Analysis of evolution of exon-intron structure of eukaryotic genes

The availability of multiple, complete eukaryotic genome sequences allows one to address many fundamental evolutionary questions on genome scale. One such important, long-standing problem is evolution of exon-intron structure of eukaryotic genes. Analysis of orthologous genes from completely sequenced genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists. The data on shared and lineage-specific intron positions were used as the starting point for evolutionary reconstruction with parsimony and maximum-likelihood approaches. Parsimony methods produce reconstructions with intron-rich ancestors but also infer lineage-specific, in many cases, high levels of intron loss and gain. Different probabilistic models gave opposite results, apparently depending on model parameters and assumptions, from domination of intron loss, with extremely intron-rich ancestors, to dramatic excess of gains, to the point of denying any true conservation of intron positions among deep eukaryotic lineages. Development of models with adequate, realistic parameters and assumptions seems to be crucial for obtaining more definitive estimates of intron gain and loss in different eukaryotic lineages. Many shared intron positions were detected in ancestral eukaryotic paralogues which evolved by duplication prior to the divergence of extant eukaryotic lineages. These findings indicate that numerous introns were present in eukaryotic genes already at the earliest stages of evolution of eukaryotes and are compatible with the hypothesis that the original, catastrophic intron invasion accompanied the emergence of the eukaryotic cells. Comparison of various features of old and younger introns starts shedding light on probable mechanisms of intron insertion, indicating that propagation of old introns is unlikely to be a major mechanism for origin of new ones. The existence and structure of ancestral protosplice sites were addressed by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and, accordingly, are not subject to selection for splicing efficiency. It was shown that introns indeed predominantly insert into or are fixed in specific protosplice sites which have the consensus sequence (A/C)AG|Gt.     

Evolution of the multifaceted eukaryotic akirin gene family

Background  

Akirins are nuclear proteins that form part of an innate immune response pathway conserved in Drosophila and mice. This studies aim was to characterise the evolution of akirin gene structure and protein function in the eukaryotes.     

Evolution of specificity in the eukaryotic endomembrane system

Two hundred years after Darwin's birth, our understanding of genetic mechanisms and cell biology has advanced to a level unimaginable in the 19th century. We now know that eukaryotic cells contain a huge variety of internal compartments, each with their own function, identity and history. For the compartments that together form the membrane-trafficking system, one of the central questions is how that identity is encoded and how it evolved. Here we review the key components involved in membrane-trafficking events, including SNAREs, Rabs, vesicle coats, and tethers and what is known about their evolutionary history. Our current understanding suggests a possible common mechanism by which the membrane-trafficking organelles might have evolved. This model of increased organellar complexity by gene duplication and co-evolution of multiple, interacting, specificity-encoding proteins could well be applicable to other non-endosymbiotic organelles as well. The application of basic evolutionary principles well beyond their original scope has been exceedingly powerful not only in reconstructing the history of cellular compartments, but for medical and applied research as well, and underlines the contributions of Darwin's ideas in modern biology.     

Computational prediction of eukaryotic protein-coding genes

The human genome sequence is the book of our life. Buried in this large volume are our genes, which are scattered as small DNA fragments throughout the genome and comprise a small percentage of the total text. Finding these indistinct 'needles' in a vast genomic 'haystack' can be extremely challenging. In response to this challenge, computational prediction approaches have proliferated in recent years that predict the location and structure of genes. Here, I discuss these approaches and explain why they have become essential for the analyses of newly sequenced genomes.     

On the chromatin structure of eukaryotic telomeres

Telomeres prevent chromosome fusions and degradation by exonucleases and are implicated in DNA repair, homologous recombination, chromosome pairing and segregation. All these functions of telomeres require the integrity of their chromatin structure, which has been traditionally considered as heterochromatic. In agreement with this idea, different studies have reported that telomeres associate with heterochromatic marks. However, these studies addressed simultaneously the chromatin structures of telomeres and subtelomeric regions or the chromatin structure of telomeres and Interstitial Telomeric Sequences (ITSs). The independent analysis of Arabidopsis telomeres, subtelomeric regions and ITSs has allowed the discovery of euchromatic telomeres. In Arabidopsis, whereas subtelomeric regions and ITSs associate with heterochromatic marks, telomeres exhibit euchromatic features. We think that this scenario could be found in other model systems if the chromatin organizations of telomeres, subtelomeric regions and ITSs are independently analyzed.     

