Amino Acid Usage Research Articles

Relationship between amino acid usage and amino acid evolution in primates

Amino acid usage varies from species to species. A previous study has found a universal trend in amino acid gain and loss in many taxa and a one-way model of amino acid evolution in which the number of new amino acids increases as the number of old amino acids decreases was proposed. Later studies showed that this pattern of amino acid gain and loss is likely to be compatible with the neutral theory. The present work aimed to further study this problem by investigating the evolutionary patterns of amino acids in 8 primates (the nucleotide and protein alignments are available online http://gattaca.nju.edu.cn/pub_data.html). First, the number of amino acids gained and lost was calculated and the evolution trend of each amino acid was inferred. These values were found to be closely related to the usage of each amino acid. Then we analyzed the mutational trend of amino acid substitution in human using SNPs, this trend is highly correlated with fixation trend only with greater variance. Finally, the trends in the evolution of 20 amino acids were evaluated in human on different time scales, and the increasing rate of 5 significantly increasing amino acids was found to decrease as a function of time elapsed since divergence, and the dS/dN ratio also found to increase as a function of time elapsed since divergence. These results suggested that the observed amino acid substitution pattern is influenced by mutation and purifying selection. In conclusion, the present study shows that usage of amino acids is an important factor capable of influencing the observed pattern of amino acid evolution, and also presented evidences suggesting that the observed universal trend of amino acid gain and loss is compatible with neutral evolution.

Three-dimensional eukaryotic genomic organization is strongly correlated with codon usage expression and function

It has been shown that the distribution of genes in eukaryotic genomes is not random; however, formerly reported relations between gene function and genomic organization were relatively weak. Previous studies have demonstrated that codon usage bias is related to all stages of gene expression and to protein function. Here we apply a novel tool for assessing functional relatedness, codon usage frequency similarity (CUFS), which measures similarity between genes in terms of codon and amino acid usage. By analyzing chromosome conformation capture data, describing the three-dimensional (3D) conformation of the DNA, we show that the functional similarity between genes captured by CUFS is directly and very strongly correlated with their 3D distance in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, mouse and human. This emphasizes the importance of three-dimensional genomic localization in eukaryotes and indicates that codon usage is tightly linked to genome architecture.

Hydrophobicity and aromaticity are primary factors shaping variation in amino acid usage of chicken proteome.

Amino acids are utilized with different frequencies both among species and among genes within the same genome. Up to date, no study on the amino acid usage pattern of chicken has been performed. In the present study, we carried out a systematic examination of the amino acid usage in the chicken proteome. Our data indicated that the relative amino acid usage is positively correlated with the tRNA gene copy number. GC contents, including GC1, GC2, GC3, GC content of CDS and GC content of the introns, were correlated with the most of the amino acid usage, especially for GC rich and GC poor amino acids, however, multiple linear regression analyses indicated that only approximately 10–40% variation of amino acid usage can be explained by GC content for GC rich and GC poor amino acids. For other intermediate GC content amino acids, only approximately 10% variation can be explained. Correspondence analyses demonstrated that the main factors responsible for the variation of amino acid usage in chicken are hydrophobicity, aromaticity and genomic GC content. Gene expression level also influenced the amino acid usage significantly. We argued that the amino acid usage of chicken proteome likely reflects a balance or near balance between the action of selection, mutation, and genetic drift.

The influence of flanking secondary structures on amino Acid content and typical lengths of 3/10 helices.

