Exon-intron Structure Of Genes Research Articles

Design of potential Ilp markers for Hordeum vulgare and H. marinum Atg2, Atg7, Atg8 genes

Aim. Design of potential intron length polymorphism markers for ATG2, ATG7, ATG8 genes. Methods. Use of classical bioinformatics methods to search for loci encoding ATG2, ATG7, ATG8 in the Hordeum marinum genome based on homology with cultivated barley genes; analysis of exon-intron structure of genes, design of ILP markers. Results. The sequences of ATG2, ATG7, ATG8 genes in H. marinum were determined. Analyzed differences in the exon-intron structure of the ATG2, ATG7, ATG8 loci in cultivated (H. vulgare) and salt-resistant wild barley (H. marinum). 25 potential ILP markers were designed for introns that showed significant interspecies length polymorphism: 13 for ATG2, 6 for ATG7, 3 for ATG8A, 2 for ATG8B and 1 for ATG8D. Conclusions. Based on gene structure comparative analysis of the autophagosome formation proteins ATG2, ATG7, ATG8 of cultivated H. vulgare and salt-tolerasnt H. marinum, a number of introns are recommended for further use as a potential source of ILP markers for the study of interspecies genetic differentiation of cereals.

Genome-Wide Analysis of Aquaporins Gene Family in Populus euphratica and Its Expression Patterns in Response to Drought, Salt Stress, and Phytohormones.

Aquaporins (AQPs) play an essential role in membrane water transport during plant responses to water stresses centered on conventional upstream signals. Phytohormones (PHs) regulate plant growth and yield, working with transcription factors to help plants withstand environmental challenges and regulate physiological and chemical processes. The AQP gene family is important, so researchers have studied its function and regulatory system in numerous species. Yet, there is a critical gap the understanding of many of their molecular features, thus our full knowledge of AQPs is far-off. In this study, we undertook a broad examination of the AQP family gene in Populus euphratica via bioinformatics tools and analyzed the expression patterns of certain members in response to drought, salt, and hormone stress. A total of 22 AQP genes were examined in P. euphratica, and were categorized into four main groups, including TIPs, PIPs, SIPs, and NIPs based on phylogenetic analysis. Comparable exon-intron gene structures were found by gene structure examination, and similarities in motif number and pattern within the same subgroup was determined by motif analysis. The PeuAQP gene family has numerous duplications, and there is a distinct disparity in how the members of the PeuAQP family react to post-translational modifications. Abiotic stress and hormone responses may be mediated by AQPs, as indicated by the abundance of stress response elements found in 22 AQP genes, as revealed by the promoter's cis-elements prediction. Expression pattern analysis reveals that selected six AQP genes from the PIP subgroup were all expressed in the leaves, stem, and roots with varying expression levels. Moreover, qRT-PCR analysis discovered that the majority of the selected AQP members were up- or down-regulated in response to hormone treatment and abiotic stress. Remarkably, PeuAQP14 and PeuAQP15 appeared to be highly responsive to drought stress and PeuAQP15 exhibited a high response to salt stress. The foliar application of the phytohormones (SA, IAA, GA3, MeJA, and ABA) were found to either activate or inhibit PeuAQP, suggesting that they may mitigate the effects of water shortage of poplar water stress. The present work enhances our knowledge of the practical roles of AQPs in stress reactions and offers fundamental information for the AQP genes in poplar species. It also highlights a direction for producing new varieties of poplar species with drought, salt, and hormone tolerance and holds substantial scientific and ecological importance, offering a potential contribution to the conservation of poplar species in arid regions.

Characterization of PYL gene family and identification of HaPYL genes response to drought and salt stress in sunflower.

