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     

Extensive Sequence Conservation Among Insect,Nematode, and Vertebrate Vitellogenins Reveals Ancient Common Ancestry

The eggs of most oviparous animals are provisioned with a class of protein called vitellogenin (Vg) which is stored as the major component of yolk. Until recently, deduced amino acid sequences were available only from vertebrate and nematode Vgs, which proved to be homologous. The sequences of several insect Vgs are now known, but early attempts at pairwise alignments with vertebrate and nematode Vgs have been problematic, leading to conflicting conclusions about how closely insect Vgs are related to the others. In this paper we demonstrate that insect Vg sequences can be confidently aligned with one another along their entire lengths and with multiple vertebrate and nematode Vg sequences along most of their spans. Although divergence is high, conservation among insect, vertebrate, and nematode Vg sequences is widespread with a preponderance of glycine, proline, and cysteine residues among strictly conserved amino acids, establishing conclusively that Vgs from the three phyla are homologous. Areas of least-certain alignment are primarily in and around insect and vertebrate polyserine domains which are not homologous. Phylogenetic reconstructions of Vgs based on sequence identities indicate that the insect lineage is the most diverged and that the mammalian serum protein, apolipoprotein B-100, arose from a Vg ancestor after the nematode/vertebrate divergence. Received: 6 May 1996 / Accepted: 27 September 1996     

Dynamic Rearrangement Within the Antheraea pernyi Silk Fibroin Gene Is Associated with Four Types of Repetitive Units

We characterized a full-length gene encoding wild silkmoth Antheraea pernyi fibroin (Ap-fibroin) to clarify the conformation of repetitive sequences. The gene consisted of a first exon encoding 14 amino acid residues, a short intron (120 bp), and a long second exon encoding 2,625 amino acid residues. Three amino acids, alanine, glycine, and serine, amounted to 81% of the Ap-fibroin sequence. The Ap-fibroin, except for 155 residues of the amino terminus, was composed of 80 tandemly arranged polyalanine-containing units (motifs). A motif was a doublet of a polyalanine block (PAB) and a nonpolyalanine block (NPAB). Seventy-eight of the 80 motifs were classified into four types based on differences in the NPAB sequences. Although respective motifs were significantly conserved, many rearrangements were observed within the second exon, i.e., the triplication of a 558-bp-long sequence and other duplication events of shorter sequences. Chi-like sequences, GCTGGAG, might contribute to the rearrangement within the gene as described in human minisatellite loci, because they were found at specific sites of NPAB-encoding sequences in three of four types of motifs. The present results support the idea that the Ap-fibroin gene is unstable like minisatellite sequences and that the evolution of this gene is strongly associated with its instability. Received: 18 February 2000 / Accepted: 30 June 2000     

Detection of Signature Sequences in Overlapping Genes and Prediction of a Novel Overlapping Gene in Hepatitis G Virus

In viruses an increased coding ability is provided by overlapping genes, in which two alternative open reading frames (ORFs) may be translated to yield two distinct proteins. The identification of signature sequences in overlapping genes is a topic of particular interest, since additional out-of-frame coding regions can be nested within known genes. In this work, a novel feature peculiar to overlapping coding regions is presented. It was detected by analysis of a sample set of 21 virus genomic sequences and consisted in the repeated occurrence of a cluster of basic amino acid residues, encoded by a frame, combined to a stretch of acidic residues, encoded by the corresponding overlapping frame. A computer scan of an additional set of virus sequences demonstrated that this feature is common to several other known overlapping ORFs and led to prediction of a novel overlapping gene in hepatitis G virus (HGV). The occurrence of a bifunctional coding region in HGV was also supported by its extremely lower rate of synonymous nucleotide substitutions compared to that observed in the other gene regions of the HGV genome. Analysis of the amino acid sequence that was deduced from the putative overlapping gene revealed a high content of basic residues and the presence of a nuclear targeting signal; these characteristics suggest that a core-like protein may be expressed by this novel ORF. Received: 21 July 1999 / Accepted: 26 October 1999     

Complete Mitochondrial Genome of a Neotropical Fruit Bat, Artibeus jamaicensis, and a New Hypothesis of the Relationships of Bats to Other Eutherian Mammals