We used 3D structures of a highly redundant set of bacterial proteins encoded by genes of high, average, and low GC-content. Four types of connecting bridges—regions situated between any of two major elements of secondary structure (alpha helices and beta strands)—containing a pure random coil were compared with connecting bridges containing 3/10 helices. We included discovered trends in the original “VVTAK Connecting Bridges” algorithm, which is able to predict more probable conformation for a given connecting bridge. The highest number of significant differences in amino acid usage was found between 3/10 helices containing bridges connecting two beta strands (they have increased Phe, Tyr, Met, Ile, Leu, Val, and His usages but decreased usages of Asp, Asn, Gly, and Pro) and those without 3/10 helices. The typical (most common) length of 3/10 helices situated between two beta strands and between beta strand and alpha helix is equal to 5 amino acid residues. The preferred length of 3/10 helices situated between alpha helix and beta strand is equal to 3 residues. For 3/10 helices situated between two alpha helices, both lengths (3 and 5 amino acid residues) are typical.

Compositional features are potentially involved in the regulation of gene expression of tumor suppressor genes in human tissues

Different mechanisms regulate the expression level of tissue specific genes in human. Here we report some compositional features such as codon usage bias, amino acid usage bias, codon frequency, and base composition which may be potentially related to mRNA amount of tissue specific tumor suppressor genes. Our findings support the possibility that structural elements in gene and protein may play an important role in the regulation of tumor suppressor genes, development, and tumorigenesis. The data presented here can open broad vistas in the understanding and treatment of a variety of human malignancies.

Analysis of the relationship between genomic GC Content and patterns of base usage, codon usage and amino acid usage in prokaryotes: similar GC content adopts similar compositional frequencies regardless of the phylogenetic lineages.

The GC contents of 2670 prokaryotic genomes that belong to diverse phylogenetic lineages were analyzed in this paper. These genomes had GC contents that ranged from 13.5% to 74.9%. We analyzed the distance of base frequencies at the three codon positions, codon frequencies, and amino acid compositions across genomes with respect to the differences in the GC content of these prokaryotic species. We found that although the phylogenetic lineages were remote among some species, a similar genomic GC content forced them to adopt similar base usage patterns at the three codon positions, codon usage patterns, and amino acid usage patterns. Our work demonstrates that in prokaryotic genomes: a) base usage, codon usage, and amino acid usage change with GC content with a linear correlation; b) the distance of each usage has a linear correlation with the GC content difference; and c) GC content is more essential than phylogenetic lineage in determining base usage, codon usage, and amino acid usage. This work is exceptional in that we adopted intuitively graphic methods for all analyses, and we used these analyses to examine as many as 2670 prokaryotes. We hope that this work is helpful for understanding common features in the organization of microbial genomes.

The mitochondrial genome of Frankliniella intonsa: Insights into the evolution of mitochondrial genomes at lower taxonomic levels in Thysanoptera

Thrips is an ideal group for studying the evolution of mitochondrial (mt) genomes in the genus and family due to independent rearrangements within this order. The complete sequence of the mitochondrial DNA (mtDNA) of the flower thrips Frankliniella intonsa has been completed and annotated in this study. The circular genome is 15,215bp in length with an A+T content of 75.9% and contains the typical 37 genes and it has triplicate putative control regions. Nucleotide composition is A+T biased, and the majority of the protein-coding genes present opposite CG skew which is reflected by the nucleotide composition, codon and amino acid usage. Although the known thrips have massive gene rearrangements, it showed no reversal of strand asymmetry. Gene rearrangements have been found in the lower taxonomic levels of thrips. Three tRNA genes were translocated in the genus Frankliniella and eight tRNA genes in the family Thripidae. Although the gene arrangements of mt genomes of all three thrips species differ massively from the ancestral insect, they are all very similar to each other, indicating that there was a large rearrangement somewhere before the most recent common ancestor of these three species and very little genomic evolution or rearrangements after then. The extremely similar sequences among the CRs suggest that they are ongoing concerted evolution. Analyses of the up and downstream sequence of CRs reveal that the CR2 is actually the ancestral CR. The three CRs are in the same spot in each of the three thrips mt genomes which have the identical inverted genes. These characteristics might be obtained from the most recent common ancestor of this three thrips. Above observations suggest that the mt genomes of the three thrips keep a single massive rearrangement from the common ancestor and have low evolutionary rates among them.

