Structural organization of lower marine nonvertebrate calmodulin genes.

The troponin C (TnC) superfamily genes generally possess five introns, and the positions where they are inserted are well conserved except for the fourth intron. Based on a structural comparison of TnC genes, we proposed that the common ancestor of TnC or TnC superfamily genes had no intron corresponding to the modern fourth intron, and therefore members of the superfamily have gained the fourth intron independently within each lineage. Here, we cloned calmodulin (CaM, one of the members of the TnC superfamily) cDNAs from two lower marine nonvertebrates, the sea anemone, Metridium senile, belonging to the Cnidaria, and the sponge, Halichondria okadai, belonging to the Porifera, and also determined their genomic organization. Chordate CaM genes generally possess five introns, but neither sea anemone nor sponge CaM has anything corresponding to the fourth intron of chordate CaMs, suggesting that the early metazoan CaM must have had only four introns. The modern fourth intron of chordate CaMs was acquired within the chordate lineage after nonvertebrate/chordate divergence. This notion concurs with our proposal explaining the evolution of the TnC superfamily genes.     

The genomic structure of the scallop, Patinopecten yessoensis, troponin C gene: a hypothesis for the evolution of troponin C

Two cDNAs encoding troponin C (TnC) isoforms are isolated from the scallop, Patinopecten yessoensis, striated adductor muscle. The sequential differences between these isoforms, named TnC(long) and TnC(short), are restricted in several residues of the C-terminal region. TnC(long) is commonly expressed in both the striated and the smooth adductor muscle; however, TnC(short) is only in the striated adductor muscle. The TnC gene is a single copy gene in the scallop, thus they are expressed through the alternative splicing from the same gene. The scallop TnC gene is constructed from five exons and four introns, and positions of introns are identical with chordate TnC genes, although the scallop TnC possesses no corresponding intron to the fourth intron of chordates. The loss of this intron is also observed in Drosophila TnC; these may be remnants of their ancestor, namely the early metazoan TnC gene might be a five exons-four introns structure. In addition, the absence of the corresponding intron is also observed among protostomian calmodulins (CaMs), a molecule closely related to TnC. This suggests that the common ancestor gene of the TnC superfamily might also be a five exons-four introns structure. Assuming this to be true, the discordance of the fourth intron positions observed among members of the family is well explained by the evolutionary independent gain of the intron on each member's lineage.     

Genomic structure of the amphioxus calcium vector protein.

Calcium vector protein (CaVP) is an EF-hand Ca(2+)-binding protein, which is unique to the protochordate, amphioxus. CaVP is supposed to act as a Ca(2+) signal transductor, but its exact function remains unknown. Not only its function but also its exact evolutionary relationship to other Ca(2+)-binding proteins is unclear. To investigate the evolution of CaVP, we have determined the complete sequences of CaVP cDNAs from two amphioxus species, Branchiostoma lanceolatum and B. floridae, whose open reading frame cDNA and amino acid sequences show 96.5 and 98.2% identity, respectively. We have also elucidated the structure of the gene of B. floridae CaVP, which is made up of seven exons and six introns. The positions of four of the six introns (introns 1, 2, 3, and 5) are identical with those of calmodulin, troponin C, and the Spec protein of the sea urchin. These latter proteins belong to the so-called troponin C superfamily (TnC superfamily) and thus CaVP likely also belongs to this family. Intron 6 is positioned in the 3' noncoding region and is unique to CaVP, so it may represent a landmark of the CaVP lineage only. The position of intron 4 is not conserved in the genes of the TnC superfamily or CaVP, and seems to result from either intron sliding or the addition of an intron (randomly inserted into or close to domain III) to the genes of the TnC superfamily during their evolution.     

The structural organization of the ascidian, Halocynthia roretzi, calmodulin genes. The vicissitude of introns during the evolution of calmodulin genes

Two distinct calmodulin (CaM) genes are isolated from the ascidian, Halocynthia roretzi, (Hr-CaM A and Hr-CaM B) and those structures are determined. There are three nucleotide substitutions, producing two amino acid differences between Hr-CaM A and Hr-CaM B, and those are corresponding to two of the known eight variable residues among metazoan CaMs. Both Hr-CaM A and Hr-CaM B are constructed from six exons and five introns, and the positions of introns are identical. The positions of introns of Hr-CaMs are also identical with those of vertebrate CaMs, except third introns. The third introns of Hr-CaMs are inserted at 28bp upstream when compared with vertebrate CaMs. Thus, sliding of the third intron might have occurred in only the ascidian lineage prior to the gene duplication that also occurred only in that lineage. In addition, with the comparison of the intron positions, we attempt to investigate the vicissitude of introns during the evolution of metazoan CaMs.     

