Mode of Action of Herbicidal Derivatives of Aminomethylenebisphosphonic Acid. Part II. Reversal of Herbicidal Action by Aromatic Amino Acids

The herbicidal action of N-pyridylaminomethylenebisphosphonic acids is accompanied by an impairment of anthocyanin biosynthesis. This suggests that they might act as inhibitors of some steps in aromatic amino acid biosynthesis. Herbicidal effects were reversed by aromatic amino acids using both bacterial and plant models, a finding that strongly supports this hypothesis. Structural features of these compounds suggest the sixth enzyme in the shikimate pathway 5-enol-pyruvoylshikimate-3-phosphate (EPSP) synthase as a possible target, since a strong structural similarity exists between aminomethylenebisphosphonic acid and an inhibitor of EPSP synthase, the herbicide glyphosate. This is, however, not the case since they did not act as inhibitors of this enzyme. Received July 29; 1996; accepted May 27, 1997     

Molecular dissection of the large mechanosensitive ion channel (MscL) of E. coli: Mutants with altered channel gating and pressure sensitivity

In the search for the essential functional domains of the large mechanosensitive ion channel (MscL) of E. coli, we have cloned several mutants of the mscL gene into a glutathione S-transferase fusion protein expression system. The resulting mutated MscL proteins had either amino acid additions, substitutions or deletions in the amphipathic N-terminal region, and/or deletions in the amphipathic central or hydrophilic C-terminal regions. Proteolytic digestion of the isolated fusion proteins by thrombin yielded virtually pure recombinant MscL proteins that were reconstituted into artificial liposomes and examined for function by the patch-clamp technique. The addition of amino acid residues to the N-terminus of the MscL did not affect channel activity, whereas N-terminal deletions or changes to the N-terminal amino acid sequence were poorly tolerated and resulted in channels exhibiting altered pressure sensitivity and gating. Deletion of 27 amino acids from the C-terminus resulted in MscL protein that formed channels similar to the wild-type, while deletion of 33 C-terminal amino acids extinguished channel activity. Similarly, deletion of the internal amphipathic region of the MscL abolished activity. In accordance with a recently proposed spatial model of the MscL, our results suggest that (i) the N-terminal portion participates in the channel activation by pressure, and (ii) the essential channel functions are associated with both, the putative central amphipathic α-helical portion of the protein and the six C-terminal residues RKKEEP forming a charge cluster following the putative M2 membrane spanning α-helix. Received: 25 September 1996/Revised: 21 November 1996     

rBAT is an Amino Acid Exchanger with Variable Stoichiometry

The rBAT protein, when expressed in Xenopus oocytes, was previously shown to reproduce the selectivity of the Na⁺-independent neutral and basic amino acid transport system called b^o,+. More recently, the capacity of rBAT to generate a transmembrane current was demonstrated when addition of neutral amino acids stimulated the efflux of cations (presumably basic amino acids) in rBAT-injected oocytes. In the present paper, aminoisobutyric acid (AIB), a neutral amino acid analogue, was shown to induce outward currents (efflux of basic amino acids) through rBAT similar to those caused by alanine in terms of affinity, maximal currents and I-V curves. Despite generating similar currents, the AIB transport rate was more than 30 times lower than that of alanine, thus challenging the assumption that rBAT functions as a classical exchanger. Experiments using a cut-open oocyte voltage clamp demonstrated that AIB was capable of stimulating rBAT-mediated currents from either side of the membrane. AIB, like alanine, was able to stimulate the efflux of radiolabeled alanine and arginine while no rBAT-mediated efflux was measurable in the absence of external rBAT substrates. These results demonstrate that (i) the presence of amino acids is required on both sides of the membrane for rBAT to mediate amino acid flux and thus rBAT must be some type of exchanger but (ii) rBAT-mediated amino acid influx is not stoichiometrically related to the efflux. A model of a ``double gated pore' is proposed to account for these properties of rBAT, which contravene standard models of exchangers and other transporters. Received: 15 June 1995/Revised: 21 September 1995     

What Amino Acid Properties Affect Protein Evolution?