Amino Acid Uptake and Metabolism of Legionella pneumophila Hosted by Acanthamoeba castellanii

Legionella pneumophila survives and replicates within a Legionella-containing vacuole (LCV) of amoebae and macrophages. Less is known about the carbon metabolism of the bacteria within the LCV. We have now analyzed the transfer and usage of amino acids from the natural host organism Acanthamoeba castellanii to Legionella pneumophila under in vivo (LCV) conditions. For this purpose, A. castellanii was 13C-labeled by incubation in buffer containing [U-(13)C(6)]glucose. Subsequently, these 13C-prelabeled amoebae were infected with L. pneumophila wild type or some mutants defective in putative key enzymes or regulators of carbon metabolism. 13C-Isotopologue compositions of amino acids from bacterial and amoebal proteins were then determined by mass spectrometry. In a comparative approach, the profiles documented the efficient uptake of Acanthamoeba amino acids into the LCV and further into L. pneumophila where they served as precursors for bacterial protein biosynthesis. More specifically, A. castellanii synthesized from exogenous [U-13C6]glucose unique isotopologue mixtures of several amino acids including Phe and Tyr, which were also observed in the same amino acids from LCV-grown L. pneumophila. Minor but significant differences were only detected in the isotopologue profiles of Ala, Asp, and Glu from the amoebal or bacterial protein fractions, respectively, indicating partial de novo synthesis of these amino acids by L. pneumophila. The similar isotopologue patterns in amino acids from L. pneumophila wild type and the mutants under study reflected the robustness of amino acid usage in the LCV of A. castellannii.

Genomic and comparative genomic analyses of Rickettsia heilongjiangensis provide insight into its evolution and pathogenesis

Rickettsia heilongjiangensis, the causative agent of far eastern spotted fever, is an obligate intracellular gram-negative bacterium that belongs to the spotted fever group rickettsiae. To understand the evolution and pathogenesis of R. heilongjiangensis, we analyzed its genome and compared it with other rickettsial genomes available in GenBank. The R. heilongjiangensis chromosome contains 1333 genes, including 1297 protein coding genes and 36 RNA coding genes. The genome also contains 121 pseudogenes, 54 insertion sequences, and 39 tandem repeats. Sixteen genes encoding the major components of the type IV secretion systems were identified in the R. heilongjiangensis genome. In total, 37 β-barrel outer membrane proteins were predicted in the genome, eight of which have been previously confirmed to be outer membrane proteins. In addition, 266 potential virulence factor genes, seven partially deleted antibiotic resistance genes, and a genomic island were identified in the genome. The codon usage in the genome is compatible with its low GC content, and the amino acid usage shows apparent bias. A comparative genomic analysis showed that R. heilongjiangensis and R. japonica share one unique fragment that may be a target sequence for a diagnostic assay. The orthologs of 37 genes of R. heilongjiangensis were found in pathogenic R. rickettsii str. Sheila Smith but not in non-pathogenic R. rickettsii str. Iowa, which may explain why R. heilongjiangensis is pathogenic. Pan-genome analysis showed that R. heilongjiangensis and 42 other rickettsiae strains share 693 core genes with a pan-genome size of 4837 genes. The pan-genome-based phylogeny showed that R. heilongjiangensis was closely related to R. japonica.

