Dynamic Insertion–Deletion of Introns in Deuterostome EF-1α Genes

To test the validity of intron–exon structure as a phylogenetic marker, the intron–exon structure of EF-1α genes was investigated for starfish, acornworms, ascidians, larvaceans, and amphioxus and compared with that of vertebrates. Of the 11 distinct intron insertion sites found within the coding regions of the deuterostome EF-1α genes, 7 are shared by several taxa, while the remainder are unique to certain taxa. Examination of the shared introns of the deuterostome EF-1α gene revealed that independent intron loss or intron insertion must have occurred in separate lineages of the deuterostome taxa. Maximum parsimony analysis of the intron–exon data matrix recovered five parsimonious trees (consistency index = 0.867). From this result, we concluded that the intron–exon structure of deuterostome EF-1α has evolved more dynamically than previously thought, rendering it unsuitable as a phylogenetic marker. We also reconstructed an evolutionary history of intron insertion–deletion events on the deuterostome phylogeny, based on several molecular phylogenetic studies. These analyses revealed that the deuterostome EF-1α gene has lost individual introns more frequently than all introns simultaneously.     

Origin and Inheritance of Group I Introns in 26S rRNA Genes of Gaeumannomyces graminis

Studies of the distribution of the three group I introns (intron A, intron T, and intron AT) in the 26S rDNA of Gaeumannomyces graminis had suggested that they were transferred to a common ancestor of G. graminis var. avenae and var. tritici after it had branched off from var. graminis. Intron AT and intron A exhibited vertical inheritance and coevolved in concert with their hosts. Intron loss could occur after its acquisition. Loss of any one of the three introns could occur in var. tritici whereas only loss of intron T had been found in the majority of var. avenae isolates. The existence of isolates of var. tritici and var. avenae with three introns suggested that intron loss could be reversed by intron acquisition and that the whole process is a dynamic one. This process of intron acquisition and intron loss reached different equilibrium points for different varieties and subgroups, which explained the irregular distribution of these introns in G. graminis. Each of the three group I introns was more closely related to other intron sequences that share the same insertion point in the 26S rDNA than to each other. These introns in distantly related organisms appeared to have a common ancestry. This system had provided a good model for studies on both the lateral transfer and common ancestry of group I introns in the 26S rRNA genes. Received: 17 May 1996 / Accepted: 14 January 1997     

Scapharca inæquivalvis Tetrameric Hemoglobin A and B Genes: Evidence for a Minigene

Vertebrate and many invertebrate globin genes have a three-exon/two-intron organization, with introns in highly conserved positions. According to the ``intron early' hypothesis, introns are the vestigial segments which flank previously independent coding sequences, thus providing evidence for the assembly of the ancient proteins by ``exon shuffling.' In this paper, we report the analysis of the genes of the bivalve mollusk Scapharca inaequivalvis tetrameric hemoglobin (HbII), which support this hypothesis, at least for the hemoglobin genes. We show the existence of ``minigenes' in the IIA and IIB globin genes, spanning part of the first and second introns, ``in frame' with the heme-binding domain coded by the second exon. Further support for the exon shuffling hypothesis can be found in the degree of identity of the ``new' translated sequences with those flanking the central protein domain of some invertebrate hemoglobins. Received: 31 July 1997 / Accepted: 12 December 1997     

Ferritin gene organization: Differences between plants and animals suggest possible kingdom-specific selective constraints

Ferritin, a protein widespread in nature, concentrates iron ∼10¹¹–10¹²-fold above the solubility within a spherical shell of 24 subunits; it derives in plants and animals from a common ancestor (based on sequence) but displays a cytoplasmic location in animals compared to the plastid in contemporary plants. Ferritin gene regulation in plants and animals is altered by development, hormones, and excess iron; iron signals target DNA in plants but mRNA in animals. Evolution has thus conserved the two end points of ferritin gene expression, the physiological signals and the protein structure, while allowing some divergence of the genetic mechanisms. Comparison of ferritin gene organization in plants and animals, made possible by the cloning of a dicot (soybean) ferritin gene presented here and the recent cloning of two monocot (maize) ferritin genes, shows evolutionary divergence in ferritin gene organization between plants and animals but conservation among plants or among animals; divergence in the genetic mechanism for iron regulation is reflected by the absence in all three plant genes of the IRE, a highly conserved, noncoding sequence in vertebrate animal ferritin mRNA. In plant ferritin genes, the number of introns (n= 7) is higher than in animals (n= 3). Second, no intron positions are conserved when ferritin genes of plants and animals are compared, although all ferritin gene introns are in the coding region; within kingdoms, the intron positions in ferritin genes are conserved. Finally, secondary protein structure has no apparent relationship to intron/exon boundaries in plant ferritin genes, whereas in animal ferritin genes the correspondence is high. The structural differences in introns/exons among phylogenetically related ferritin coding sequences and the high conservation of the gene structure within plant or animal kingdoms suggest that kingdom-specific functional constraints may exist to maintain a particular intron/exon pattern within ferritin genes. In the case of plants, where ferritin gene intron placement is unrelated to triplet codons or protein structure, and where ferritin is targeted to the plastid, the selection pressure on gene organization may relate to RNA function and plastid/nuclear signaling. Received: 25 July 1995 / Accepted: 3 October 1995     