We studied 10 protein-coding mitochondrial genes from 19 mammalian species to evaluate the effects of 10 amino acid properties on the evolution of the genetic code, the amino acid composition of proteins, and the pattern of nonsynonymous substitutions. The 10 amino acid properties studied are the chemical composition of the side chain, two polarity measures, hydropathy, isoelectric point, volume, aromaticity, aliphaticity, hydrogenation, and hydroxythiolation. The genetic code appears to have evolved toward minimizing polarity and hydropathy but not the other seven properties. This can be explained by our finding that the presumably primitive amino acids differed much only in polarity and hydropathy, but little in the other properties. Only the chemical composition (C) and isoelectric point (IE) appear to have affected the amino acid composition of the proteins studied, that is, these proteins tend to have more amino acids with typical C and IE values, so that nonsynonymous mutations tend to result in small differences in C and IE. All properties, except for hydroxythiolation, affect the rate of nonsynonymous substitution, with the observed amino acid changes having only small differences in these properties, relative to the spectrum of all possible nonsynonymous mutations. Received: 2 January 1998 / Accepted: 25 April 1998     

The Molecular Evolution of Visual Pigments of Freshwater Crayfishes (Decapoda: Cambaridae)

This study examines the diverse maximum wavelength absorption (λ_max) found in crayfishes (Decapoda: Cambaridae and Parastacidae) and the associated genetic variation in their opsin locus. We measured the wavelength absorption in the photoreceptors of six species that inhabit environments of different light intensities (i.e., burrows, streams, standing waters, and subterranean waters). Our results indicate that there is relatively little variation in λ_max (522–530 nm) among species from different genera and families. The existing variation did not correlate with the habitat differences of the crayfishes studied. We simultaneously sequenced the rhodopsin gene to identify the amino acid replacements that affect shifts in maximum wavelength absorption. We then related these to changes that correlated with shifts in λ_max by reconstructing ancestral character states using a maximum-likelihood approach. Using amino acid sequences obtained from five species (all were 301 amino acids in length), we identified a number of candidates for producing shifts of 4 to 8 nm in λ_max. These amino acid replacements occurred in similar regions to those involved in spectral shifts in vertebrates. Received: 12 March 1997 / Accepted: 3 June 1997     

Energy from Redox Disproportionation of Sugar Carbon Drives Biotic and Abiotic Synthesis

To identify the energy source that drives the biosynthesis of amino acids, lipids, and nucleotides from glucose, we calculated the free energy change due to redox disproportionation of the substrate carbon of (1) 26-carbon fermentation reactions and (2) the biosynthesis of amino acids and lipids of E. coli from glucose. The free energy (cal/mmol of carbon) of these reactions was plotted as a function of the degree of redox disproportionation of carbon (disproportionative electron transfers (mmol)/mmol of carbon). The zero intercept and proportionality between energy yield and degree of redox disporportionation exhibited by this plot demonstrate that redox disproportionation is the principal energy source of these redox reactions (slope of linear fit =−10.4 cal/mmol of disproportionative electron transfers). The energy and disproportionation values of E. coli amino acid and lipid biosynthesis from glucose lie near this linear curve fit with redox disproportionation accounting for 84% and 96% (and ATP only 6% and 1%) of the total energy of amino acid and lipid biosynthesis, respectively. These observations establish that redox disproportionation of carbon, and not ATP, is the primary energy source driving amino acid and lipid biosynthesis from glucose. In contrast, we found that nucleotide biosynthesis involves very little redox disproportionation, and consequently depends almost entirely on ATP for energy. The function of sugar redox disproportionation as the major source of free energy for the biosynthesis of amino acids and lipids suggests that sugar disproportionation played a central role in the origin of metabolism, and probably the origin of life. Received: 18 April 1996 / Accepted: 31 October 1996     

Sequential Amino Acid Exchange across b0,+-like System in Chicken Brush Border Jejunum