미세조류 추출물 및 MAAs 함유 자외선차단제의 자외선차단지수(SPF) 및 자외선 A 차단지수(PFA) 평가

천연물 유래 자외선차단제 개발을 위해 미세조류 추출물 및 mycosporine-like amino acids(MAAs)의 활용가능성을 연구하였다. Chlamycomonas hedleyi 추출물과 추출물 분획물인 MAAs가 각각 7% 포함된 자외선차단크림을 제조하고 인체 적용시험을 실시하였다. 이를 통하여 자외선차단지수(SPF) 및 자외선 A차단지수(PFA)를 평가하였다. Ch. hedleyi 추출물과 MAAs 함유 자외선 차단 크림의 자외선 차단지수는 각각 9.07 및 9.42로 나타났으며, 자외선 A차단지수는 각각 2.43과 2.41로 측정되었다. 자외선 A차단지수가 모두 2 상으로 측정되어 두가지 자외선 차단크림의 자외선 A차단등급은 [PA+]으로 분류되었다. 이상의 연구결과는 미세조류 추출물 및 MAAs 함유 자외선차단제가 자외선을 효과적으로 막지 못하지만, 무기자외선 차단제가 혼합되면 만족할만한 자외선 차단효과를 보일 수 있을 것으로 판단된다. For the sunscreen development from natural resources, a possible usage of microalgal extracts or mycosporine-like amino acids (MAAs) was investigated. Sunscreens containing 7% of microalgal extracts or MAAs derived from microalgae, Chlamydomonas hedleyi, were prepared and they were applied to human research. Through this clinical research, the values of Sun Protection Factors, Sun Protector Factor (SPF) and Protection Factor of UVA (PFA), of sunscreen containing microalgal extracts or MAAs were determined: SPF values of microalgal extracts and MAAs indicated 9.07 and 9.42, respectively, while PFA ones did 2.43 and 2.41. Due to more than 2 of PFA value in both sunscreens, they can be classified into [PA+]. Taken together, although sunscreen containing microalgae-derived extracts or MAAs does not effectively protect UV irradiation, its capacity can be satisfied if inorganic UV-protecting compounds are added.

Protein languages differ depending on microorganism lifestyle.

Few quantitative measures of genome architecture or organization exist to support assumptions of differences between microorganisms that are broadly defined as being free-living or pathogenic. General principles about complete proteomes exist for codon usage, amino acid biases and essential or core genes. Genome-wide shifts in amino acid usage between free-living and pathogenic microorganisms result in fundamental differences in the complexity of their respective proteomes that are size and gene content independent. These differences are evident across broad phylogenetic groups–a result of environmental factors and population genetic forces rather than phylogenetic distance. A novel comparative analysis of amino acid usage–utilizing linguistic analyses of word frequency in language and text–identified a global pattern of higher peptide word repetition in 376 free-living versus 421 pathogen genomes across broad ranges of genome size, G+C content and phylogenetic ancestry. This imprint of repetitive word usage indicates free-living microorganisms have a bias for repetitive sequence usage compared to pathogens. These findings quantify fundamental differences in microbial genomes relative to life-history function.

Hydrophobicity is a hallmark of immunogenic MHC class I T cell epitopes (APP2P.111)

Abstract The molecular mechanisms underlying MHC class I-peptide immunogenicity are not well defined. Here, we investigated the biochemical properties that are associated with immunogenicity from immunogenic (n=5035) and non-immunogenic (n=4853) MHC-I peptides curated from the Immune Epitope Database (IEDB). Using a bioinformatic comparative analysis, we evaluated three amino acid properties (hydrophobicity, side chain mass, and polarity) for an association with immunogenicity within the pMHC dataset. We found a strong bias towards hydrophobic amino acid usage within immunogenic peptides (Spearman ρ = 0.71, P = 4.24 x 10^-4); this difference in hydrophobicity is due to preferential usage of non-polar hydrophobic amino acids in TCR-contact residues for peptides restricted by HLA-A2, H-2Db, H-2Kb (P-values < 0.01). We used amino acid hydrophobicity to design an artificial neural network (ANN) model of immunogenicity for MHC-I peptides. We trained this model to predict mouse H2Db-restricted immunogenic epitopes for 4 different antigens from LCMV, Adenovirus, and Influenza A virus. Our findings suggest that hydrophobicity of TCR contact residues may be a fundamental biochemical hallmark that favors immunogenicity of MHC/peptide complexes. Incorporating hydrophobicity into epitope prediction algorithms may improve prediction of immunogenic CTL epitopes.

