Beginner's Guide on the Use of PAML to Detect Positive Selection.

The CODEML program in the PAML package has been widely used to analyze protein-coding gene sequences to estimate the synonymous and nonsynonymous rates (dS and dN) and to detect positive Darwinian selection driving protein evolution. For users not familiar with molecular evolutionary analysis, the program is known to have a steep learning curve. Here, we provide a step-by-step protocol to illustrate the commonly used tests available in the program, including the branch models, the site models, and the branch-site models, which can be used to detect positive selection driving adaptive protein evolution affecting particular lineages of the species phylogeny, affecting a subset of amino acid residues in the protein, and affecting a subset of sites along prespecified lineages, respectively. A data set of the myxovirus (Mx) genes from ten mammal and two bird species is used as an example. We discuss a new feature in CODEML that allows users to perform positive selection tests for multiple genes for the same set of taxa, as is common in modern genome-sequencing projects. The PAML package is distributed at https://github.com/abacus-gene/paml under the GNU license, with support provided at its discussion site (https://groups.google.com/g/pamlsoftware). Data files used in this protocol are available at https://github.com/abacus-gene/paml-tutorial.

The chloroplast genome of Farsetia hamiltonii Royle, phylogenetic analysis, and comparative study with other members of Clade C of Brassicaceae.

BACKGROUND: Farsetia hamiltonii Royle is a medicinally important annual plant from the Cholistan desert that belongs to the tribe Anastaticeae and clade C of the Brassicaceae family. We provide the entire chloroplast sequence of F.hamiltonii, obtained using the Illumina HiSeq2500 and paired-end sequencing. We compared F. hamiltonii to nine other clade C species, including Farsetia occidentalis, Lobularia libyca, Notoceras bicorne, Parolinia ornata, Morettia canescens, Cochlearia borzaeana, Megacarpaea polyandra, Biscutella laevigata, and Iberis amara. We conducted phylogenetic research on the 22 Brassicaceae species, which included members from 17 tribes and six clades. RESULTS: The chloroplast genome sequence of F.hamiltonii of 154,802 bp sizes with 36.30% GC content and have a typical structure comprised of a Large Single Copy (LSC) of 83,906 bp, a Small Single Copy (SSC) of 17,988 bp, and two copies of Inverted Repeats (IRs) of 26,454 bp. The genomes of F. hamiltonii and F. occidentalis show shared amino acid frequencies and codon use, RNA editing sites, simple sequence repeats, and oligonucleotide repeats. The maximum likelihood tree revealed Farsetia as a monophyletic genus, closely linked to Morettia, with a bootstrap score of 100. The rate of transversion substitutions (Tv) was higher than the rate of transition substitutions (Ts), resulting in Ts/Tv less than one in all comparisons with F. hamiltonii, indicating that the species are closely related. The rate of synonymous substitutions (Ks) was greater than non-synonymous substitutions (Ka) in all comparisons with F. hamiltonii, with a Ka/Ks ratio smaller than one, indicating that genes underwent purifying selection. Low nucleotide diversity values range from 0.00085 to 0.08516, and IR regions comprise comparable genes on junctions with minimal change, supporting the conserved status of the selected chloroplast genomes of the clade C of the Brassicaceae family. We identified ten polymorphic regions, including rps8-rpl14, rps15-ycf1, ndhG-ndhI, psbK-psbI, ccsA-ndhD, rpl36-rps8, petA-psbJ, ndhF-rpl32, psaJ-rpl3, and ycf1 that might be exploited to construct genuine and inexpensive to solve taxonomic discrepancy and understand phylogenetic relationship amongst Brassicaceae species. CONCLUSION: The entire chloroplast sequencing of F. hamiltonii sheds light on the divergence of genic chloroplast sequences among members of the clade C. When other Farsetia species are sequenced in the future, the full F. hamiltonii chloroplast will be used as a source for comprehensive taxonomical investigations of the genus. The comparison of F. hamiltonii and other clade C species adds new information to the phylogenetic data and evolutionary processes of the clade. The results of this study will also provide further molecular uses of clade C chloroplasts for possible plant genetic modifications and will help recognise more Brassicaceae family species.