Comparative evolutionary rates of introns and exons in murine rodents

Analysis of DNA sequences of 132 introns and 140 exons from 42 pairs of orthologous genes of mouse and rat was used to compare patterns of evolutionary change between introns and exons. The mean of the absolute difference in length (measured in base pairs) between the two species was nearly five times as high in the case of introns as in the case of exons. The average rate of nucleotide substitution in introns was very similar to the rate of synonymous substitution in exons, and both were about three times the rate of substitution at nonsynonymous sites in exons. G+C content of introns and exons of the same gene were correlated; but mean G+C content at the third positions of exons was significantly higher than that of introns or positions 1–2 of exons from the same gene. G+C content was conserved over evolutionary time, as indicated by strong correlations between mouse and rat; but the change in G+C content was greatest at position 3 of exons, intermediate in introns, and lowest at positions 1–2 in introns. Received: 23 December 1996 / Accepted: 1 April 1997     

Evolution of Duplicated <Emphasis Type="BoldItalic">reggie</Emphasis> Genes in Zebrafish and Goldfish

Invertebrates, tetrapod vertebrates, and fish might be expected to differ in their number of gene copies, possibly due the occurrence of genome duplication events during animal evolution. Reggie (flotillin) genes code for membrane-associated proteins involved in growth signaling in developing and regenerating axons. Until now, there appeared to be only two reggie genes in fruitflies, mammals, and fish. The aim of this research was to search for additional copies of reggie genes in fishes, since a genome duplication might have increased the gene copy number in this group. We report the presence of up to four distinct reggie genes (two reggie-1 and two reggie-2 genes) in the genomes of zebrafish and goldfish. Phylogenetic analyses show that the zebrafish and goldfish sequence pairs are orthologous, and that the additional copies could have arisen through a genome duplication in a common ancestor of bony fish. The presence of novel reggie mRNAs in fish embryos indicates that the newly discovered gene copies are transcribed and possibly expressed in the developing and regenerating nervous system. The intron/exon boundaries of the new fish genes characterized here correspond with those of human genes, both in location and phase. An evolutionary scenario for the evolution of reggie intron-exon structure, where loss of introns appears to be a distinctive trait in invertebrate reggie genes, is presented. Received: 24 January 2001 / Accepted: 27 July 2001     

The Archaea Monophyly Issue: A Phylogeny of Translational Elongation Factor G(2) Sequences Inferred from an Optimized Selection of Alignment Positions

A global alignment of EF-G(2) sequences was corrected by reference to protein structure. The selection of characters eligible for construction of phylogenetic trees was optimized by searching for regions arising from the artifactual matching of sequence segments unique to different phylogenetic domains. The spurious matchings were identified by comparing all sections of the global alignment with a comprehensive inventory of significant binary alignments obtained by BLAST probing of the DNA and protein databases with representative EF-G(2) sequences. In three discrete alignment blocks (one in domain II and two in domain IV), the alignment of the bacterial sequences with those of Archaea–Eucarya was not retrieved by database probing with EF-G(2) sequences, and no EF-G homologue of the EF-2 sequence segments was detected by using partial EF-G(2) sequences as probes in BLAST/FASTA searches. The two domain IV regions (one of which comprises the ADP-ribosylatable site of EF-2) are almost certainly due to the artifactual alignment of insertion segments that are unique to Bacteria and to Archaea–Eucarya. Phylogenetic trees have been constructed from the global alignment after deselecting positions encompassing the unretrieved, spuriously aligned regions, as well as positions arising from misalignment of the G′ and G″ subdomain insertion segments flanking the ``fifth' consensus motif of the G domain (?varsson, 1995). The results show inconsistencies between trees inferred by alternative methods and alternative (DNA and protein) data sets with regard to Archaea being a monophyletic or paraphyletic grouping. Both maximum-likelihood and maximum-parsimony methods do not allow discrimination (by log-likelihood difference and difference in number of inferred substitutions) between the conflicting (monophyletic vs. paraphyletic Archaea) topologies. No specific EF-2 insertions (or terminal accretions) supporting a crenarchaeal–eucaryal clade are detectable in the new EF-G(2) sequence alignment.     