Analysis of human TCRβ repertoire by a RARE-sequencing method reveals distinct features of TCRβ diversity and distribution and their alterations with age (IRM10P.758)

Abstract The T cell receptor (TCR) repertoire (diversity and distribution) is a vital parameter of the overall function of T cells. Reduction of TCR diversity and alteration of TCR distribution is believed to occur with age and acts as a major contributor for age-associated decline of immune function. However, neither the actual size of the TCR repertoire nor its precise age-related change have been directly determined. Applying a RACE-next generation sequencing (RAP-Seq) method, we assessed the TCRβ repertoire of blood CD4+ and CD8+ T cells of 13 adults (21-94 years old) including 6 adults with longitudinal samples. We found that TCRβ diversity of CD4+ T cells is 3-4 times greater than CD8+ T cells. TCRβ CDR3 was distinct between CD4+ and CD8+ T cells with preferential amino acid usage. Reduced TCRβ diversity and expanded highly abundant TCRβ clones occurred from middle to old subjects (45-75 years old) but not in very old subjects (80-94 years old). Together, these findings reveal the distinct features and actual age-associated changes of TCRβ repertoire, which could serve as a measure of the immune competency and a guide for the clinical intervention in older humans.

Differences in the Composition of the Human Antibody Repertoire by B Cell Subsets in the Blood

The vast initial diversity of the antibody repertoire is generated centrally by means of a complex series of V(D)J gene rearrangement events, variation in the site of gene segment joining, and TdT catalyzed N-region addition. Although the diversity is great, close inspection has revealed distinct and unique characteristics in the antibody repertoires expressed by different B cell developmental subsets. In order to illustrate our approach to repertoire analysis, we present an in-depth comparison of V(D)J gene usage, hydrophobicity, length, DH reading frame, and amino acid usage between heavy chain repertoires expressed by immature, transitional, mature, memory IgD+, memory IgD−, and plasmacytes isolated from the blood of a single individual. Our results support the view that in both human and mouse, the H chain repertoires expressed by individual, developmental B cell subsets appear to differ in sequence content. Sequencing of unsorted B cells from the blood is thus likely to yield an incomplete or compressed view of what is actually happening in the immune response of the individual. Our findings support the view that studies designed to correlate repertoire expression with diseases of immune function will likely require deep sequencing of B cells sorted by subset.

Selection of peptides binding to metallic borides by screening M13 phage display libraries

BackgroundMetal borides are a class of inorganic solids that is much less known and investigated than for example metal oxides or intermetallics. At the same time it is a highly versatile and interesting class of compounds in terms of physical and chemical properties, like semiconductivity, ferromagnetism, or catalytic activity. This makes these substances attractive for the generation of new materials. Very little is known about the interaction between organic materials and borides. To generate nanostructured and composite materials which consist of metal borides and organic modifiers it is necessary to develop new synthetic strategies. Phage peptide display libraries are commonly used to select peptides that bind specifically to metals, metal oxides, and semiconductors. Further, these binding peptides can serve as templates to control the nucleation and growth of inorganic nanoparticles. Additionally, the combination of two different binding motifs into a single bifunctional phage could be useful for the generation of new composite materials.ResultsIn this study, we have identified a unique set of sequences that bind to amorphous and crystalline nickel boride (Ni3B) nanoparticles, from a random peptide library using the phage display technique. Using this technique, strong binders were identified that are selective for nickel boride. Sequence analysis of the peptides revealed that the sequences exhibit similar, yet subtle different patterns of amino acid usage. Although a predominant binding motif was not observed, certain charged amino acids emerged as essential in specific binding to both substrates. The 7-mer peptide sequence LGFREKE, isolated on amorphous Ni3B emerged as the best binder for both substrates. Fluorescence microscopy and atomic force microscopy confirmed the specific binding affinity of LGFREKE expressing phage to amorphous and crystalline Ni3B nanoparticles.ConclusionsThis study is, to our knowledge, the first to identify peptides that bind specifically to amorphous and to crystalline Ni3B nanoparticles. We think that the identified strong binding sequences described here could potentially serve for the utilisation of M13 phage as a viable alternative to other methods to create tailor-made boride composite materials or new catalytic surfaces by a biologically driven nano-assembly synthesis and structuring.