The complete mitochondrial genome was obtained from a microchiropteran bat, Artibeus jamaicensis. The presumptive amino acid sequence for the protein-coding genes was compared with predicted amino acid sequences from several representatives of other mammalian orders. Data were analyzed using maximum parsimony, maximum likelihood, and neighbor joining. All analyses placed bats as the sister group of carnivores, perissodactyls, artiodactyls, and cetaceans (e.g., 100% bootstrap value with both maximum parsimony and neighbor joining). The data strongly support a new hypothesis about the origin of bats, specifically a bat/ferungulate grouping. None of the analyses supported the superorder Archonta (bats, flying lemurs, primates, and tree shrews). Our hypothesis regarding the relationship of bats to other eutherian mammals is concordant with previous molecular studies and contrasts with hypotheses based solely on morphological criteria and an incomplete fossil record. The A. jamaicensis mitochondrial DNA control region has a complex pattern of tandem repeats that differs from previously reported chiropteran control regions. Received: 22 January 1998 / Accepted: 3 June 1998     

Expression of the Dentin Matrix Protein 1 Gene in Birds

The emergence of jawed vertebrates was predicated on the appearance of several innovations, including tooth formation. The development of teeth requires the participation of several specialized genes, in particular, those necessary for the formation of hard tissues—dentin, enamel, and cementum. Some vertebrates, most conspicuously birds, secondarily lost the tooth-forming ability. To determine the fate of some of the tooth-forming genes in the birds, we tested a domestic fowl cDNA library for the expression of the dentin matrix protein 1 (DMP1) gene. The library was prepared from the poly(A⁺) RNA isolated from the jaws of 11- to 13-day-old embryos and the testing was carried out by the polymerase chain reaction with degenerate primers designed on the basis of the available mammalian and reptile sequences. A chicken homologue of the DMP1 gene identified by this approach was shown to be expressed in the jaws and long bones, the same two tissues as in mammals. The chicken DMP1 gene has an exon/intron organization similar to that of its mammalian and reptile counterparts. The chicken gene contains three short highly conserved segments, the rest of the gene being poorly alignable or not alignable with its mammalian or reptilian homologues. The distribution of similarities and dissimilarities along the gene is indicative of a mode of evolution in which only short segments are kept constant, while the rest of the gene is relatively free to vary as long as the proportion of certain amino acid residues is retained in the encoded polypeptide. The DMP1 gene may have been retained in birds because of its involvement in bone formation. Received: 5 April 1999 / Accepted: 9 August 1999     

Distinct Stages of Protein Evolution as Suggested by Protein Sequence Analysis

Evolution of proteins encoded in nucleotide sequences began with the advent of the triplet code. The chronological order of the appearance of amino acids on the evolution scene and the steps in the evolution of the triplet code have been recently reconstructed (Trifonov, 2000b) on the basis of 40 different ranking criteria and hypotheses. According to the consensus chronology, the pair of complementary GGC and GCC codons for the amino acids alanine and glycine appeared first. Other codons appeared as complementary pairs as well, which divided their respective amino acids into two alphabets, encoded by triplets with either central purines or central pyrimidines: G, D, S, E, N, R, K, Q, C, H, Y, and W (Glycine alphabet G) and A, V, P, S, L, T, I, F, and M (Alanine alphabet A). It is speculated that the earliest polypeptide chains were very short, presumably of uniform length, belonging to two alphabet types encoded in the two complementary strands of the earliest mRNA duplexes. After the fusion of the minigenes, a mosaic of the alphabets would form. Traces of the predicted mosaic structure have been, indeed, detected in the protein sequences of complete prokaryotic genomes in the form of weak oscillations with the period 12 residues in the form of alteration of two types of 6 residue long units. The next stage of protein evolution corresponded to the closure of the chains in the loops of the size 25–30 residues (Berezovsky et al., 2000). Autocorrelation analysis of proteins of 23 complete archaebacterial and eubacterial genomes revealed that the preferred distances between valine, alanine, glycine, leucine, and isoleucine along the sequences are in the same range of 25–30 residues, indicating that the loops are primarily closed by hydrophobic interactions between the ends of the loops. The loop closure stage is followed by the formation of typical folds of 100–200 amino acids, via end-to-end fusion of the genes encoding the loop-size chains. This size was apparently dictated by the optimal ring closure for DNA. In both cases the closure into the ring (loop) rendered evolutionarily advantageous stability to the respective structures. Further gene fusions lead to the formation of modern multidomain proteins. Recombinational gene splicing is likely to have appeared after the DNA circularization stage. Received: 21 December 2000 / Accepted: 28 February 2001     

