correlate the codon variability and amino acid availability. The fluctuation in amino acids such as Arg, Ala, His, Asp, Gly, Leu and Glu in α and β subunits was observed at G1C1 position; however, fluctuation in other amino acids such as Ser, Thr, Cys and Trp was observed at G2C2 position. The coding selection pressure of amino acids such as Ala, Thr, Tyr, Asp, Gly, Ile, Leu, Asn, and Ser in α and β subunits of PBPs was more elaborated at G3C3 position. In this study, we observed that each subunit of PBPs is codon specific for particular amino acid. These results suggest that genomic constraint linked with GC constituents selects the codon for particular amino acids and furthermore, the codon level study may be a novel approach to explore many problems associated with genomics and proteomics of cyanobacteria.     

Plant protein hydrolysates as fish fry feed in aquaculture. Hydrolysis of rapeseed proteins by an enzyme complex from king crab hepatopancreas

Rapeseed proteins were processed by an enzyme complex isolated from king crab hepatopancreas in order to obtain a hydrolysate for use as fish fry feed. The amino acid composition of the obtained protein preparation was close to the amino acid composition of fishmeal traditionally used in the production of fish feed. SDS-PAGE, HPLC, and mass spectrometric analysis of the products of enzymatic hydrolysis of rapeseed proteins showed that the proteins were hydrolyzed to a high degree. The composition of the hydrolysates depended on the hydrolysis time and included free amino acids (27% of the total weight of the protein mix after 3 h of hydrolysis and 56% after 21 h of hydrolysis), short peptides (2 to 20 amino acid residues), and small amounts of protein fragments with a molecular weight of approximately 14 kDa, as shown by by SDS-PAG electrophoresis.     

Quantitative microdetermination of DNA by two-dimensional electron capture gas chromatography of the thymine constituent

A highly sensitive and specific two-dimensional electron-capture gas chromatographic method has been developed for determining small amounts of DNA by measuring its thymine content. The method can also be used to measure RNA based on uracil content. The nucleic acids were hydrolyzed and released constituents were separated and detected as their chloromethyldimethylsilyl ethers. The minimal amount detected was 5 pg of each base. Standard curves were linear from 5 to 200 pg. This method allowed quantitative determination of 2 ng of DNA (routinely detectable quantity) after hydrolysis of biological material in formic acid, even in the presence of large amounts of RNA and/or protein. For example, this method has been shown to be successful in determination of the DNA contents of manually isolated nucleic such as from amphibian oocytes. Besides being accurate, the procedure was rapid: after maximal hydrolysis (usually about 45 min) the derivatization and gas chromatographic analysis was completed in another 15 min. The procedure described represents a direct biochemical alternative to cytophotometric estimation of nuclear contents and has the advantage of providing values for absolute DNA content per nucleus.     

鼠曲草的氨基酸含量的测定及营养评价

用氨基酸分析仪测定了鼠曲草中各种氨基酸的组成,并对其进行营养评价。结果表明,鼠曲草样品(茎、花)经酸水解 处理,含有苏氨酸、缬氨酸、蛋氨酸、苯丙氨酸、异亮氨酸、亮氨酸和赖氨酸等17种氨基酸,茎中总氨基酸质量分数为18.25%、花 中为14.44%;其中包含人体必需的8种氨基酸,配比合理:E/(E+N)=0.38(茎)或0.37(花),E/N=0.62(茎)或0.59(花), 与WHO/FAO提出的E/(E+N)应为0.4左右,E/N应为0.6左右的参考蛋白质模式接近,说明鼠曲草在医学和营养学上都 有很高的研究价值,值得大力开发利用。     

Hydrolysis of Soybean Proteins with Kamchatka Crab Hepatopancreas Enzyme Complex

To perform hydrolysis with the enzyme complex from the hepatopancreas of the Kamchatka crab, a protein mixture was isolated from soybean meal by extraction at alkaline pH values. Extractable low-molecular impurities were removed by ultrafiltration and precipitation of proteins with alcohol. The amino acid composition of the obtained protein extract turned out to be similar to the composition of the fish meal traditionally used in the production of fish feeds. Analysis of the products of fermentolysis by DDS-electrophoresis, HPLC, and mass spectrometry showed a high degree of hydrolysis of soybean proteins. Depending on the time of fermentolysis, the hydrolysates contained up to 60% (18 h of hydrolysis) of free amino acids (the fraction of the weight of the hydrolyzed protein mixture) and short peptides (2–20 amino acid residues).     

Which Amino Acids Should Be Used in Prebiotic Chemistry Studies?

The adsorption of amino acids on minerals and their condensation under conditions that resemble those of prebiotic earth is a well studied subject. However, which amino acids should be used in these experiments is still an open question. The main goal of this review is to attempt to answer this question. There were two sources of amino acids for the prebiotic earth: (1) exogenous -- meaning that the amino acids were synthesized outside the earth and delivered to our planet by interplanetary dust particles (IDPs), meteorites, comets, etc. and (2) endogenous -- meaning that they were synthesized on earth in atmospheric mixtures, hydrothermal vents, etc. For prebiotic chemistry studies, the use of a mixture of amino acids from both endogenous and exogenous sources is suggested. The exogenous contribution of amino acids to this mixture is very different from the average composition of proteins, and contains several non-protein amino acids. On the other hand, the mixture of amino acids from endogenous sources is seems to more closely resemble the amino acid composition of terrestrial proteins.