HIV-1 Lethality and Loss of Env Protein Expression Induced by Single Synonymous Substitutions in the Virus Genome Intronic-Splicing Silencer.

Synonymous genome recoding has been widely used to study different aspects of virus biology. Codon usage affects the temporal regulation of viral gene expression. In this study, we performed synonymous codon mutagenesis to investigate whether codon usage affected HIV-1 Env protein expression and virus viability. We replaced the codons AGG, GAG, CCU, ACU, CUC, and GGG of the HIV-1 env gene with the synonymous codons CGU, GAA, CCG, ACG, UUA, and GGA, respectively. We found that recoding the Env protein gp120 coding region (excluding the Rev response element [RRE]) did not significantly affect virus replication capacity, even though we introduced 15 new CpG dinucleotides. In contrast, changing a single codon (AGG to CGU) located in the gp41 coding region (HXB2 env position 2125 to 2127), which was included in the intronic splicing silencer (ISS), completely abolished virus replication and Env expression. Computational analyses of this mutant revealed a severe disruption in the ISS RNA secondary structure. A variant that restored ISS secondary RNA structure also reestablished Env production and virus viability. Interestingly, this codon variant prevented both virus replication and Env translation in a eukaryotic expression system. These findings suggested that disrupting mRNA splicing was not the only means of inhibiting translation. Our findings indicated that synonymous gp120 recoding was not always deleterious to HIV-1 replication. Importantly¸ we found that disrupting an external ISS loop strongly affected HIV-1 replication and Env translation.IMPORTANCE Synonymous substitutions can influence virus phenotype, replication capacity, and virulence. In this study, we explored how synonymous codon mutations impacted HIV-1 Env protein expression and virus replication capacity. We changed a single codon, AGG to CGU, which was located in the gp41 coding region (env nucleotide residues 2125 to 2127) and was included in the HIV-1 intronic splicing silencer. This change completely abolished virus replication and Env expression. We also found that changing codon usage in the gp120 region by including an increased number of CpG dinucleotides did not significantly affect Env expression or virus viability. Our findings showed that synonymous recoding was useful for altering viral phenotype and exploring virus biology.

Gauging the trends of pseudogenes in plants.

Pseudogenes, the debilitated parts of ancient genes, were previously scrapped off as junk or discarded genes with no functional significance. Pseudogenes have come under scrutiny for their functionality, since recent studies have unveiled their importance in the regulation of their corresponding parent genes and various biological mechanisms. Despite the enormous occurrence of pseudogenes in plants, the lack of experimental validation has contributed toward their unresolved roles in gene regulation. Contrarily, most of the studies associated with gene regulation have been mainly reported for humans, mice, and other mammalian genomes. Consequently, in order to present a cumulative report on plant-based pseudogenes research, an attempt has been made to assemble multiple studies presenting the pseudogene classification, the prediction and the determination of comparative accuracies of various computational pipelines, and recent trends in analyzing their biological functions, and regulatory mechanisms. This review represents the classical, as well as the recent advances on pseudogene identification and their potential roles in transcriptional regulation, which could possibly invigorate the quality of genome annotation, evolutionary analysis, and complexity surrounding the regulatory pathways in plants. Thus, when the ambiguous boundary girdling the pseudogenes eventually recedes on account of their explicit orchestration role, research in flora would no longer saunter compared to that on fauna.

Large-Scale Comparative Analysis of Codon Models Accounting for Protein and Nucleotide Selection.