In the context of global climate change, drought and soil salinity are some of the most devastating abiotic stresses affecting agriculture today. PYL proteins are essential components of abscisic acid (ABA) signaling and play critical roles in responding to abiotic stressors, including drought and salt stress. Although PYL genes have been studied in many species, their roles in responding to abiotic stress are still unclear in the sunflower. In this study, 19 HaPYL genes, distributed on 15 of 17 chromosomes, were identified in the sunflower. Fragment duplication is the main cause of the expansion of PYL genes in the sunflower genome. Based on phylogenetic analysis, HaPYL genes were divided into three subfamilies. Members in the same subfamily share similar protein motifs and gene exon-intron structures, except for the second subfamily. Tissue expression patterns suggested that HaPYLs serve different functions when responding to developmental and environmental signals in the sunflower. Exogenous ABA treatment showed that most HaPYLs respond to an increase in the ABA level. Among these HaPYLs, HaPYL2a, HaPYL4d, HaPYL4g, HaPYL8a, HaPYL8b, HaPYL8c, HaPYL9b, and HaPYL9c were up-regulated with PEG6000 treatment and NaCl treatment. This indicates that they may play a role in resisting drought and salt stress in the sunflower by mediating ABA signaling. Our findings provide some clues to further explore the functions of PYL genes in the sunflower, especially with regards to drought and salt stress resistance.

Comparative analysis of the Squamosa Promoter Binding-Like (SPL) gene family in Nicotiana benthamiana and Nicotiana tabacum

SQUAMOSA PROMOTER BINDING-LIKE (SPL) proteins constitute a large family of transcription factors known to play key roles in growth and developmental processes, including juvenile-to-adult and vegetative-to-reproductive phase transitions. This makes SPLs interesting targets for precision breeding in plants of the Nicotiana genus used as e.g. recombinant biofactories. We report the identification of 49 SPL genes in Nicotiana tabacum cv. K326 and 43 SPL genes in Nicotiana benthamiana LAB strain, which were classified into eight phylogenetic groups according to the SPL classification in Arabidopsis. Exon-intron gene structure and DNA-binding domains were highly conserved between homeologues and orthologues. Thirty of the NbSPL genes and 33 of the NtSPL genes were found to be possible targets of microRNA 156. The expression of SPL genes in leaves was analysed by RNA-seq at three different stages, revealing that genes not under miR156 control were in general constitutively expressed at high levels, whereas miR156-regulated genes showed lower expression, often developmentally regulated. We selected the N. benthamiana SPL13_1a gene as target for a CRISPR/Cas9 knock-out experiment. We show here that a full knock-out in this single gene leads to a significant delay in flowering time, a trait that could be exploited to increase biomass for recombinant protein production.

Genome-Wide Identification and Expression Profiles of C-Repeat Binding Factor Transcription Factors in Betula platyphylla under Abiotic Stress.

CBF (C-repeat binding factor) transcription factor subfamily belongs to AP2/ERF (Apetala 2/ethylene-responsive factor) transcription factor family, known for playing a vital role in plant abiotic stress response. Although some CBF transcription factors have been identified in several species, such as Arabidopsis, tobacco, tomato and poplar, research of CBF focus mainly on model plant Arabidopsis and have not been reported in Betula platyphylla yet. In this study, a total of 20 BpCBF subfamily members were identified. The conserved domains, physicochemical properties, exon-intron gene structure and the structure of conserved protein motifs of BpCBFs were analyzed via bioinformatic tools. The collinearity analysis of CBF genes was performed between Betula platyphylla and Arabidopsis thaliana, Betula platyphylla, and Populus trichocarpa. The cis-acting elements in the promoter region of BpCBFs were identified, which were mainly environmental stress-related and hormone-related element components. In this case, the expression patterns of the 20 BpCBFs upon ABA or salt treatment were investigated. Most of these transcription factors were responsive to ABA or salt stress in different plant tissues. The up-regulation trend upon cold treatment of the six cold-responsive genes validated by qRT-PCR was consistent with the result of RNA-seq. BpCBF7 showed transcription activating activity. This study sheds light on the responses of BpCBFs to abiotic stress and provides a reference for further study of CBF transcription factors in woody plants.