On the chromatin structure of eukaryotic telomeres

Telomeres prevent chromosome fusions and degradation by exonucleases and are implicated in DNA repair, homologous recombination, chromosome pairing and segregation. All these functions of telomeres require the integrity of their chromatin structure, which has been traditionally considered as heterochromatic. In agreement with this idea, different studies have reported that telomeres associate with heterochromatic marks. However, these studies addressed simultaneously the chromatin structures of telomeres and subtelomeric regions or the chromatin structure of telomeres and Interstitial Telomeric Sequences (ITSs). The independent analysis of Arabidopsis telomeres, subtelomeric regions and ITSs has allowed the discovery of euchromatic telomeres. In Arabidopsis, whereas subtelomeric regions and ITSs associate with heterochromatic marks, telomeres exhibit euchromatic features. We think that this scenario could be found in other model systems if the chromatin organizations of telomeres, subtelomeric regions and ITSs are independently analyzed.Key words: telomeres, subtelomeres, euchromatin, heterochromatin, ChIP, immunolocalizationTelomeric DNA usually contains tandem repeats of a short GC rich motif. The number of repeats and, therefore, the length of telomeres is subject to regulation and influences relevant biological processes like aging and cancer.^1–3 In situ hybridization studies have revealed that telomeric repeats are also present at interstitial chromosomal loci.^4,5 An analysis of the genome sequence from different eukaryotes indicates that ITSs have a widespread distribution in different model systems including zebrafish, chicken, opossum, mouse, dog, cattle, horse, human, rice, poplar or Arabidopsis (see Fig. 1 for an example; www.ncbi.nlm.nih.gov/mapview). These ITSs have been related to chromosomal aberrations, fragile sites, hot spots for recombination and diseases caused by genomic instability, although their functions remain unknown.⁶Open in a separate windowFigure 1Distribution of the main telomeric repeat arrays in the genome of several model organisms. These representations have been performed by using the megaBLAST program and the all assemblies genomic databases at NCBI (www.ncbi.nlm.nih.gov/mapview). Searches for homology with 100 tandem telomeric repeats were done using the default parameters except that the expected threshold was set to 10 and the filters were turned off. Chromosomes are represented as vertical bars and numbered at the bottom. The horizontal bars represent the telomeric repeat arrays. Colors indicate the BLAST scores (red ≥200; pink 80–200; green 50–80).Telomeres and ITSs have probably cross talk through evolution. In some instances, ITSs could have been generated by telomeric fusions. Pioneering studies performed by Hermann J. Muller in Drosophila and Barbara McClintock in maize showed that newly formed chromosome ends tend to fuse giving rise to the so-called breakage-fusion-bridge cycle.^7,8 This cycle can lead to stable chromosomal reorganizations after healing of the broken ends. In addition, Muller and McClintock found that, unlike these newly formed broken chromosome ends, natural chromosomal ends are quite stable and do not tend to fuse.⁹ It is currently known that telomere dysfunction due to mutations that cause telomeric shortening or abolish the expression of certain telomeric proteins can lead to telomeric fusions, anaphase bridges and genome reorganizations.^1–3,10,11 Therefore, telomeric shortening or alterations of telomeric chromatin structure might be expected to generate ITSs through evolution by promoting telomeric fusions.¹² ITSs might also originate through the activity of telomerase during the repair process of double strand breaks or by recombination.^13–16 In addition, telomerase activity might lead to the formation of new telomeres by healing of chromosome breaks within internal telomeric repeats and even within other sequences.^17–19 This process of healing involves the acquisition of telomeric chromatin structure.DNA folds into two major chromatin organizations inside the cell nucleus: heterochromatin and euchromatin. Heterochromatin is highly condensed in interphase nuclei and is usually associated with repetitive and silent DNA. By contrast, euchromatin has an open conformation and is often related to the capacity to be transcribed. Both kinds of chromatin exhibit defined epigenetic modifications that influence their biochemical behavior. Thus, the study of these epigenetic marks is an issue of major interest.The chromatin structures of telomeres and ITSs might be different. Therefore, they should be studied independently. Chromatin structure analyses are usually performed by immunocytolocalization or by chromatin immunoprecipitation (ChIP).