There are numerous sources of variation in the rate of synonymous substitutions inside genes, such as direct selection on the nucleotide sequence, or mutation rate variation. Yet scans for positive selection rely on codon models which incorporate an assumption of effectively neutral synonymous substitution rate, constant between sites of each gene. Here we perform a large-scale comparison of approaches which incorporate codon substitution rate variation and propose our own simple yet effective modification of existing models. We find strong effects of substitution rate variation on positive selection inference. More than 70% of the genes detected by the classical branch-site model are presumably false positives caused by the incorrect assumption of uniform synonymous substitution rate. We propose a new model which is strongly favored by the data while remaining computationally tractable. With the new model we can capture signatures of nucleotide level selection acting on translation initiation and on splicing sites within the coding region. Finally, we show that rate variation is highest in the highly recombining regions, and we propose that recombination and mutation rate variation, such as high CpG mutation rate, are the two main sources of nucleotide rate variation. Although we detect fewer genes under positive selection in Drosophila than without rate variation, the genes which we detect contain a stronger signal of adaptation of dynein, which could be associated with Wolbachia infection. We provide software to perform positive selection analysis using the new model.

Unbiased Estimate of Synonymous and Nonsynonymous Substitution Rates with Nonstationary Base Composition.

The measurement of synonymous and nonsynonymous substitution rates (dS and dN) is useful for assessing selection operating on protein sequences or for investigating mutational processes affecting genomes. In particular, the ratio dNdS is expected to be a good proxy for ω, the ratio of fixation probabilities of nonsynonymous mutations relative to that of neutral mutations. Standard methods for estimating dN, dS, or ω rely on the assumption that the base composition of sequences is at the equilibrium of the evolutionary process. In many clades, this assumption of stationarity is in fact incorrect, and we show here through simulations and analyses of empirical data that nonstationarity biases the estimate of dN, dS, and ω. We show that the bias in the estimate of ω can be fixed by explicitly taking into consideration nonstationarity in the modeling of codon evolution, in a maximum likelihood framework. Moreover, we propose an exact method for estimating dN and dS on branches, based on stochastic mapping, that can take into account nonstationarity. This method can be directly applied to any kind of codon evolution model, as long as neutrality is clearly parameterized.

Dating Whole Genome Duplication in Ceratopteris thalictroides and Potential Adaptive Values of Retained Gene Duplicates.

Whole-genome duplications (WGDs) are widespread in plants and frequently coincide with global climatic change events, such as the Cretaceous-Tertiary (KT) extinction event approximately 65 million years ago (mya). Ferns have larger genomes and higher chromosome numbers than seed plants, which likely resulted from multiple rounds of polyploidy. Here, we use diploid and triploid material from a model fern species, Ceratopteris thalictroides, for the detection of WGDs. High-quality RNA-seq data was used to infer the number of synonymous substitutions per synonymous site (Ks) between paralogs; Ks age distribution and absolute dating approach were used to determine the age of WGD events. Evidence of an ancient WGD event with a Ks peak value of approximately 1.2 was obtained for both samples; however, the Ks frequency distributions varied significantly. Importantly, we dated the WGD event at 51-53 mya, which coincides with the Paleocene-Eocene Thermal Maximum (PETM), when the Earth became warmer and wetter than any other period during the Cenozoic. Duplicate genes were preferentially retained for specific functions, such as environment response, further support that the duplicates may have promoted quick adaption to environmental changes and potentially resulted in evolutionary success, especially for pantropical species, such as C. thalictroides, which exhibits higher temperature tolerance.

VHICA, a New Method to Discriminate between Vertical and Horizontal Transposon Transfer: Application to the Mariner Family within Drosophila.