English

Barbiturases exist solely in bacteria and encompass an undistinguished protein family. s-Triazine compound introduction into the environment owing to recent industrial practices have revitalized barbiturases. Codon usage patterns were analysed in this study for 17 barbiturase genes encoded by 16 bacterial species. Multivariate and correspondence study of amino acid and codon usage was employed for detecting the cause of variation in the gene content. GC3 of synonymously variable codons, RSCU, NC and CAI were estimated with statistical softwares. Examination of DNA composition along with codon usage was done to reveal dynamics of gene evolution and expression of this enzyme. Keywords: Codon usage, barbiturase gene, multivariate statistical analysis African Journal of Biotechnology , Vol. 13(2), pp. 194-201, 8 January, 2014

VH Replacement Footprint Analyzer-I, a Java-Based Computer Program for Analyses of Immunoglobulin Heavy Chain Genes and Potential VH Replacement Products in Human and Mouse

VH replacement occurs through RAG-mediated secondary recombination between a rearranged VH gene and an upstream unrearranged VH gene. Due to the location of the cryptic recombination signal sequence (cRSS, TACTGTG) at the 3′ end of VH gene coding region, a short stretch of nucleotides from the previous rearranged VH gene can be retained in the newly formed VH–DH junction as a “footprint” of VH replacement. Such footprints can be used as markers to identify Ig heavy chain (IgH) genes potentially generated through VH replacement. To explore the contribution of VH replacement products to the antibody repertoire, we developed a Java-based computer program, VH replacement footprint analyzer-I (VHRFA-I), to analyze published or newly obtained IgH genes from human or mouse. The VHRFA-1 program has multiple functional modules: it first uses service provided by the IMGT/V-QUEST program to assign potential VH, DH, and JH germline genes; then, it searches for VH replacement footprint motifs within the VH–DH junction (N1) regions of IgH gene sequences to identify potential VH replacement products; it can also analyze the frequencies of VH replacement products in correlation with publications, keywords, or VH, DH, and JH gene usages, and mutation status; it can further analyze the amino acid usages encoded by the identified VH replacement footprints. In summary, this program provides a useful computation tool for exploring the biological significance of VH replacement products in human and mouse.

Abstract A85: Peptides specific for chronic lymphocytic leukemic cells: Characterization and use in targeted drug delivery.