Genome-wide analysis of wheat Di19 gene family and functional analysis of TaDi19-7 in transgenic Arabidopsis

Di19 (Drought-induced 19) proteins play an important role in the regulation of plant growth and stress response. Although Di19 genes have been well-studied in plants, they have not been sufficiently investigated in wheat. Here, we identified 18 Di19 genes (TaDi19s) in wheat and categorized them into three groups using phylogenetic analyses compatible with gene exon-intron structures and protein motif compositions. Genome-wide duplication analysis of the Di19 gene family between wheat and other species showed that there were more duplication events that occurred between wheat and Brachypodium distachyon than Oryza sativa and Arabidopsis thaliana. Further cis-regulatory elements screening revealed that TaDi19 genes may be involved in plant development and stress response. Gene expression analysis revealed diverse expression patterns of TaDi19 genes in different tissue types and stress conditions. Moreover, TaDi19–7 overexpression promoted flowering and branching in transgenic Arabidopsis. Additionally, TaDi19–7 overexpressing lines were more sensitive to drought and salt stresses compared to wild-type plants. Finally, we found that TaDi19–7 affects flowering and abiotic stress response by binding and regulating the expression levels of FT, SOS2, and DREB2A in Arabidopsis. Overall, our study characterized the Di19 gene family in wheat and investigated the biological functions of TaDi19–7 in plant growth and stress tolerance, therefore providing a basis for functional study of TaDi19s in wheat development and abiotic stress response.

Genome-Wide Analysis of the Homeobox Gene Family and Identification of Drought-Responsive Members in Populus trichocarpa.

Homeobox (HB) genes play critical roles in the regulation of plant morphogenesis, growth and development. Here, we identified a total of 156 PtrHB genes from the Populus trichocarpa genome. According to the topologies and taxonomy of the phylogenetic tree constructed by Arabidopsis thaliana HB members, all PtrHB proteins were divided into six subgroups, namely HD-ZIP, ZF-HD, HB-PHD, TALE, WOX and HB-OTHERS. Multiple alignments of conserved homeodomains (HDs) revealed the conserved loci of each subgroup, while gene structure analysis showed similar exon–intron gene structures, and motif analysis indicated the similarity of motif number and pattern in the same subgroup. Promoter analysis indicated that the promoters of PtrHB genes contain a series of cis-acting regulatory elements involved in responding to various abiotic stresses, indicating that PtrHBs had potential functions in these processes. Collinearity analysis revealed that there are 96 pairs of 127 PtrHB genes mainly distributing on Chromosomes 1, 2, and 5. We analyzed the spatio-temporal expression patterns of PtrHB genes, and the virus-induced gene silencing (VIGS) of PtrHB3 gene resulted in the compromised tolerance of poplar seedlings to mannitol treatment. The bioinformatics on PtrHB family and preliminary exploration of drought-responsive genes can provide support for further study of the family in woody plants, especially in drought-related biological processes. It also provides a direction for developing new varieties of poplar with drought resistance. Overall, our results provided significant information for further functional analysis of PtrHB genes in poplar and demonstrated that PtrHB3 is a dominant gene regulating tolerance to water stress treatment in poplar seedlings.

Characterization of the Liriodendron Chinense MYB Gene Family and Its Role in Abiotic Stress Response.

Liriodendron chinense (Lchi) is a Magnoliaceae plant, which is a basic angiosperm left behind by the Pleistocene and mainly distributed in the south of the Yangtze River. Liriodendron hybrids has good wood properties and is widely used in furniture and in other fields. It is not clear if they can adapt to different environmental conditions, such as drought and high and low temperatures, and the molecular mechanisms for this adaptation are unknown. Among plant transcription factors (TFs), the MYB gene family is one of the largest and is often involved in stress or adversity response signaling, growth, and development. Therefore, studying the role of MYBTFs in regulating abiotic stress signaling, growth, and development in Lchi is helpful to promote afforestation in different environments. In our research, a genome-wide analysis of the LchiMYB gene family was performed, including the phylogenetic relationship tree, gene exon-intron structure, collinearity, and chromosomal position. According to the evolutionary tree, 190 LchiMYBs were divided into three main branches. LchiMYBs were evenly distributed across 19 chromosomes, with their collinearity, suggesting that segment duplication events may have contributed to LchiMYB gene expansion. Transcriptomes from eight tissues, 11 stages of somatic embryogenesis, and leaves after cold, heat, and drought stress were used to analyze the function of the MYB gene family. The results of tissue expression analysis showed that most LchiMYB genes regulated bark, leaf, bud, sepal, stigma, and stamen development, as well as the four important stages (ES3, ES4, ES9, and PL) of somatic embryogenesis. More than 60 LchiMYBs responded to heat, cold, and drought stress; some of which underwent gene duplication during evolution. LchiMYB3 was highly expressed under all three forms of stress, while LchiMYB121 was strongly induced by both cold and heat stress. Eight genes with different expression patterns were selected and verified by quantitative real-time PCR (qRT-PCR) experiments. The results suggested that these LchiMYBs may regulate Lchi growth development and resistance to abiotic stress. This study shows the cross-regulatory function of LchiMYBs in the growth and development, asexual reproduction, and abiotic resistance of Lchi. This information will prove pivotal to directing further studies on the biological function of Lchi MYBTFs in genetic improvement and abiotic stress response.