Transposable elements (TEs) are genomic repeated sequences that display complex evolutionary patterns. They are usually inherited vertically, but can occasionally be transmitted between sexually independent species, through so-called horizontal transposon transfers (HTTs). Recurrent HTTs are supposed to be essential in life cycle of TEs, which are otherwise destined for eventual decay. HTTs also impact the host genome evolution. However, the extent of HTTs in eukaryotes is largely unknown, due to the lack of efficient, statistically supported methods that can be applied to multiple species sequence data sets. Here, we developed a new automated method available as a R package "vhica" that discriminates whether a given TE family was vertically or horizontally transferred, and potentially infers donor and receptor species. The method is well suited for TE sequences extracted from complete genomes, and applicable to multiple TEs and species at the same time. We first validated our method using Drosophila TE families with well-known evolutionary histories, displaying both HTTs and vertical transmission. We then tested 26 different lineages of mariner elements recently characterized in 20 Drosophila genomes, and found HTTs in 24 of them. Furthermore, several independent HTT events could often be detected within the same mariner lineage. The VHICA (Vertical and Horizontal Inheritance Consistence Analysis) method thus appears as a valuable tool to analyze the evolutionary history of TEs across a large range of species.

Analyses of transcriptome sequences reveal multiple ancient large-scale duplication events in the ancestor of Sphagnopsida (Bryophyta).

The goal of this research was to investigate whether there has been a whole-genome duplication (WGD) in the ancestry of Sphagnum (peatmoss) or the class Sphagnopsida, and to determine if the timing of any such duplication(s) and patterns of paralog retention could help explain the rapid radiation and current ecological dominance of peatmosses. RNA sequencing (RNA-seq) data were generated for nine taxa in Sphagnopsida (Bryophyta). Analyses of frequency plots for synonymous substitutions per synonymous site (Ks ) between paralogous gene pairs and reconciliation of 578 gene trees were conducted to assess evidence of large-scale or genome-wide duplication events in each transcriptome. Both Ks frequency plots and gene tree-based analyses indicate multiple duplication events in the history of the Sphagnopsida. The most recent WGD event predates divergence of Sphagnum from the two other genera of Sphagnopsida. Duplicate retention is highly variable across species, which might be best explained by local adaptation. Our analyses indicate that the last WGD could have been an important factor underlying the diversification of peatmosses and facilitated their rise to ecological dominance in peatlands. The timing of the duplication events and their significance in the evolutionary history of peat mosses are discussed.

Are Synonymous Substitutions in Flowering Plant Mitochondria Neutral?

Angiosperm mitochondrial genes appear to have very low mutation rates, while non-gene regions expand, diverge, and rearrange quickly. One possible explanation for this disparity is that synonymous substitutions in plant mitochondrial genes are not truly neutral and selection keeps their occurrence low. If this were true, the explanation for the disparity in mutation rates in genes and non-genes needs to consider selection as well as mechanisms of DNA repair. Rps14 is co-transcribed with cob and rpl5 in most plant mitochondrial genomes, but in some genomes, rps14 has been duplicated to the nucleus leaving a pseudogene in the mitochondria. This provides an opportunity to compare neutral substitution rates in pseudogenes with synonymous substitution rates in the orthologs. Genes and pseudogenes of rps14 have been aligned among different species and the mutation rates have been calculated. Neutral substitution rates in pseudogenes and synonymous substitution rates in genes are significantly different, providing evidence that synonymous substitutions in plant mitochondrial genes are not completely neutral. The non-neutrality is not sufficient to completely explain the exceptionally low mutation rates in land plant mitochondrial genomes, but selective forces appear to play a small role.

Gene expression, chromosome heterogeneity and the fast-X effect in mammals.

The higher rate of non-synonymous over synonymous substitutions (dN/dS) of the X chromosome compared with autosomes is often interpreted as a consequence of X hemizygosity. However, other factors, such as gene expression, are also known to vary between X and autosomes. Analysing 4800 orthologues in six mammals, we found that gene expression levels, associated with GC content, fully account for the variation in dN/dS between X and autosomes with no detectable effect of hemizygosity. We also report an extensive variance in dN/dS and gene expression between autosomes.

The impact of local genomic properties on the evolutionary fate of genes.