Abstract The ability to selectively target drugs to Chronic Lymphocytic Leukemia (CLL) cells may significantly improve the efficacy of current chemotherapy protocols for this disease. Our studies in model leukemic systems have already demonstrated that a strategy involving Peptide-Drug Conjugates (PDCs) may achieve this goal. In a previous preliminary study, we used a phage display peptide library to isolate a series of peptides that bind CLL cells. The current research has expanded on this work and began by absorbing a 7-mer linear and 7-mer cyclic phage display peptide libraries on a series of normal human or murine primary cells. Rigorous pre-absorption reduces the original vast phage library population (1011 clones) and enhances the ability to select peptides specific for the target CLL cells. The cells used included endothelial, heart, kidney, lung, liver, skin, fibroblasts and several subpopulations of blood cells. These absorptions resulted in a 7-log reduction in the size of the phage libraries. The stock libraries were also injected into mice. Recovery of blood circulating phage 24hrs later showed that in vivo absorption reduced the pool size of viable phage by approximately 8-logs. These "absorbed" libraries now form the basis for isolating CLL cell-bound and cell-internalizing phage. Target cells include murine cell lines derived from spontaneously-developed aggressive CLL (BCL-1 and A20) and human patient CLL cells. Analysis of peptide sequences of A-20 binding or internalizing phage clones revealed overlap in amino acid usage but not one consistent consensus sequence, suggesting that these peptides target different CLL-specific membrane components, some of which can induce phage internalization and should be applicable for PDCs. Using a similar approach, we are isolating peptides for BCL-1 cells as well as peptides to cells from an initial cohort of CLL patients. The bioinformatic and biochemical results for all these peptides will be presented. Peptides selected on the basis of uptake kinetics will be conjugated to cytotoxic drugs using our dendrimer technology. This technology has already allowed us to demonstrate improved efficacy of known CLL drugs. Progress on this stage of the project will also be presented. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A85. Citation Format: Michael A. Firer, Bat-Chen Lubin, Sharon Cohen, Galia Luboshits. Peptides specific for chronic lymphocytic leukemic cells: Characterization and use in targeted drug delivery. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A85.

Evolution of tryptophan biosynthetic pathway in microbial genomes: a comparative genetic study

Biosynthetic pathway evolution needs to consider the evolution of a group of genes that code for enzymes catalysing the multiple chemical reaction steps leading to the final end product. Tryptophan biosynthetic pathway has five chemical reaction steps that are highly conserved in diverse microbial genomes, though the genes of the pathway enzymes show considerable variations in arrangements, operon structure (gene fusion and splitting) and regulation. We use a combined bioinformatic and statistical analyses approach to address the question if the pathway genes from different microbial genomes, belonging to a wide range of groups, show similar evolutionary relationships within and between them. Our analyses involved detailed study of gene organization (fusion/splitting events), base composition, relative synonymous codon usage pattern of the genes, gene expressivity, amino acid usage, etc. to assess inter- and intra-genic variations, between and within the pathway genes, in diverse group of microorganisms. We describe these genetic and genomic variations in the tryptophan pathway genes in different microorganisms to show the similarities across organisms, and compare the same genes across different organisms to find the possible variability arising possibly due to horizontal gene transfers. Such studies form the basis for moving from single gene evolution to pathway evolutionary studies that are important steps towards understanding the systems biology of intracellular pathways.

Putative anticodons in mitochondrial tRNA sidearm loops: Pocketknife tRNAs?

The hypothesis that tRNA sidearm loops bear anticodons assumes crossovers between anticodon and sidearms, or translation by expressed aminoacylated tRNA halves forming single stem-loops. Only the latter might require ribosomal adaptations. Drosophila mitochondrial codon usages coevolve with sidearm numbers bearing matching putative anticodons (comparing different codon families in one genome, macroevolution) and when comparing different genomes for single codon families (microevolution). Coevolution between Drosophila and yeast mitochondrial antisense tRNAs and codon usages partly confounds microevolutionary patterns for putative sidearm anticodons. Some tRNA sidearm loops have more than seven nucleotides, putative expanded anticodons potentially matching quadruplet codons (tetracodons, codons expanded by a fourth silent position, forming tetragenes (predicted by alignment analyses of Drosophila mitochondrial genomes)). Tetracodon numbers coevolve with expanded tRNA sidearm loops. Sidearm coevolution with amino acid usages and tetragenes occurs for putative anticodons in 5′ and 3′ sidearms loops (D and TΨC loops, respectively), are stronger for the D-loop. Results slightly favour isolated stem-loops upon crossover hypotheses. An alternative hypothesis, that patterns observed for sidearm ‘anticodons’ do not imply translational activity, but recognition signals for tRNA synthetases that aminoacylate tRNAs, is incompatible with tetracodon/tetra-anticodon coevolution. Hence analyses strengthen translational hypotheses for tRNA sidearm anticodons, tetragenes, and antisense tRNAs.