In the small intestine, cationic amino acids are transported by y⁺-like and b^0,+-like systems present in the luminal side of the epithelium. Here, we report the characterization of a b^0,+-like system in the apical membrane of the chicken jejunum, and its properties as an amino acid exchanger. Analysis of the brush border membrane by Western blot points out the presence of rBAT (protein related to b^0,+ amino acid transport system) in these membranes. A functional mechanism for amino acid exchange across this system was established by kinetic analysis measuring fluxes at varying substrate concentrations both in internal (in) and external (out) vesicle compartments. This intestinal b^0,+-like system functions for l-arginine as an obligatory exchanger since its transport capacity increases 100–200 fold in exchange conditions, thus suggesting an important role in the intestinal absorption of cationic amino acids. The kinetic analysis of Arg_in efflux velocities is compatible with the formation of a ternary complex and excludes a model involving a ping-pong mechanism. The binding affinity of Arg_out is higher than that of Arg_in, suggesting a possible order of binding (Arg_out first) for the formation of the ternary complex during the exchange cycle. A model of double translocation pathways with alternating access is discussed. Received: 23 March 2000/Revised: 29 December 2000     

Selection and Methionine Accumulation in the Fat Body Protein 2 Gene (FBP2), a Duplicate of the Drosophila Alcohol Dehydrogenase (ADH) Gene

The Drosophila fat body protein 2 gene (Fbp2) is an ancient duplication of the alcohol dehydrogenase gene (Adh) which encodes a protein that differs substantially from ADH in its methionine content. In D. melanogaster, there is one methionine in ADH, while there are 51 (20% of all amino acids) in FBP2. Methionine is involved in 46% of amino acid replacements when Fbp2 DNA sequences are compared between D. melanogaster and D. pseudoobscura. Methionine accumulation does not affect conserved residues of the ADH-ADH^r-FBP2 multigene family. The multigene family has evolved by replacement of mildly hydrophobic amino acids by methionine with no apparent reversion. Its short-term evolution was compared between two Drosophila species, while its long-term evolution was compared between two genera belonging respectively to acalyptrate and calyptrate Diptera, Drosophila and Sarcophaga. The pattern of nucleotide substitution was consistent with an independent accumulation of methionines at the Fbp2 locus in each lineage. Under a steady-state model, the rate of methionine accumulation was constant in the lineage leading to Drosophila, and was twice as fast as that in the calyptrate lineage. Substitution rates were consistent with a slight positive selective advantage for each methionine change in about one-half of amino acid sites in Drosophila. This shows that selection can potentially account for a large proportion of amino acid replacements in the molecular evolution of proteins. Received: 12 December 1994 / Accepted: 15 April 1996     

The Binding Specificity of Amino Acid Transport System y+L in Human Erythrocytes is Altered by Monovalent Cations

System y⁺L is a broad-scope amino acid transporter which binds and translocates cationic and neutral amino acids. Na⁺ replacement with K⁺ does not affect lysine transport, but markedly decreases the affinity of the transporter for l-leucine and l-glutamine. This observation suggests that the specificity of system y⁺L varies depending on the ionic composition of the medium. Here we have studied the interaction of the carrier with various amino acids in the presence of Na⁺, K⁺, Li⁺ and guanidinium ion. In agreement with the prediction, the specificity of system y⁺L was altered by the monovalent cations. In the presence of Na⁺, l-leucine was the neutral amino acid that interacted more powerfully. Elongation of the side chain (glycine - l-norleucine) strengthened binding. In contrast, bulkiness at the level of the β carbon was detrimental. In K⁺, the carrier behaved as a cationic amino acid specific carrier, interacting weakly with neutral amino acids. Li⁺ was found to potentiate neutral amino acid binding and in general the apparent affinities were higher than in Na⁺; elongation of the nonpolar side chain made a more important contribution to binding and the carrier was more tolerant towards β carbon substitution. Guanidinium stimulated the interaction of the carrier with neutral amino acids, but the effect was restricted to certain analogues (e.g., l-leucine, l-glutamine, l-methionine). Thus, in the presence of guanidinium, the carrier discriminates sharply among different neutral amino acids. The results suggest that the monovalent cations stabilize different carrier conformations. Received: 22 January 1996/Revised: 26 April 1996     