Evolutionary dynamics of spliceosomal intron revealed by <Emphasis Type="Italic">in silico</Emphasis> analyses of the P-Type ATPase superfamily genes

It has been long debated whether spliceosomal introns originated in the common ancestor of eukaryotes and prokaryotes. In this study, we tested the possibility that extant introns were inherited from the common ancestor of eukaryotes and prokaryotes using in silico simulation. We first identified 21 intron positions that are shared among different families of the P-Type ATPase superfamily, some of which are known to have diverged before the separation of prokaryotes and eukaryotes. Theoretical estimates of the expected number of intron positions shared by different genes suggest that the introns at those 21 positions were inserted independently. There seems to be no intron that arose from before the diversification of the P-Type ATPase superfamily. Namely, the present introns were inserted after the separation of eukaryotes and prokaryotes.     

Intron structure of the human antithrombin III gene differs from that of other members of the serine protease inhibitor superfamily

Antithrombin III (ATIII) plays an integral role in the coagulation system by inhibiting thrombin and several other activated clotting factors. Inherited deficiency of ATIII is quite common and can result in life-threatening thrombotic complications. In order to understand the basis of ATIII deficiency, we have isolated and characterized the normal human ATIII gene from a recombinant Charon 4A bacteriophage genomic library. The ATIII gene contains six exons and five introns distributed over approximately 19 kilobases of DNA. The positions of introns in the ATIII gene were compared with other members of the serine protease inhibitor family which share 17-31% amino acid homology. When aligned to achieve maximal protein homology, only one of the ATIII introns corresponded to the four introns of rat angiotensinogen or human alpha 1-antitrypsin. Similarly, only one ATIII intron was homologous to the seven introns of chicken ovalbumin. We present two testable models to explain the discrepancy in intron positions among members of the serine protease inhibitor superfamily of genes.     

Complete sequence of a bovine type I cytokeratin gene: conserved and variable intron positions in genes of polypeptides of the same cytokeratin subfamily

The complete sequence of a bovine gene encoding an epidermal cytokeratin of mol. wt. 54 500 (No VIb) of the acidic (type I) subfamily is presented, including an extended 5' upstream region. The gene (4377 bp, seven introns) which codes for a representative of the glycine-rich subtype of cytokeratins of this subfamily, is compared with genes coding for: another subtype of type I cytokeratin; a basic (type II) cytokeratin gene; and vimentin, a representative of another intermediate filament (IF) protein class. The positions of the five introns located within the highly homologous alpha-helix-rich rod domain are identical or equivalent, i.e., within the same triplet, in the two cytokeratin I genes. Four of these intron positions are also identical with intron sites in the vimentin gene, and three of these intron positions are identical or similar in the type I and type II cytokeratin subfamilies. On the other hand, the gene organization of both type I cytokeratins differs from that of the type II cytokeratin in the rod region in five intron positions and in the introns located in the carboxy-terminal tail region, with the exception of one position at the rod-tail junction. Remarkably, the two type I cytokeratins also differ from each other in the positions of two introns located at and in the region coding for the hypervariable, carboxy-terminal portion. The introns and the 5' upstream regions of the cytokeratin VIb gene do not display notable sequence homologies with the other IF protein genes, but sequences identical with--or very similar to--certain viral and immunoglobulin enhancers have been identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intron loss and gain during evolution of the catalase gene family in angiosperms.