Functionally indispensable genes are likely to be retained and otherwise to be lost during evolution. This evolutionary fate of a gene can also be affected by factors independent of gene dispensability, including the mutability of genomic positions, but such features have not been examined well. To uncover the genomic features associated with gene loss, we investigated the characteristics of genomic regions where genes have been independently lost in multiple lineages. With a comprehensive scan of gene phylogenies of vertebrates with a careful inspection of evolutionary gene losses, we identified 813 human genes whose orthologs were lost in multiple mammalian lineages: designated 'elusive genes.' These elusive genes were located in genomic regions with rapid nucleotide substitution, high GC content, and high gene density. A comparison of the orthologous regions of such elusive genes across vertebrates revealed that these features had been established before the radiation of the extant vertebrates approximately 500 million years ago. The association of human elusive genes with transcriptomic and epigenomic characteristics illuminated that the genomic regions containing such genes were subject to repressive transcriptional regulation. Thus, the heterogeneous genomic features driving gene fates toward loss have been in place and may sometimes have relaxed the functional indispensability of such genes. This study sheds light on the complex interplay between gene function and local genomic properties in shaping gene evolution that has persisted since the vertebrate ancestor.

Non-synonymous to synonymous substitutions suggest that orthologs tend to keep their functions, while paralogs are a source of functional novelty.

Orthologs separate after lineages split from each other and paralogs after gene duplications. Thus, orthologs are expected to remain more functionally coherent across lineages, while paralogs have been proposed as a source of new functions. Because protein functional divergence follows from non-synonymous substitutions, we performed an analysis based on the ratio of non-synonymous to synonymous substitutions (dN/dS), as proxy for functional divergence. We used five working definitions of orthology, including reciprocal best hits (RBH), among other definitions based on network analyses and clustering. The results showed that orthologs, by all definitions tested, had values of dN/dS noticeably lower than those of paralogs, suggesting that orthologs generally tend to be more functionally stable than paralogs. The differences in dN/dS ratios remained suggesting the functional stability of orthologs after eliminating gene comparisons with potential problems, such as genes with high codon usage biases, low coverage of either of the aligned sequences, or sequences with very high similarities. Separation by percent identity of the encoded proteins showed that the differences between the dN/dS ratios of orthologs and paralogs were more evident at high sequence identity, less so as identity dropped. The last results suggest that the differences between dN/dS ratios were partially related to differences in protein identity. However, they also suggested that paralogs undergo functional divergence relatively early after duplication. Our analyses indicate that choosing orthologs as probably functionally coherent remains the right approach in comparative genomics.

Substitutions in Spike and Nucleocapsid proteins of SARS-CoV-2 circulating in South America.

SARS-CoV-2 is a new member of the genus Betacoronavirus, responsible for the COVID-19 pandemic. The virus crossed the species barrier and established in the human population taking advantage of the spike protein high affinity for the ACE receptor to infect the lower respiratory tract. The Nucleocapsid (N) and Spike (S) are highly immunogenic structural proteins and most commercial COVID-19 diagnostic assays target these proteins. In an unpredictable epidemic, it is essential to know about their genetic variability. The objective of this study was to describe the substitution frequency of the S and N proteins of SARS-CoV-2 in South America. A total of 504 amino acid and nucleotide sequences of the S and N proteins of SARS-CoV-2 from seven South American countries (Argentina, Brazil, Chile, Ecuador, Peru, Uruguay, and Colombia), reported as of June 3, and corresponding to samples collected between March and April 2020, were compared through substitution matrices using the Muscle algorithm. Forty-three sequences from 13 Colombian departments were obtained in this study using the Oxford Nanopore and Illumina MiSeq technologies, following the amplicon-based ARTIC network protocol. The substitutions D614G in S and R203K/G204R in N were the most frequent in South America, observed in 83% and 34% of the sequences respectively. Strikingly, genomes with the conserved position D614 were almost completely replaced by genomes with the G614 substitution between March to April 2020. A similar replacement pattern was observed with R203K/G204R although more marked in Chile, Argentina and Brazil, suggesting similar introduction history and/or control strategies of SARS-CoV-2 in these countries. It is necessary to continue with the genomic surveillance of S and N proteins during the SARS-CoV-2 pandemic as this information can be useful for developing vaccines, therapeutics and diagnostic tests.