Genome-Wide Identification of MATE Gene Family in Potato (Solanum tuberosum L.) and Expression Analysis in Heavy Metal Stress.

A genome-wide identification and expression analysis of multidrug and toxic compound extrusion (MATE) gene family in potato was carried out to explore the response of MATE proteins to heavy meta stress. In this study, we identified 64 MATE genes from potato genome, which are located on 12 chromosomes, and are divided into I–IV subfamilies based on phylogenetic analysis. According to their order of appearance on the chromosomes, they were named from StMATE1–64. Subcellular location prediction showed that 98% of them are located on the plasma membrane as transporters. Synteny analysis showed that five pairs of collinearity gene pairs belonged to members of subfamily I and subfamily II had two pairs indicating that the duplication is of great significance to the evolution of genes in subfamilies I and II. Gene exon–intron structures and motif composition are more similar in the same subfamily. Every StMATE gene contained at least one cis-acting element associated with regulation of hormone transport. The relative expression levels of eight StMATE genes were significantly upregulated under Cu2+ stress compared with the non-stress condition (0 h). After Cd2+ stress for 24 h, the expression levels of StMATE33 in leaf tissue were significantly increased, indicating its crucial role in the process of Cd2+ stress. Additionally, StMATE18/60/40/33/5 were significantly induced by Cu2+ stress, while StMATE59 (II) was significantly induced by Ni2+ stress. Our study initially explores the biological functions of StMATE genes in the regulation of heavy metal stress, further providing a theoretical basis for studying the subsequent molecular mechanisms in detail.

Identification of Candidate Auxin Response Factors Involved in Pomegranate Seed Coat Development.

Auxin response factors (ARFs) are transcription factors, regulating the auxin signaling pathways involved in plant development and related processes. In this study, we performed the genome-wide identification and characterization of ARFs in pomegranate and compared them with ARFs from three other species. Seventeen PgrARFs were identified and clustered into four groups, according to their phylogenetic relationship with the remaining 59 ARFs. A recent whole-genome duplication event in pomegranate may have contributed to the expansion and diversification of PgrARFs. Genomic truncation and variant splicing mechanisms contributed to the divergence of PgrARFs, a conclusion that was supported by different exon-intron structures of genes and incomplete conserved domains of PgrARFs in a specific phylogenetic group (group III). Interestingly, the absence of motifs from certain PgrARF genes corresponded to their low transcription levels, which contrasted to the highly expressed PgrARFs with intact motifs. Specifically, PgrARF1 and PgrARF2 highly expressed in both inner and outer seed coat, and phylogenetically related to Arabidopsis orthologs which mediates cell divisions in seed coat. We infer these two PgrARFs might involve in seed coat development through cell divisions in response to auxin regulation. These findings provided information on the characteristics and evolutionary relationships of PgrARFs, but also shed lights on their potential roles during seed coat development in pomegranate.