Angiosperms (flowering plants), including both monocots and dicots, contain small catalase gene families. In the dicot, Arabidopsis thaliana, two catalase (CAT) genes, CAT1 and CAT3, are tightly linked on chromosome 1 and a third, CAT2, which is more similar to CAT1 than to CAT3, is unlinked on chromosome 4. Comparison of positions and numbers of introns among 13 angiosperm catalase genomic sequences indicates that intron positions are conserved, and suggests that an ancestral catalase gene common to monocots and dicots contained seven introns. Arabidopsis CAT2 has seven introns; both CAT1 and CAT3 have six introns in positions conserved with CAT2, but each has lost a different intron. We suggest the following sequence of events during the evolution of the Arabidopsis catalase gene family. An initial duplication of an ancestral catalase gene gave rise to CAT3 and CAT1. CAT1 then served as the template for a second duplication, yielding CAT2. Intron losses from CAT1 and CAT3 followed these duplications. One subclade of monocot catalases has lost all but the 5''-most and 3''-most introns, which is consistent with a mechanism of intron loss by replacement of an ancestral intron-containing gene with a reverse-transcribed DNA copy of a fully spliced mRNA. Following this event of concerted intron loss, the Oryza sativa (rice, a monocot) CAT1 lineage acquired an intron in a novel position, consistent with a mechanism of intron gain at proto-splice sites.     

Pushing back the expansion of introns in animal genomes

In a recent paper in Science, surveyed the position of introns in 30 genes of a marine annelid and showed that over 60% of the introns occupy positions identical to those in human homologs. In contrast, both human and marine annelid genes share only 30% of their introns with other invertebrates. These observations suggest that the common ancestor of most animal phyla had intron-rich genes and reinforce the notion that introns proliferated early in the evolutionary history of eukaryotes.     

Generation of a database containing discordant intron positions in eukaryotic genes (MIDB)

MOTIVATION: Intron sliding is the relocation of intron-exon boundaries over short distances and is often also referred to as intron slippage or intron migration or intron drift. We have generated a database containing discordant intron positions in homologous genes (MIDB--Mismatched Intron DataBase). Discordant intron positions are those that are either closely located in homologous genes (within a window of 10 nucleotides) or an intron position that is present in one gene but not in any of its homologs. The MIDB database aims at systematically collecting information about mismatched introns in the genes from GenBank and organizing it into a form useful for understanding the genomics and dynamics of introns thereby helping understand the evolution of genes. RESULTS: Intron displacement or sliding is critically important for explaining the present distribution of introns among orthologous and paralogous genes. MIDB allows examining of intron movements and allows mapping of intron positions from homologous proteins onto a single sequence. The database is of potential use for molecular biologists in general and for researchers who are interested in gene evolution and eukaryotic gene structure. Partial analysis of this database allowed us to identify a few putative cases of intron sliding. AVAILABILITY: http://intron.bic.nus.edu.sg/midb/midb.html     

Compartmentalized isozyme genes and the origin of introns

Summary Both the mouse cytosolic malate dehydrogenase gene and its mitochondrial counterpart contain eight introns, of which two are present at identical positions between the isozyme genes. The probability that the two intron positions coincide by chance between the two genes has been shown to be significantly small (=1.3×10^–3), suggesting that the conservation of the intron positions has a biological significance. On the basis of a rooted phylogenetic tree inferred from a comparison of these isozymes and lactate dehydrogenases, we have shown that the origins of the conserved introns are very old, possibly going back to a date before the divergence of eubacteria, archaebacteria, and eukaryotes. In the aspartate aminotransferase isozyme genes, five of the introns are at identical places. The origins of the five conserved introns, however, are not obvious at present. It remains possible that some or all of the conserved introns have evolved after the divergence of eubacteria and eukaryotes.     

Nucleotide sequence of the triosephosphate isomerase gene from Aspergillus nidulans: implications for a differential loss of introns

A functional cDNA from Aspergillus nidulans encoding triosephosphate isomerase (TPI) was isolated by its ability to complement a tpi1 mutation in Saccharomyces cerevisiae. This cDNA was used to obtain the corresponding gene, tpiA. Alignment of the cDNA and genomic DNA nucleotide sequences indicated that tpiA contains five introns. The intron positions in the tpiA gene were compared with those in the TPI genes of human, chicken, and maize. One intron is present at an identical position in all four organisms, two other introns are located in similar positions in A. nidulans and maize, and the remaining two introns are unique to A. nidulans. These Aspergillus-specific introns are located in regions of the protein that were predicted to be interrupted by introns based on analysis of a Go plot of chicken TPI. These comparisons are discussed in relation to the evolution of introns within TPI genes.     