Investigating the genomic landscape of novel coronavirus (2019-nCoV) to identify non-synonymous mutations for use in diagnosis and drug design.

Novel coronavirus has wrecked medical and health care facilities claiming ∼5% death tollsglobally. All efforts to contain the pathogenesis either using inhibitory drugs or vaccines largelyremained futile due to a lack of better understanding of the genomic feature of this virus. In thepresent study, we compared the 2019-nCoV with other coronaviruses, which indicated that batSARS like coronavirus could be a probable ancestor of the novel coronavirus. The proteinsequence similarity of pangolin-hCoV and bat-hCoV with human coronavirus was higher ascompared to their nucleotide similarity denoting the occurrence of more synonymous mutationsin the genome. Phylogenetic and alignment analysis of 591 novel coronaviruses of differentclades from Group I to Group V revealed several mutations and concomitant amino acidchanges. Detailed investigation on nucleotide substitution unfolded 100 substitutions in thecoding region of which 43 were synonymous and 57 were of non-synonymous type. The nonsynonymous substitutions resulting into 57 amino acid changes were found to be distributed overdifferent hCoV proteins with maximum on spike protein. An important di-amino acid change RGto KR was observed in ORF9 protein. Additionally, several interesting features of the novelcoronavirus genome have been highlighted in respect to various other human infecting viruseswhich may explain extreme pathogenicity, infectivity and simultaneously the reason behindfailure of the antiviral therapies. SUMMARY: This study presents a comprehensive phylogenetic analysis of SARS-CoV2 isolates to understand discrete mutations that are occurring between patient samples. The analysis unravel various amino acid mutations in the viral proteins which may provide an explanation for varying treatment efficacies of different inhibitory drugs and a future direction towards a combinatorial treatment therapies based on the kind of mutation in the viral genome.

Non-synonymous Substitutions in HIV-1 GAG Are Frequent in Epitopes Outside the Functionally Conserved Regions and Associated With Subtype Differences.

In 2019, 38 million people lived with HIV-1 infection resulting in 690,000 deaths. Over 50% of this infection and its associated deaths occurred in Sub-Saharan Africa. The West African region is a known hotspot of the HIV-1 epidemic. There is a need to develop an HIV-1 vaccine if the HIV epidemic would be effectively controlled. Few protective cytotoxic T Lymphocytes (CTL) epitopes within the HIV-1 GAG (HIV_gagconsv) have been previously identified to be functionally conserved among the HIV-1 M group. These epitopes are currently the focus of universal HIV-1 T cell-based vaccine studies. However, these epitopes' phenotypic and genetic properties have not been observed in natural settings for HIV-1 strains circulating in the West African region. This information is critical as the usefulness of universal HIV-1 vaccines in the West African region depends on these epitopes' occurrence in strains circulating in the area. This study describes non-synonymous substitutions within and without HIV_gagconsv genes isolated from 10 infected Nigerians at the early stages of HIV-1 infection. Furthermore, we analyzed these substitutions longitudinally in five infected individuals from the early stages of infection till after seroconversion. We identified three non-synonymous substitutions within HIV_gagconsv genes isolated from early HIV infected individuals. Fourteen and nineteen mutations outside the HIV_gagconsv were observed before and after seroconversion, respectively, while we found four mutations within the HIV_gagconsv. These substitutions include previously mapped CTL epitope immune escape mutants. CTL immune pressure likely leaves different footprints on HIV-1 GAG epitopes within and outside the HIV_gagconsv. This information is crucial for universal HIV-1 vaccine designs for use in the West African region.

Compositional Analysis of Flatworm Genomes Shows Strong Codon Usage Biases Across All Classes.