Molecular Evolution of the Photolyase–Blue-Light Photoreceptor Family

The photolyase–blue-light photoreceptor family is composed of cyclobutane pyrimidine dimer (CPD) photolyases, (6-4) photolyases, and blue-light photoreceptors. CPD photolyase and (6-4) photolyase are involved in photoreactivation for CPD and (6-4) photoproducts, respectively. CPD photolyase is classified into two subclasses, class I and II, based on amino acid sequence similarity. Blue-light photoreceptors are essential light detectors for the early development of plants. The amino acid sequence of the receptor is similar to those of the photolyases, although the receptor does not show the activity of photoreactivation. To investigate the functional divergence of the family, the amino acid sequences of the proteins were aligned. The alignment suggested that the recognition mechanisms of the cofactors and the substrate of class I CPD photolyases (class I photolyases) are different from those of class II CPD photolyases (class II photolyases). We reconstructed the phylogenetic trees based on the alignment by the NJ method and the ML method. The phylogenetic analysis suggested that the ancestral gene of the family had encoded CPD photolyase and that the gene duplication of the ancestral proteins had occurred at least eight times before the divergence between eubacteria and eukaryotes. Received: 23 October 1996 / Accepted: 1 April 1997     

Evolution of N-terminal sequences of the vertebrate HOXA13 protein

While the the role of the homeodomain in HOX function has been evaluated extensively, little attention has been given to the non-homeodomain portions of the HOX proteins. To investigate the evolution of the HOXA13 protein and to identify conserved residues in the N-terminal region of the protein with potential functional significance, N-terminal Hoxa13 coding sequences were PCR-amplified from fish, amphibian, reptile, chicken, and marsupial and eutherian mammal genomic DNA. Compared with fish HOXA13, the mammalian protein has increased in size by 35% primarily owing to the accumulation of alanine repeats and flanking segments rich in proline, glycine, or serine within the first 215 amino acids. Certain residues and amino acid motifs were strongly conserved, and several HOXA13 N-terminal domains were also shared in the paralogous HOXB13 and HOXD13 genes; however, other conserved regions appear to be unique to HOXA13. Two domains highly conserved in HOXA13 orthologs are shared with Drosophila AbdB and other vertebrate AbdB-like proteins. Marsupial and eutherian mammalian HOXA13 proteins have three large homopolymeric alanine repeats of 14, 12, and 17–18 residues that are absent in reptiles, birds, and fish. Thus, the repeats arose after the divergence of reptiles from the lineage that would give rise to the mammals. In contrast, other short homopolymeric alanine repeats in mammalian HOXA13 have remained virtually the same length, suggesting that forces driving or limiting repeat expansion are context dependent. Consecutive stretches of identical third-base usage in alanine codons within the large repeats were found, supporting replication slippage as a mechanism for their generation. However, numerous species-specific base substitutions affecting third-base alanine repeat codon positions were observed, particularly in the largest repeat. Therefore, if the large alanine repeats were present prior to eutherian mammal development as is suggested by the opossum data, then a dynamic process of recurring replication slippage and point mutation within alanine repeat codons must be considered to reconcile these observations. This model might also explain why the alanine repeats are flanked by proline, serine, and glycine-rich sequences, and it reveals a biological mechanism that promotes increases in protein size and, potentially, acquisition of new functions. Received: 8 June 1999 / Accepted: 23 September 1999     