Globin gene family evolution and functional diversification in annelids

Globins are the most common type of oxygen-binding protein in annelids. In this paper, we show that circulating intracellular globin (Alvinella pompejana and Glycera dibranchiata), noncirculating intracellular globin (Arenicola marina myoglobin) and extracellular globin from various annelids share a similar gene structure, with two conserved introns at canonical positions B12.2 and G7.0. Despite sequence divergence between intracellular and extracellular globins, these data strongly suggest that these three globin types are derived from a common ancestral globin-like gene and evolved by duplication events leading to diversification of globin types and derived functions. A phylogenetic analysis shows a distinct evolutionary history of annelid extracellular hemoglobins with respect to intracellular annelid hemoglobins and mollusc and arthropod extracellular hemoglobins. In addition, dehaloperoxidase (DHP) from the annelid, Amphitrite ornata, surprisingly exhibits close phylogenetic relationships to some annelid intracellular globins. We have characterized the gene structure of A. ornata DHP to confirm assumptions about its homology with globins. It appears that it has the same intron position as in globin genes, suggesting a common ancestry with globins. In A. ornata, DHP may be a derived globin with an unusual enzymatic function.     

Divergent Histories of rDNA Group I Introns in the Lichen Family Physciaceae

The wide but sporadic distribution of group I introns in protists, plants, and fungi, as well as in eubacteria, likely resulted from extensive lateral transfer followed by differential loss. The extent of horizontal transfer of group I introns can potentially be determined by examining closely related species or genera. We used a phylogenetic approach with a large data set (including 62 novel large subunit [LSU] rRNA group I introns) to study intron movement within the monophyletic lichen family Physciaceae. Our results show five cases of horizontal transfer into homologous sites between species but do not support transposition into ectopic sites. This is in contrast to previous work with Physciaceae small subunit (SSU) rDNA group I introns where strong support was found for multiple ectopic transpositions. This difference in the apparent number of ectopic intron movements between SSU and LSU rDNA genes may in part be explained by a larger number of positions in the SSU rRNA, which can support the insertion and/or retention of group I introns. In contrast, we suggest that the LSU rRNA may have fewer acceptable positions and therefore intron spread is limited in this gene. Reviewing Editor: Dr. W. Ford Doolittle     

Conservation versus parallel gains in intron evolution

Orthologous genes from distant eukaryotic species, e.g. animals and plants, share up to 25–30% intron positions. However, the relative contributions of evolutionary conservation and parallel gain of new introns into this pattern remain unknown. Here, the extent of independent insertion of introns in the same sites (parallel gain) in orthologous genes from phylogenetically distant eukaryotes is assessed within the framework of the protosplice site model. It is shown that protosplice sites are no more conserved during evolution of eukaryotic gene sequences than random sites. Simulation of intron insertion into protosplice sites with the observed protosplice site frequencies and intron densities shows that parallel gain can account but for a small fraction (5–10%) of shared intron positions in distantly related species. Thus, the presence of numerous introns in the same positions in orthologous genes from distant eukaryotes, such as animals, fungi and plants, appears to reflect mostly bona fide evolutionary conservation.     

Intron-exon organization and phylogeny in a large superfamily, the paralogous cytochrome P450 genes of Arabidopsis thaliana

The cytochrome P450 gene superfamily is represented by 80 genes in animal genomes and perhaps more than 300 genes in plant genomes. We analyzed about half of all Arabidopsis P450 genes, a very large dataset of truly paralogous genes. Sequence alignments were used to draw phylogenetic trees, and this information was compared with the intron-exon organization of each P450 gene. We found 60 unique intron positions, of which 37 were phase 0 introns. Our results confirm the polyphyletic origin of plant P450 genes. One group of these genes, the A-type P450s, are plant specific and characterized by a simple organization, with one highly conserved intron. Closely related A-type P450 genes are often clustered in the genome with as many as a dozen genes (e.g., of the CYP71 subfamily) on a short stretch of chromosome. The other P450 genes (non-A-type) form several distinct clades and are characterized by numerous introns. One such clade contains the two CYP51 genes, which are thought to encode obtusifoliol 14a demethylase. The two CYP51 genes have a single intron that is not shared with CYP51 genes from vertebrates or fungi, or with any other Arabidopsis P450 gene. Only a few of the Arabidopsis P450 genes are intronless (e.g., the CYP710A and CYP96A subfamilies). There was a relatively good correlation between intron conservation and phylogenetic relationships between members of the P450 subfamilies. Gene organization appears to be a useful tool in establishing the evolutionary relatedness of P450 genes, which may help in predictions of P450 function.     