^20–23 Special care should be taken when the epigenetic status of telomeres is analyzed by immunocytolocalization. This technique does not allow differentiating between telomeres and subtelomeric regions. Since subtelomeric regions are known to be heterochromatic in many eukaryotic organisms, heterochromatic marks should be immunolocalized at the chromosome ends of these organisms. However, these marks could correspond to subtelomeric regions and not to telomeres.The ChIP technique implies the immunoprecipitation of chromatin with specific antibodies and the further analysis of the immunoprecipitated DNA. DNA sequences immunoprecipitated by a specific antibody are thought to associate in vivo with the feature recognized by this antibody. Whereas the enrichment of single copy sequences in the immunoprecipitated DNA has been usually analyzed by quantitative PCR, the analyses of repetitive DNA sequences have been often performed by hybridization. Thus, multiple telomeric chromatin structure analyses have been performed by hybridizing immunoprecipitated DNA with a telomeric probe. However, these analyses displayed simultaneously the chromatin structures of telomeres and ITSs. High throughput sequencing analyses of the immunoprecipitated DNA might help overcome this problem. Nevertheless, since the reads obtained with these techniques at present are short, it is still difficult to ascertain whether the enrichment of immunoprecipitated telomeric sequences corresponds to telomeres or to ITSs. Third-generation long-read accurate technologies and new algorithms that discriminate between telomeres and ITSs should solve the problem.In principle, the combination of immunocytolocalization and ChIP experiments should help to differentiate between telomeres and ITSs. However, since subtelomeric regions are known to influence telomere function and contain degenerated ITSs, at least in some organisms like humans or Arabidopsis, this may not be necessarily true.⁶ A specific epigenetic mark might be required for telomere function, found associated with telomeric repeats by ChIP and with the end of chromosomes by immunocytolocalization and still not associate with true telomeres but with subtelomeric regions and ITSs or just with subtelomeric ITSs.An alternative way to analyze the chromatin structure of telomeres by ChIP involves the use of frequently cutting restriction enzymes. The chromatin structures of Arabidopsis telomeres and ITSs have been independently studied by using Tru9I, a restriction enzyme that recognizes the sequence TTAA.²⁴ Since telomeres in Arabidopsis and in other model systems are composed of perfect telomeric repeat arrays, they remain uncut after digestion with Tru9I.²⁵ In contrast, Arabidopsis ITSs are frequently cut because they are composed of short arrays of perfect telomeric repeats interspersed with degenerated repeats.^25–28 Thus, when Arabidopsis genomic DNA is digested with Tru9I and hybridized with a telomeric probe, most of the signals corresponding to ITSs disappear.²⁵ The use of Tru9I has made possible to discover that Arabidopsis telomeres exhibit euchromatic features. In contrast, Arabidopsis ITSs and subtelomeric regions are heterochromatic.²⁴ In Arabidopsis, heterochromatin is characterized by cytosine methylation, which can be targeted at CpG, CpNpG or CpNpN residues (where N is any nucleotide), and by H3K9me1,2, H3K27me1,2 and H4K20me1. In turn, Arabidopsis euchromatin is characterized by H3K4me1,2,3, H3K36me1,2,3, H4K20me2,3 and by histones acetylation.²⁹ ChIP experiments processed with Tru9I have revealed that Arabidopsis telomeres have high levels of euchromatic marks (H3K4me2, H3K9 and H4K16 acetylation) and low levels of heterochromatic marks (H3K9me2, H3K27me1 and DNA methylation).²⁴ Therefore, Arabidopsis telomeres exhibit epigenetic modifications characteristic of euchromatin.Different studies in mice, humans or Arabidopsis have reported that telomeres are heterochromatic based on the existence of siRNAs containing telomeric sequences, on the association of telomeric sequences with telomeric and with heterochromatin proteins, on the methylation of telomeric sequences or on the histones modifications associated with telomeric sequences.^30–34 However, the experiments presented in those studies addressed simultaneously the chromatin organizations of telomeres and subtelomeric regions or of telomeres and ITSs. Telomeres have also been reported to be heterochromatic based on the existence of the so-called TElomeric Repeat containing RNAs (TERRA), which are present in different eukaryotes.³⁵ At telomeric regions, TERRA are transcribed from subtelomeric promoters towards chromosome ends. Since human subtelomeric TERRA are mostly composed of subtelomeric sequences, with only about 200 bp of telomeric sequences at their 3′ ends, they might be related to subtelomeric heterochromatin formation rather than to the formation of telomeric chromatin. Nevertheless, TERRA interact with human telomeric proteins and influence telomere function. In addition, TERRA might also be related to ITSs heterochromatinization.^34,35We believe that the scenario found in Arabidopsis could also be found in other model systems if the chromatin structures of telomeres, subtelomeric regions and ITSs are independently analyzed. Several reports have described the presence of histone H3.3 at mice telomeres.^36–39 Since this histone variant has been previously associated with active chromatin, these studies are compatible with a euchromatic organization of telomeres. However, again in these reports, the experiments shown addressed simultaneously the chromatin organization of telomeres and subtelomeric regions or of telomeres and ITSs. In general terms, we believe that a clear distinction between telomeres and ITSs should be established when future ChIP experiments are analyzed. The use of third generation high throughput sequencing technologies or of frequently cutting restriction enzymes might help in this task.As mentioned above, the epigenetic modifications associated with telomeric regions are known to be important for telomere function. These modifications are required to provide genome stability.³³ In this context, it will be relevant to ascertain how the function of Arabidopsis telomeres is influenced by their euchromatic marks and by the presence of heterochromatin at subtelomeric regions.     