The Historical Factor: The Biosynthetic Relationships Between Amino Acids and Their Physicochemical Properties in the Origin of the Genetic Code

Two forces are in general, hypothesized to have influenced the origin of the organization of the genetic code: the physicochemical properties of amino acids and their biosynthetic relationships. In view of this, we have considered a model incorporating these two forces. In particular, we have studied the optimization level of the physicochemical properties of amino acids in the set of amino acid permutation codes that respects the biosynthetic relationships between amino acids. Where the properties of amino acids are represented by polarity and molecular volume we obtain indetermination percentages in the organization of the genetic code of approximately 40%. This indicates that the contingent factor played a significant role in structuring the genetic code. Furthermore, this result is in agreement with the genetic code coevolution hypothesis, which attributes a merely ancillary role to the properties of amino acids while it suggests that it was their biosynthetic relationships that organized the code. Furthermore, this result does not favor the stereochemical models proposed to explain the origin of the genetic code. On the other hand, where the properties of amino acids are represented by polarity alone, we obtain an indetermination percentage of at least 21.5%. This might suggest that the polarity distances played an important role and would therefore provide evidence in favor of the physicochemical hypothesis of genetic code origin. Although, overall, the analysis might have given stronger support to the latter hypothesis, this did not actually occur. The results are therefore discussed in the context of the different theories proposed to explain the origin of the genetic code. Received: 10 September 1996 / Accepted: 3 March 1997     

Archaeabacterial Seryl-tRNA Synthetases: Adaptation to Extreme Environments and Evolutionary Analysis

The aminoacyl-tRNA synthetases are ubiquitous enzymes which catalyze a crucial step of the cell life, the specific attachment of amino acids to their cognate tRNA. The amino acid sequences of three archaeal seryl-tRNA synthetases (SerRS) from Haloarcula marismortui and Methanococcus jannaschii, both belonging to the group of Euryarchaeota, and from Sulfolobus solfataricus, of the group of Crenarchaeota, were aligned with other eubacterial and eukaryal available SerRS sequences. In an attempt to identify some features of adaptation to extreme environments of these organisms, amino acid composition and amino acid substitutions between mesophilic and thermophilic SerRS were analyzed. In addition, universal phylogenetic trees of SerRS including the three known archaeal sequences, rooted by the threonyl-tRNA synthetases were inferred. Amino acid analyses of the SerRS revealed two ways of adaptation to thermophilic environments between the Eubacteria and the Archaea; most of the usually described amino acid substitutions were nonsignificant in the case of archaeal thermophilic SerRS and most amino acid composition biases seemed to be linked to the genome G+C content pressure. The phylogenetic analysis of the SerRS showed the Archaea to be paraphyletic, H. marismortui emerging with the Gram-positive Bacteria, M. jannaschii being near the root of the tree, and S. solfataricus branching with Eucarya. Received: 30 March 1998 / Accepted: 14 July 1998     

The Rates of Molecular Evolution in Rodent and Primate Mitochondrial DNA

A higher rate of molecular evolution in rodents than in primates at synonymous sites and, to a lesser extent, at amino acid replacement sites has been reported previously for most nuclear genes examined. Thus in these genes the average ratio of amino acid replacement to synonymous substitution rates in rodents is lower than in primates, an observation at odds with the neutral model of molecular evolution. Under Ohta's mildly deleterious model of molecular evolution, these observations are seen as the consequence of the combined effects of a shorter generation time (driving a higher mutation rate) and a larger effective population size (resulting in more effective selection against mildly deleterious mutations) in rodents. The present study reports the results of a maximum-likelihood analysis of the ratio of amino acid replacements to synonymous substitutions for genes encoded in mitochondrial DNA (mtDNA) in these two lineages. A similar pattern is observed: in rodents this ratio is significantly lower than in primates, again consistent only with the mildly deleterious model. Interestingly the lineage-specific difference is much more pronounced in mtDNA-encoded than in nuclear-encoded proteins, an observation which is shown to run counter to expectation under Ohta's model. Finally, accepting certain fossil divergence dates, the lineage-specific difference in amino acid replacement-to-synonymous substitution ratio in mtDNA can be partitioned and is found to be entirely the consequence of a higher mutation rate in rodents. This conclusion is consistent with a replication-dependent model of mutation in mtDNA. Received: 24 September 1999 / Accepted: 18 September 2000     