The evolutionary gain of spliceosomal introns: sequence and phase preferences

Theories regarding the evolution of spliceosomal introns differ in the extent to which the distribution of introns reflects either a formative role in the evolution of protein-coding genes or the adventitious gain of genetic elements. Here, systematic methods are used to assess the causes of the present-day distribution of introns in 10 families of eukaryotic protein-coding genes comprising 1,868 introns in 488 distinct alignment positions. The history of intron evolution inferred using a probabilistic model that allows ancestral inheritance of introns, gain of introns, and loss of introns reveals that the vast majority of introns in these eukaryotic gene families were not inherited from the most recent common ancestral genes, but were gained subsequently. Furthermore, among inferred events of intron gain that meet strict criteria of reliability, the distribution of sites of gain with respect to reading-frame phase shows a 5:3:2 ratio of phases 0, 1 and 2, respectively, and exhibits a nucleotide preference for MAG GT (positions -3 to +2 relative to the site of gain). The nucleotide preferences of intron gain may prove to be the ultimate cause for the phase bias. The phase bias of intron gain is sufficient to account quantitatively for the well-known 5:3:2 bias in phase frequencies among extant introns, a conclusion that holds even when taxonomic heterogeneity in phase patterns is considered. Thus, intron gain accounts for the vast majority of extant introns and for the bias toward phase 0 introns that previously was interpreted as evidence for ancient formative introns.     

Investigation of loss and gain of introns in the compact genomes of pufferfishes (Fugu and Tetraodon)

We have investigated intron evolution in the compact genomes of 2 closely related species of pufferfishes, Fugu rubripes and Tetraodon nigroviridis, that diverged about 32 million years ago (MYA). Analysis of 148,028 aligned intron positions in 13,547 gene pairs using human as an outgroup identified 57 and 24 intron losses in Tetraodon and fugu lineages, respectively, and no gain in either lineage. For comparison, we analyzed 144,545 intron positions in 12,866 orthologous pairs of genes in human and mouse that diverged about 61 MYA using dog as an outgroup and identified 51 intron losses in mouse and 3 losses in human and no gain. The rate of intron loss in Tetraodon is higher than that in fugu, mouse, and human but lower than the previous estimates for other eukaryotes. The introns lost in pufferfishes and mammals are significantly shorter than the mean size of introns in the genome. One intron deleted in fugu and another in Tetraodon have left behind 6 and 3 nucleotides, respectively, suggesting that they were lost due to genomic deletions. Such losses of introns are likely to be the result of a higher rate of DNA deletions experienced by the genomes of pufferfishes compared with mammals. The shorter generation time of Tetraodon compared with fugu, and the rich diversity and higher activity of transposable elements in pufferfishes compared with mammals, may be responsible for the higher rate of intron loss in Tetraodon. Our findings indicate that overall very little intron turnover has occurred in pufferfishes and mammals during recent evolution and that intron gain is an extremely rare event in vertebrate evolution.     

Intron distribution in Plantae: 500 million years of stasis during land plant evolution

Little is known about the evolution of the intron-exon organization in the more primitive groups of land plants, and the intron distribution among Plantae (glauco-, rhodo-, chloro- and streptophytes) has not been investigated so far. The present study is focused on some key species such as the liverwort Marchantia polymorpha, representing the most ancient lineage of land plants, and the streptophycean green alga Mesostigma viride, branching prior to charophycean green algae and terrestrial plants. The intron distribution of six genes for sugar phosphate metabolism was analyzed including four different glyceraldehyde-3-phosphate dehydrogenases (GAPDH), the sedoheptulose-1,7-bisphosphatase (SBP) and the glucose-6-phosphate isomerase (GPI). We established 15 new sequences including three cDNA and twelve genomic clones with up to 24 introns per gene, which were identified in the GPI of Marchantia. The intron patterns of all six genes are completely conserved among seed plants, lycopods, mosses and even liverworts. This intron stasis without any gain of novel introns seem to last for nearly 500 million years and may be characteristic for land plants in general. Some unique intron positions in Mesostigma document that a uniform distribution is no common trait of all streptophytes, but it may correlate with the transition to terrestrial habitats. However, the respective genes of chlorophycean green algae display largely different patterns, thus indicating at least one phase of massive intron rearrangement in the green lineage. We moreover included rhodophyte and glaucophyte reference sequences in our analyses and, even if the well documented monophyly of Plantae is not reflected by a uniform intron distribution, at least one GPI intron is strictly conserved for 1.5 billion years.     

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.