Evolution of the Sry genes

Existing DNA sequence data on the Sry gene, the mammalian sex- determining locus in the Y chromosome, were analyzed for primates, rodents, and bovids. In all three taxonomic groups, the terminal sequences evolved faster than the HMG (high mobility group) boxes, and this applies both to synonymous (Ks) and nonsynonymous (Ka) nucleotide substitutions. Similar intragenic correlation between synonymous and nonsynonymous substitution rates was not found either in other mammalian genes that contain a conservative box (Sox, Msx) or in the MADS-box genes of plants. The rate of nonsynonymous substitutions exceeds significantly that of synonymous substitutions in the terminal Sry sequences of apes. We did not find good support for the hypothesis that the high evolutionary rate of Sry would be associated with a promiscuous mating system.      

Evolution of prokaryotic and eukaryotic virulence effectors

Coevolutionary interactions between plants and their bacterial and eukaryotic pathogens are mediated by virulence effectors. These effectors face the daunting challenge of carrying out virulence functions, while also potentially exposing the pathogen to host defense systems. Very strong selective pressures are imposed by these competing roles, and the subsequent genetic changes leave their footprints in the extant allelic variation. This review examines the evolutionary processes that drive pathogen-host interactions as revealed by the genetic signatures left in virulence effectors, and speculate on the different pressures imposed on bacterial versus eukaryotic pathogens. We find numerous instances of positive selection for new allelic forms, and diversifying selection for genetic variability, which results in altered host-pathogen interactions. We also describe how the genetic structure of both bacterial and eukaryotic virulence effectors may contribute to their rapid generation and turnover.     

Cloning of eukaryotic genes in single-strand phage vectors: the human interferon genes

Using oligonucleotide probes with defined sequences, we have selected clones from a human lymphocyte cDNA library which represent human leukocyte (HuIFN-α) and fibroblast (HuIFN-β) interferon gene sequences. Double-stranded f1 phage DNA was used as the vector for initial cloning of cDNA. Clones carrying interferon gene sequences were identified by hybridization with the oligonucleotide probes. The same oligonucleotide probes were used as primers for dideoxy chain termination sequencing of the clones. One HuIFN-α clone, 201, has a nucleotide sequence different from published HuIFN-α sequences. Under control of the lacUV5 promoter, the 201 gene has been used to express biologically active HuIFN-α in Escherichia coli.     