Phylogenetic,Structural and Functional Characteristics of the Na-K-Cl Cotransporter Family

Summary Bumetanide-sensitive Na-K-Cl cotransporters and thiazide-sensitive Na-Cl cotransporters comprise a family of integral membrane transport proteins, the Na-K-Cl cotransporter (NKCC) family. Each of the members of this family is over 1,000 amino acids in length. We have multiply aligned the ten currently sequenced members of this family from human, rabbit, rodent, shark, flounder, moth, worm and yeast sources. Phylogenetic analyses suggest the presence of at least six isoforms of these full length proteins in eukaryotes. Average hydropathy and average similarity plots have been derived revealing that each of these proteins possesses a central, well conserved, hydrophobic domain of almost invariant length, possibly consisting of twelve transmembrane α-helical spanners, an N-terminal, poorly conserved, hydrophilic domain of variable length, and a C-terminal, moderately conserved, hydrophilic domain of moderately constant length. A functionally uncharacterized homologue of this family occurs in the cyanobacterium Synechococcus sp. Limited sequence similarity of these proteins with members of a family of basic amino acid transporters suggests that the NKCC family may be distantly related to the previously characterized, ubiquitous, amino acid-polyamine-choline (APC) family of facilitators. These observations suggest that the NKCC family is an old family that has its roots in the prokaryotic kingdom. Received: 27 July 1995/Revised: 8 November 1995     

Positional Dependence, Cliques, and Predictive Motifs in the bHLH Protein Domain

Quantitative analyses were carried out on a large number of proteins that contain the highly conserved basic helix–loop–helix domain. Measures derived from information theory were used to examine the extent of conservation at amino acid sites within the bHLH domain as well as the extent of mutual information among sites within the domain. Using the Boltzmann entropy measure, we described the extent of amino acid conservation throughout the bHLH domain. We used position association (pa) statistics that reflect the joint probability of occurrence of events to estimate the ``mutual information content' among distinct amino acid sites. Further, we used pa statistics to estimate the extent of association in amino acid composition at each site in the domain and between amino acid composition and variables reflecting clade and group membership, loop length, and the presence of a leucine zipper. The pa values were also used to describe groups of amino acid sites called ``cliques' that were highly associated with each other. Finally, a predictive motif was constructed that accurately identifies bHLH domain-containing proteins that belong to Groups A and B. Received: 15 December 1997 / Accepted: 1 October 1998     

The Genetic Code: What Is It Good For? An Analysis of the Effects of Selection Pressures on Genetic Codes

How did the ``universal' genetic code arise? Several hypotheses have been put forward, and the code has been analyzed extensively by authors looking for clues to selection pressures that might have acted during its evolution. But this approach has been ineffective. Although an impressive number of properties has been attributed to the universal code, it has been impossible to determine whether selection on any of these properties was important in the code's evolution or whether the observed properties arose as a consequence of selection on some other characteristic. Therefore we turned the question around and asked, what would a genetic code look like if it had evolved in response to various different selection pressures? To address this question, we constructed a genetic algorithm. We found first that selecting on a particular measure yields codes that are similar to each other. Second, we found that the universal code is far from minimized with respect to the effects of mutations (or translation errors) on the amino acid compositions of proteins. Finally, we found that the codes that most closely resembled real codes were those generated by selecting on aspects of the code's structure, not those generated by selecting to minimize the effects of amino acid substitutions on proteins. This suggests that the universal genetic code has been selected for a particular structure—a structure that confers an important flexibility on the evolution of genes and proteins—and that the particular assignments of amino acids to codons are secondary. Received: 29 December 1998 / Accepted: 8 July 1999     