In the present work, we performed a comparative genome-wide analysis of 22 species representative of the main clades and lifestyles of the phylum Platyhelminthes. We selected a set of 700 orthologous genes conserved in all species, measuring changes in GC content, codon, and amino acid usage in orthologous positions. Values of 3rd codon position GC spanned over a wide range, allowing to discriminate two distinctive clusters within freshwater turbellarians, Cestodes and Trematodes respectively. Furthermore, a hierarchical clustering of codon usage data differs remarkably from the phylogenetic tree. Additionally, we detected a synonymous codon usage bias that was more dramatic in extreme GC-poor or GC-rich genomes, i.e., GC-poor Schistosomes preferred to use AT-rich terminated synonymous codons, while GC-rich M. lignano showed the opposite behavior. Interestingly, these biases impacted the amino acidic usage, with preferred amino acids encoded by codons following the GC content trend. These are associated with non-synonymous substitutions at orthologous positions. The detailed analysis of the synonymous and non-synonymous changes provides evidence for a two-hit mechanism where both mutation and selection forces drive the diverse coding strategies of flatworms.

Cancer evolution and heterogeneity.

Undoubtedly, intratumor heterogeneity (ITH) is one of the causes of the intractability of cancers. Recently, technological innovation in genomics has promoted studies on ITH in solid tumors and on the pattern and level of diversity, which varies among malignancies. We profiled the genome in multiple regions of nine colorectal cancer (CRC) cases. The most impressive finding was that in the late phase, a parental clone branched into numerous subclones. We found that minor mutations were dominant in advanced CRC named neutral evolution; that is, driver gene aberrations were observed with high proportion in the early-acquired phase, but low in the late-acquired phase. Then, we validated that neutral evolution could cause ITH in advanced CRC by super-computational analysis. According to the clinical findings, we explored a branching evolutionary process model in cancer evolution, which assumes that each tumor cell has cellular automaton. According to the model, we verified factors to foster ITH with neutral evolution in advanced CRC. In this review, we introduce recent advances in the field of ITH including the general component of ITH, clonal selective factors that consolidate the evolutionary process, and a representative clinical application of ITH.

The fate of retrotransposed processed genes in Arabidopsis thaliana.

Processed genes are functional genes that have arisen as a result of the retrotransposition of mRNA molecules. We found 6 genes that generated processed genes in the common ancestor of five Brassicaceae species (Arabidopsis thaliana, Arabidopsis lyrata, Capsella rubella, Brassica rapa and Thellungiella parvula). These processed genes have therefore been kept for at least 30millionyears. Analyses of the Ka/Ks ratio of these genes, and of those having given rise to them, show that they evolve relatively slowly and suggest that the processed genes maintained the same function as that of their parental gene. There is a significant negative correlation between the number of ESTs and transcripts produced and the Ka/Ks ratios of the parental genes but not of the processed genes. This suggests that selection has not yet adapted the selective pressure the processed genes experience to their expression level. However, the A. thaliana processed genes tend to be expressed in the same tissues as that of their parental genes. Furthermore, most have a CAATT-box, a TATA-box and are located about 1kb from another protein-coding gene. Altogether, our results suggest that the processed genes found in the A. thaliana genome have been kept to produce more of the same product, and in the same tissues, as that encoded by their parental gene.

A Case of IL-7R Deficiency Caused by a Novel Synonymous Mutation and Implications for Mutation Screening in SCID Diagnosis.

Reported synonymous substitutions are generally non-pathogenic, and rare pathogenic synonymous variants may be disregarded unless there is a high index of suspicion. In a case of IL7 receptor deficiency severe combined immunodeficiency (SCID), the relevance of a non-reported synonymous variant was only suspected through the use of additional in silico computational tools, which focused on the impact of mutations on gene splicing. The pathogenic nature of the variant was confirmed using experimental validation of the effect on mRNA splicing and IL7 pathway function. This case reinforces the need to use additional experimental methods to establish the functional impact of specific mutations, in particular for cases such as SCID where prompt diagnosis can greatly impact on diagnosis, treatment, and survival.