SimSpliceEvol: alternative splicing-aware simulation of biological sequence evolution

BackgroundIt is now well established that eukaryotic coding genes have the ability to produce more than one type of transcript thanks to the mechanisms of alternative splicing and alternative transcription. Because of the lack of gold standard real data on alternative splicing, simulated data constitute a good option for evaluating the accuracy and the efficiency of methods developed for splice-aware sequence analysis. However, existing sequence evolution simulation methods do not model alternative splicing, and so they can not be used to test spliced sequence analysis methods.ResultsWe propose a new method called SimSpliceEvol for simulating the evolution of sets of alternative transcripts along the branches of an input gene tree. In addition to traditional sequence evolution events, the simulation also includes gene exon-intron structure evolution events and alternative splicing events that modify the sets of transcripts produced from genes. SimSpliceEvol was implemented in Python. The source code is freely available at https://github.com/UdeS-CoBIUS/SimSpliceEvol.ConclusionsData generated using SimSpliceEvol are useful for testing spliced RNA sequence analysis methods such as methods for spliced alignment of cDNA and genomic sequences, multiple cDNA alignment, orthologous exons identification, splicing orthology inference, transcript phylogeny inference, which requires to know the real evolutionary relationships between the sequences.

Annotation of the WC1 gene family in Sus scrofa and evaluation of individual SRCR domain affinity for Mycobacterium bovis and Leptospira spp

Abstract γδ T cells respond to a variety of proteins and non-proteinaceous molecules independent of MHC presentation, making them less susceptible to immune evasion and thus; an attractive target for next-generation vaccines. Unfortunately, definitive methods to prime these cells remain elusive. WC1, a member of the group B Scavenger Receptor Cysteine-Rich (SRCR) superfamily, is expressed exclusively on the γδ T cells of swine and ruminants and functions as hybrid co-receptor and pattern recognition receptor for the γδ TCR. It is known that there are thirteen unique WC1 genes expressed in cattle, each containing up to eleven extracellular SRCR domains. Bovine WC1+ γδ T cells share restriction in their TCR gene usage, yet respond to different pathogens based on which WC1 molecule(s) they express. This can be attributed to the ability of the expressed WC1 molecule to directly bind whole pathogens via its SRCR domains. Because WC1 is expressed as a multigene array, we hypothesize that each WC1 gene has co-evolved with a different set of pathogens, and may be exploited to generate a protective immune response from WC1+ γδ T-cells. We sought to identify and characterize WC1 genes expressed in swine, and evaluate their pathogen binding potential. We have obtained cDNA evidence for ten swine WC1 genes, containing up to six extracellular SRCR domains. Through annotation with MAKER, we have confirmed the presence of seven of these genes in the current assembly, Sus scrofa 11.1, and identified four unique exon-intron gene structures. We have shown that SRCR domains from multiple WC1 genes differentially bind Leptospira spp, and Mycobacterium bovis BCG Danish and Pasteur strains.

The exon-intron gene structure upstream of the initiation codon predicts translation efficiency.

Introns in mRNA leaders are common in complex eukaryotes, but often overlooked. These introns are spliced out before translation, leaving exon-exon junctions in the mRNA leaders (leader EEJs). Our multi-omic approach shows that the number of leader EEJs inversely correlates with the main protein translation, as does the number of upstream open reading frames (uORFs). Across the five species studied, the lowest levels of translation were observed for mRNAs with both leader EEJs and uORFs (29%). This class of mRNAs also have ribosome footprints on uORFs, with strong triplet periodicity indicating uORF translation. Furthermore, the positions of both leader EEJ and uORF are conserved between human and mouse. Thus, the uORF, in combination with leader EEJ predicts lower expression for nearly one-third of eukaryotic proteins.

Phylloxerids share ancestral carotenoid biosynthesis genes of fungal origin with aphids and adelgids.

Gene transfer among reproductively isolated organisms can lead to novel phenotypes and increased fitness. Among the Sternorrhyncha, a suborder of plant sap-feeding hemipteran insects, both aphids and adelgids acquired carotenoid biosynthesis genes from a fungal donor that result in ecologically relevant pigmentation. Phylloxerids form another family that are closely related to aphids and adelgids and share similar pigmentation, but are largely uncharacterized for their presence and number of pigment genes that have duplicated among aphids. Here, we examined the transcriptomes of nine phylloxerid species, and performed PCR to amplify carotenoid genes from their genomic DNA. We identified carotenoid cyclase/synthase and desaturase genes in each species and demonstrated that they share the common fungal origin as those of aphids and adelgids based on their exon-intron gene structures and phylogenetic relationships. The phylogenetic analyses also indicated that carotenoid genes evolved following the differentiation of aphids, adelgids, and phylloxerids at the levels of family, genus, and species. Unlike aphids that duplicated these genes in their genomes, phylloxerids maintained only single copies, and some species may lack expression of certain genes. These results suggest that the phylloxerid lifestyle undergoes reduced selection pressure to expand carotenoid synthesis genes, and provides insight into these gene functions in insects.

Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution.

AP1/FUL, SEP, AGL6, and FLC subfamily genes play important roles in flower development. The phylogenetic relationships among them, however, have been controversial, which impedes our understanding of the origin and functional divergence of these genes. One possible reason for the controversy may be the problems caused by changes in the exon-intron structure of genes, which, according to recent studies, may generate non-homologous sites and hamper the homology-based sequence alignment. In this study, we first performed exon-by-exon alignments of these and three outgroup subfamilies (SOC1, AG, and STK). Phylogenetic trees reconstructed based on these matrices show improved resolution and better congruence with species phylogeny. In the context of these phylogenies, we traced evolutionary changes of exon-intron structures in each subfamily. We found that structural changes have occurred frequently following gene duplication and speciation events. Notably, exons 7 and 8 (if present) suffered more structural changes than others. With the knowledge of exon-intron structural changes, we generated more reasonable alignments containing all the focal subfamilies. The resulting trees showed that the SEP subfamily is sister to the monophyletic group formed by AP1/FUL and FLC subfamily genes and that the AGL6 subfamily forms a sister group to the three abovementioned subfamilies. Based on this topology, we inferred the evolutionary history of exon-intron structural changes among different subfamilies. Particularly, we found that the eighth exon originated before the divergence of AP1/FUL, FLC, SEP, and AGL6 subfamilies and degenerated in the ancestral FLC-like gene. These results provide new insights into the origin and evolution of the AP1/FUL, FLC, SEP, and AGL6 subfamilies.

Computer analysis of protein functional sites projection on exon structure of genes in Metazoa

BackgroundStudy of the relationship between the structural and functional organization of proteins and their coding genes is necessary for an understanding of the evolution of molecular systems and can provide new knowledge for many applications for designing proteins with improved medical and biological properties. It is well known that the functional properties of proteins are determined by their functional sites. Functional sites are usually represented by a small number of amino acid residues that are distantly located from each other in the amino acid sequence. They are highly conserved within their functional group and vary significantly in structure between such groups. According to this facts analysis of the general properties of the structural organization of the functional sites at the protein level and, at the level of exon-intron structure of the coding gene is still an actual problem.ResultsOne approach to this analysis is the projection of amino acid residue positions of the functional sites along with the exon boundaries to the gene structure. In this paper, we examined the discontinuity of the functional sites in the exon-intron structure of genes and the distribution of lengths and phases of the functional site encoding exons in vertebrate genes. We have shown that the DNA fragments coding the functional sites were in the same exons, or in close exons. The observed tendency to cluster the exons that code functional sites which could be considered as the unit of protein evolution. We studied the characteristics of the structure of the exon boundaries that code, and do not code, functional sites in 11 Metazoa species. This is accompanied by a reduced frequency of intercodon gaps (phase 0) in exons encoding the amino acid residue functional site, which may be evidence of the existence of evolutionary limitations to the exon shuffling.ConclusionsThese results characterize the features of the coding exon-intron structure that affect the functionality of the encoded protein and allow a better understanding of the emergence of biological diversity.

Cloning and expression analysis of Chitinase genes from Populus canadensis

Plant chitinases play a key role in conferring resistance to environmental stresses, including attack by fungal pathogens. In the present study, we employed rapid amplification of cDNA ends (RACE) to identify five chitinase genes in Populus canadensis Moench. Sequence alignment revealed that these genes belong to five subfamilies of chitinase genes. The full-length cDNAs of these genes ranged in size from 991 to 1358 bp and encoded proteins with mol wts from 29.5 to 40.3 kD. Five genes were grouped into three major clades based on amino acid sequences of encoded proteins. Exon-intron gene structure and protein domain analysis further supported the designation. A three-dimensional structure comparison showed the high similarity between five P. canadensis chitinases and three well-studied chitinases from other species. The expression levels of all five genes were up-regulated during Populus infection with the pathogenic fungus Marssonina brunnea, and four of them were highly induced by salt and drought stresses. Furthermore, such factors as elicitors, wounding, and low temperature also elevated the expression of these chitinase genes to varying extents. We postulated that these chitinase genes may be involved in pathways of the defense against fungal infection and function in response to various abiotic stresses.