New Glycoprotein-Associated Amino Acid Transporters

The L-type amino acid transporter LAT1 has recently been identified as being a disulfide-linked ``light chain' of the ubiquitously expressed glycoprotein 4F2hc/CD98. Several LAT1-related transporters have been identified, which share the same putative 12-transmembrane segment topology and also associate with the single transmembrane domain 4F2hc protein. They display differing amino acid substrate specificities, transport kinetics and localizations such as, for instance, y⁺LAT1 which is localized at the basolateral membrane of transporting epithelia, and the defect of which causes lysinuric protein intolerance. The b^0,+AT transporter which associates with the 4F2hc-related rBAT protein to form the luminal high-affinity diamino acid transporter defective in cystinuria, belongs to the same family of glycoprotein-associated amino acid transporters (gpaATs). These glycoprotein-associated transporters function as amino acid exchangers. They extend the specificity range of vectorial amino acid transport when located in the same membrane as carriers that unidirectionally transport one of the exchanged substrates. gpaATs belong to a phylogenetic cluster within the amino acid/polyamine/choline (APC) superfamily of transporters. This cluster, which we designate the LAT family (named after its first vertebrate member), includes some members from nematodes, yeast and bacteria. The latter of these proteins presumably lack association with a second subunit. In this review, we focus on the animal members of the LAT cluster that form, together with some of the nematode members, the family of glycoprotein-associated amino acid transporters (gpaAT family). Received: 20 July 1999/Revised: 7 September 1999     

Internal Zinc Accumulation Is Correlated with Increased Growth in Rice Suspension Culture

A high zinc concentration of 520 μm, approximately 100 times that used most often in standard plant tissue culture media, was found to be superior in liquid callus cultures of japonica rice, increasing growth to 146% compared with standard N6 medium. At the same time, the internal zinc concentration increased 40 times in fast growing cells; soluble protein doubled, and free amino acids decreased. Under zinc-free conditions the cultures slowed in growth, and several free amino acids such as aspartic acid, glutamic acid, asparagine, and glutamine accumulated. We suggest that zinc acts as a direct regulatory factor in inducing auxin activity, but not auxin levels, making high internal zinc accumulation mandatory if high auxin concentrations are required as in rice callus cultures. Received July 16, 1997; accepted September 22, 1997     

Volume-regulatory Amino Acid Release from the Protozoan Parasite Crithidia luciliae

The unicellular protozoan parasite, Crithidia luciliae, responded to osmotic swelling by undergoing a regulatory volume decrease. This process was accompanied by the efflux of amino acids (predominantly alanine, proline and glycine). The relative loss of the electroneutral amino acids proline, valine, alanine and glycine was greater than that for the anionic amino acid, glutamate; there was negligible loss of the cationic amino acids, lysine, arginine and ornithine. The characteristics of amino acid release were investigated using a radiolabeled form of the nonmetabolized alanine analogue α-aminoisobutyrate. α-Aminoisobutyrate efflux was activated within a few seconds of a reduction of the osmolality, and inactivated rapidly (again within a few seconds) on restoration of isotonicity. The initial rate of efflux of α-aminoisobutyrate from cells in hypotonic medium was unaffected by the extracellular amino acid concentration. Hypotonically activated α-aminoisobutyrate efflux (as well as the associated regulatory volume decrease) was inhibited by the sulfhydryl reagent N-ethylmaleimide but was not inhibited by a range of anion transport blockers. As in the efflux experiments, unidirectional influx rates for α-aminoisobutyrate increased markedly following reduction of the osmolality, consistent with the swelling-activated amino acid release mechanism allowing the flux of solutes in both directions. Hypotonically activated α-aminoisobutyrate influx showed no tendency to saturate up to an extracellular concentration of 50 mm. The functional characteristics of the amino acid release mechanism are those of a channel, with a preference for electroneutral and anionic amino acids over cationic amino acids. However, the pharmacology of the system differs from that of the anion-selective channels that are thought to mediate the volume-regulatory efflux of organic osmolytes from vertebrate cells. Received: 13 May 1996/Revised: 9 July 1996