Silica, Alumina, and Clay-Catalyzed Alanine Peptide Bond Formation

The peptide bond formation of alanine (ala), ala + glycine (gly), ala + diglycine (gly₂), and ala + gly cyclic anhydride (cyc-gly₂) in drying/wetting cycles at 80°C was studied. Silica, alumina, and representative smectites—montmorillonite and hectorite—were used as catalysts, and the dependence of reaction yields on the available amount of water in the reaction systems was evaluated. Silica and alumina catalyze the formation of oligopeptide mainly in temperature fluctuation experiments, whereas higher amounts of water in the reaction system support clay-catalyzed reactions. Silica and alumina are much more efficient for amino acid dimerization than clays. Whereas only 0.1% of ala oligomerized on hectorite and no reaction proceeded on montmorillonite, about 0.9 and 3.8% alanine converted into its dimer and cyclic anhydride on silica and alumina, respectively. Clay minerals, on the other hand, seem to more efficiently catalyze peptide chain elongation than amino acid dimerization. The reaction yields of ala-gly-gly and gly-gly-ala from ala + gly₂ and ala + cyc-gly₂ reached about 0.3% on montmorillonite and 1.0% on hectorite. The possible mechanisms of these reactions and the relevance of the results for prebiotic chemistry are discussed. Received: 15 December 1996 / Accepted: 1 May 1997     

Trends in Codon and Amino Acid Usage in Thermotoga maritima

The usage of synonymous codons and the frequencies of amino acids were investigated in the complete genome of the bacterium Thermotoga maritima using a multivariate statistical approach. The GC3 content of each gene was the most prominent source of variation of codon usage. Surprisingly the usage of UGU and UGC (synonymous triplets coding for Cys, the least frequent amino acid in this species) was detected as the second most prominent source of variation. However, this result is probably an artifact due to the very low frequency of Cys together with the nonbiased composition of this genome. The third trend was related to the preferential usage of a subset of codons among highly expressed genes, and these triplets are presumed to be translationally optimal. Concerning the amino acid usage, the hydropathy level of each protein (and therefore the frequency of charged residues) was the main trend, while the second factor was related to the frequency of usage of the smaller residues, suggesting that the cell economy strongly influences the architecture of the proteins. The third axis of the analysis discriminated the usage of Phe, Tyr, Trp (aromatic residues) plus Cys, Met, and His. These six residues have in common the property of being the preferential targets of reactive oxygen species, and therefore the anaerobic condition of T. maritima is an important factor for the amino acid frequencies. Finally, the Cys content of each protein was the fourth trend. Received: 22 June 2001 / Accepted: 1 October 2001     

Synonymous and Nonsynonymous Substitutions in Mammalian Genes: Intragenic Correlations

Previous investigations indicated that synonymous and nonsynonymous substitution rates are correlated in mammalian genes. In the present work, this correlation has been studied at the intragenic level using a dataset of 48 orthologous genes from species belonging to at least four different mammalian orders. The results obtained show that the intragenic variability in synonymous rates is correlated with that of nonsynonymous rates. Moreover, the variation in GC level (and especially of C level) of silent positions along each gene is correlated with the variation in synonymous rate. These results reinforce the previous conclusions that synonymous and nonsynonymous rates as well as GC levels of silent positions are to some extent under common selective constraints. Received: 10 July 1997 / Accepted: 13 August 1997     

Phylogenetic Analysis of the Friedreich Ataxia GAA Trinucleotide Repeat

Friedreich ataxia is an autosomal recessive neurodegenerative disorder associated with a GAA repeat expansion in the first intron of the gene (FRDA) encoding a novel, highly conserved, 210 amino acid protein known as frataxin. Normal variation in repeat size was determined by analysis of more than 600 DNA samples from seven human populations. This analysis showed that the most frequent allele had nine GAA repeats, and no alleles with fewer than five GAA repeats were found. The European and Syrian populations had the highest percentage of alleles with 10 or more GAA repeats, while the Papua New Guinea population did not have any alleles carrying more than 10 GAA repeats. The distributions of repeat sizes in the European, Syrian, and African American populations were significantly different from those in the Asian and Papua New Guinea populations (p < 0.001). The GAA repeat size was also determined in five nonhuman primates. Samples from 10 chimpanzees, 3 orangutans, 1 gorilla, 1 rhesus macaque, 1 mangabey, and 1 tamarin were analyzed. Among those primates belonging to the Pongidae family, the chimpanzees were found to carry three or four GAA repeats, the orangutans had four or five GAA repeats, and the gorilla carried three GAA repeats. In primates belonging to the Cercopithecidae family, three GAA repeats were found in the mangabey and two in the rhesus macaque. However, an AluY subfamily member inserted in the poly(A) tract preceding the GAA repeat region in the rhesus macaque, making the amplified sequence approximately 300 bp longer. The GAA repeat was also found in the tamarin, suggesting that it arose at least 40 million years ago and remained relatively small throughout the majority of primate evolution, with a punctuated expansion in the human genome. Received: 18 August 2000 / Accepted: 10 November 2000     