Whence genes in pieces: reconstruction of the exon–intron gene structures of the last eukaryotic common ancestor and other ancestral eukaryotes

In eukaryotes, protein-coding sequences are interrupted by non-coding sequences known as introns. During mRNA maturation, introns are excised by the spliceosome and the coding regions, exons, are spliced to form the mature coding region. The intron densities widely differ between eukaryotic lineages, from 6 to 7 introns per kb of coding sequence in vertebrates, some invertebrates and green plants, to only a few introns across the entire genome in many unicellular eukaryotes. Evolutionary reconstructions using maximum likelihood methods suggest intron-rich ancestors for each major group of eukaryotes. For the last common ancestor of animals, the highest intron density of all extant and extinct eukaryotes was inferred, at 120-130% of the human intron density. Furthermore, an intron density within 53-74% of the human values was inferred for the last eukaryotic common ancestor. Accordingly, evolution of eukaryotic genes in all lines of descent involved primarily intron loss, with substantial gain only at the bases of several branches including plants and animals. These conclusions have substantial biological implications indicating that the common ancestor of all modern eukaryotes was a complex organism with a gene architecture resembling those in multicellular organisms. Alternative splicing most likely initially appeared as an inevitable result of splicing errors and only later was employed to generate structural and functional diversification of proteins.

Statistical analysis of exon lengths in various eukaryotes

correspondence: Alexander Bolshoy Department of evolutionary and environmental Biology, University of haifa, haifa 39105, israel Tel +972 4824 0382 Fax +972 4824 0382 email bolshoy@research.haifa.ac.il Purpose: The principal goals of this research were to investigate correlations between certain properties of exons in a gene (ie, between exon density and the corresponding protein length) and to compare genomic trees obtained with different approaches of clustering based on exonic parameters. The aim was a better understanding of exon–intron structures and their origin and development. The exon–intron structures of eukaryote genes are quite different from each other, and the evolution of such structures raises many problematic questions. As a preliminary attempt to address some of these questions, we performed a statistical analysis of gene exon–intron structures. Methods: Taking whole genomes of eukaryotes, we went through all the protein-coding genes in each chromosome separately and calculated the portion of intron-containing genes and average values of the net length of all the exons in a gene, the number of the exons, and the average length of an exon. Comparing those chromosomal and genomic averages, we developed a technique of clustering based on characteristics of the exon–intron structure. This technique of clustering separates different species, grouping them according to eukaryote taxonomy. Conclusion: Our conclusion is that the best approach is based on distances among four principal components obtained by factor analysis and followed by application of clustering algorithms, such as neighbor-joining, k-means, and partitioning around medoids.

How are exons encoding transmembrane sequences distributed in the exon-intron structure of genes?

The exon-intron structure of eukaryotic genes raises a question about the distribution of transmembrane regions in membrane proteins. Were exons that encode transmembrane regions formed simply by inserting introns into preexisting genes or by some kind of exon shuffling? To answer this question, the exon-per-gene distribution was analyzed for all genes in 40 eukaryotic genomes with a particular focus on exons encoding transmembrane segments. In 21 higher multicellular eukaryotes, the percentage of multi-exon genes (those containing at least one intron) within all genes in a genome was high (>70%) and with a mean of 87%. When genes were grouped by the number of exons per gene in higher eukaryotes, good exponential distributions were obtained not only for all genes but also for the exons encoding transmembrane segments, leading to a constant ratio of membrane proteins independent of the exon-per-gene number. The positional distribution of transmembrane regions in single-pass membrane proteins showed that they are generally located in the amino or carboxyl terminal regions. This nonrandom distribution of transmembrane regions explains the constant ratio of membrane proteins to the exon-per-gene numbers because there are always two terminal (i.e., the amino and carboxyl) regions - independent of the length of sequences.