Evolution of prokaryotic homologues of the eukaryotic SEFIR protein domain

SEF/IL17 receptor (SEFIR) domains are mainly found in IL17 receptors (IL17Rs) and their adaptor proteins CIKS (connection to IKK and SAPK/JNK), which exert a host defense role in numbers of infectious diseases and promote inflammatory pathology in autoimmunity. Exploring the evolutionary pathway of SEFIR domains will provide further insight into their functions. Here, we have identified 84 SEFIR domain-containing proteins from more than 1400 prokaryotic genomes. As most SEFIR domain-containing bacterial genomes possess a single SEFIR encoding gene and the SEFIR protein domain forms homodimeric complexes like the Toll/IL1 receptor (TIR) domain, the single bacterial SEFIR proteins may receive binding partners from other organisms. Through comparative and phylogenetic sequence analyses, we show that bacterial SEFIR domain is more similar to that of vertebrate CIKS than IL17R, and it possibly emerges via a lateral gene transfer (LGT) from animals. In addition, our secondary and three-dimensional structural predictions of SEFIR domains reveal that human and pathogenic bacterial SEFIR domains share similar structural and electrostatic features. Our findings provide important clues for further experimental researches on determining the functions of SEFIR proteins in pathogenic prokaryotes.     

Immunochemical analysis of the structure of eukaryotic ribosomes

Summary Antibodies were prepared in rabbits and sheep to rat liver ribosomes, ribosomal subunits, and to mixtures of proteins from the particles. The antisera were characterized by quantitative immunoprecipitation, by passive hemagglutination, by immunodiffusion on Ouchterlony plates, and by immunoelectrophoresis. While all the antisera contained antibodies specific for ribosomal proteins, none had precipitating antibodies against ribosomal RNA. Rat liver ribosomal proteins were more immunogenic in sheep than rabbits, and the large ribosomal subunit and its proteins were more immunogenic than those of the 40S subparticle. Antisera specific for one or the other ribosomal subunit could be prepared; thus it is unlikely that there are antigenic determinants common to the proteins of the two subunits. When ribosomes, ribosomal subunits, or mixtures of proteins were used as antigens the sera contained antibodies directed against a large number of the ribosomal proteins.Abbreviations TP total proteins—used to designate mixtures of proteins from ribosomal particles, hence TP80 is a mixtures of all the proteins from 80S ribosomes - TP60 the proteins from 60S subunits - TP40 the proteins from 40S particles     

Evolution of the eukaryotic protein kinases as dynamic molecular switches

Protein kinases have evolved in eukaryotes to be highly dynamic molecular switches that regulate a plethora of biological processes. Two motifs, a dynamic activation segment and a GHI helical subdomain, distinguish the eukaryotic protein kinases (EPKs) from the more primitive eukaryotic-like kinases. The EPKs are themselves highly regulated, typically by phosphorylation, and this allows them to be rapidly turned on and off. The EPKs have a novel hydrophobic architecture that is typically regulated by the dynamic assembly of two hydrophobic spines that is usually mediated by the phosphorylation of an activation loop phosphate. Cyclic AMP-dependent protein kinase (protein kinase A (PKA)) is used as a prototype to exemplify these features of the PKA superfamily. Specificity in PKA signalling is achieved in large part by packaging the enzyme as inactive tetrameric holoenzymes with regulatory subunits that then are localized to macromolecular complexes in close proximity to dedicated substrates by targeting scaffold proteins. In this way, the cell creates discrete foci that most likely represent the physiological environment for cyclic AMP-mediated signalling.     

Molecular evolution and structure of eukaryotic nuclear RNA polymerase subunits in light of the exon-intron organization of their genes

Analysis of literary data (for Saccharomyces cerevisiae, Caenorhabditis elegans, Arabidopsis thaliana, Homo sapiens, and some other Eucarya) and our data (for Schizosaccharomyces pombe) on the exon-intron organization of the genes encoding subunits of nuclear RNA polymerases showed that introns in the orthologous genes from different organisms are arranged nonrandomly, namely, their positions, if projected on the map of the comparison of the amino acid sequences of the orthologous subunits, not infrequently coincide in evolutionarily distant species. As a rule, intron positions correspond to the boundaries of the structurally conserved regions (domains) or to the sites of possible turns of the polypeptide chain. For example, introns flank the secondary structure elements in the Rpb8 subunit with the known three-dimensional structure or the structure-function modules in subunits Rpb10 and Rpc10. These facts are in agreement with the idea of the ancient origin of introns, and with the notion of evolution of ancient protein sequences through the assembly of their genes from short protoexons selected by the nature as far back as the RNA world times. Comparative analysis of the primary structures of the subunits of eukaryotic RNA polymerases allowed us to reveal a nuclear localization signal in subunit Rpb10 and some hypothetical archaeal homologues of subunit Rpc10.