Nucleotide Composition Bias Affects Amino Acid Content in Proteins Coded by Animal Mitochondria

We show that in animal mitochondria homologous genes that differ in guanine plus cytosine (G + C) content code for proteins differing in amino acid content in a manner that relates to the G + C content of the codons. DNA sequences were analyzed using square plots, a new method that combines graphical visualization and statistical analysis of compositional differences in both DNA and protein. Square plots divide codons into four groups based on first and second position A + T (adenine plus thymine) and G + C content and indicate differences in amino acid content when comparing sequences that differ in G + C content. When sequences are compared using these plots, the amino acid content is shown to correlate with the nucleotide bias of the genes. This amino acid effect is shown in all protein-coding genes in the mitochondrial genome, including cox I, cox II, and cyt b, mitochondrial genes which are commonly used for phylogenetic studies. Furthermore, nucleotide content differences are shown to affect the content of all amino acids with A + T- and G + C-rich codons. We speculate that phylogenetic analysis of genes so affected may tend erroneously to indicate relatedness (or lack thereof) based only on amino acid content. Received: 3 July 1996 / Accepted: 6 November 1996     

Coordinated Amino Acid Changes in the Evolution of Mammalian Defensins

The mammalian defensin molecule is a short, highly cationic peptide cytotoxic to both microbial and mammalian cells which is cleaved from a precursor including a signal peptide and a highly anionic propiece. A phylogenetic analysis of 28 complete sequences from five mammalian species (mouse, rat, guinea pig, rabbit, and human) showed species-specific clusters of sequences, indicating that the genes duplicated after divergence of these species. Comparison of rates of synonymous and nonsynonymous nucleotide substitution suggested that gene duplication has often been followed by a period in which diversification of the mature defensins at the amino acid level has been selectively favored. In some comparisons, it appeared that amino acid differences in this region have appeared in a nonrandom fashion so as to change the pattern of residue charges. Because it has been hypothesized that the negative charge in the propiece serves to balance the positive charge in the mature defensin and thus to prevent cytotoxicity prior to cleavage, we used a maximum likelihood method of reconstructing ancestral states in order to test whether this balance has been maintained over evolutionary time in spite of rapid diversification of the mature defensin at the amino acid level. Reconstructed ancestral sequences always maintained a charge balance between mature defensin and propiece, and changes in the net positive charge of the mature defensin were balanced by corresponding changes in the propiece. The results support the hypothesis that, in the evolution of these proteins, amino acid changes have occurred in a coordinated fashion so as to preserve an adaptive phenotype. Received: 23 October 1996 / Accepted: 7 January 1997     

Reduction of wobble-position GC bases in Corynebacteria genes and enhancement of PCR and heterologous expression

Corynebacteria codon usage exhibits an overall GC content of 67%, and a wobble-position GC content of 88%. Escherichia coli, on the other hand has an overall GC content of 51%, and a wobble-position GC content of 55%. The high GC content of Corynebacteria genes results in an unfavorable codon preference for heterologous expression, and can present difficulties for polymerase-based manipulations due to secondary-structure effects. Since these characteristics are due primarily to base composition at the wobble-position, synthetic genes can, in principle, be designed to eliminate these problems and retain the wild-type amino acid sequence. Such genes would obviate the need for special additives or bases during in vitro polymerase-based manipulation and mutant host strains containing uncommon tRNA's for heterologous expression. We have evaluated synthetic genes with reduced wobble-position G/C content using two variants of the enzyme 2,5-diketo-D-gluconic acid reductase (2,5-DKGR A and B) from Corynebacterium. The wild-type genes are refractory to polymerase-based manipulations and exhibit poor heterologous expression in enteric bacteria. The results indicate that a subset of codons for five amino acids (alanine, arginine, glutamate, glycine and valine) contribute the greatest contribution to reduction in G/C content at the wobble-position. Furthermore, changes in codons for two amino acids (leucine and proline) enhance bias for expression in enteric bacteria without affecting the overall G/C content. The synthetic genes are readily amplified using polymerase-based methodologies, and exhibit high levels of heterologous expression in E. coli.