A possible molecular ancestor for mollusk APGWamide,insect adipokinetic hormone,and crustacean red pigment concentrating hormone

Precursor structures of various members of the neuropeptide family adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) of mandibular arthropods and the APGWamide family of mollusks were compared. Amino acid alignments showed a common overall architecture (signal peptide, active peptide, related peptide), with a similar α helix–random coil secondary structure. DNA sequence alignments revealed close similarities between the genes encoding for the peptides of the two families. The APGWamide genes are larger than the AKH/RPCH genes. The sequence environment occupied by introns is similar in AKH/RPCH and APGWamide genes. Such similarities suggest that these peptide families might have been originated by gene rearrangements from a common ancestor having either an AKH/RPCH/APGWamide-like structure or both an AKH/RPCH-like and an APGWamide-like structures. In the former model, DNA fragments could have been gained when the ancestor evolved to mollusks and it could have lost nucleotides when the progression to mandibular arthropods took place. In the second model, AKH/RPCH-like structures could have been fused during evolution toward mandibular arthropods, whereas in mollusks they could have been lost with the possible amplification of the APGWamide-like structure. Loss of domains in exon 1 may have originated the signal peptide and the first codon of the active RPCH. In exon 2, loss of domains possibly determined the junctions of codons 2 to 5 with the loss of a APGWamide copy; exon 3 underwent fewer variations. The similarity of the mollusk APGWamide precursors is closer to that of the RPCH family than the insect AKH family, indicating an earlier evolutionary departure.     

Gene Conversion and Evolution of Daphniid Hemoglobins (Crustacea, Cladocera)

The extracellular hemoglobins of cladocerans derive from the aggregation of 12 two-domain globin subunits that are apparently encoded by four genes. This study establishes that at least some of these genes occur as a tandem array in both Daphnia magna and Daphnia exilis. The genes share a uniform structure; a bridge intron separates two globin domains which each include three exons and two introns. Introns are small, averaging just 77 bp, but a longer sequence (2.2–3.2 kb) separates adjacent globin genes. A survey of structural diversity in globin genes from other daphniids revealed three independent cases of intron loss, but exon lengths were identical, excepting a 3-bp insertion in exon 5 of Simocephalus. Heterogeneity in the extent of nucleotide divergence was marked among exons, largely as a result of the pronounced diversification of the terminal exon. This variation reflected, in part, varying exposure to concerted evolution. Conversion events were frequent in exons 1–4 but were absent from exons 5 and 6. Because of this difference, the results of phylogenetic analyses were strongly affected by the sequences employed in this construction. Phylogenies based on total nucleotide divergence in exons 1–4 revealed affinities among all genes isolated from a single species, reflecting the impact of gene conversion events. In contrast, phylogenies based on total nucleotide divergence in exons 5 and 6 revealed affinities among orthologous genes from different taxa. Received: 8 March 1999 / Accepted: 14 July 1999     

Molecular evolution of calmodulin-like domain protein kinases (CDPKs) in plants and protists

Many genes for calmodulin-like domain protein kinases (CDPKs) have been identified in plants and Alveolate protists. To study the molecular evolution of the CDPK gene family, we performed a phylogenetic analysis of CDPK genomic sequences. Analysis of introns supports the phylogenetic analysis; CDPK genes with similar intron/exon structure are grouped together on the phylogenetic tree. Conserved introns support a monophyletic origin for plant CDPKs, CDPK-related kinases, and phosphoenolpyruvate carboxylase kinases. Plant CDPKs divide into two major branches. Plant CDPK genes on one branch share common intron positions with protist CDPK genes. The introns shared between protist and plant CDPKs presumably originated before the divergence of plants from Alveolates. Additionally, the calmodulin-like domains of protist CDPKs have intron positions in common with animal and fungal calmodulin genes. These results, together with the presence of a highly conserved phase zero intron located precisely at the beginning of the calmodulin-like domain, suggest that the ancestral CDPK gene could have originated from the fusion of protein kinase and calmodulin genes facilitated by recombination of ancient introns. Received: 11 July 2000 / Accepted: 18 April 2001     

Intron Dynamics and the Evolution of Integrin β-Subunit Genes: Maintenance of an Ancestral Gene Structure in the Coral, Acropora millepora

We have determined the genomic structure of an integrin β-subunit gene from the coral, Acropora millepora. The coding region of the gene contains 26 introns, spaced relatively uniformly, and this is significantly more than have been found in any integrin β-subunit genes from higher animals. Twenty-five of the 26 coral introns are also found in a β-subunit gene from at least one other phylum, indicating that the coral introns are ancestral. While there are some suggestions of intron gain or sliding, the predominant theme seen in the homologues from higher animals is extensive intron loss. The coral baseline allows one to infer that a number of introns found in only one phylum of higher animals result from frequent intron loss, as opposed to the seemingly more parsimonious alternative of isolated intron gain. The patterns of intron loss confirm results from protein sequences that most of the vertebrate genes, with the exception of β4, belong to one of two β subunit families. The similarity of the patterns within each of the β1,2,7 and β3,5,6,8 groups indicates that these gene structures have been very stable since early vertebrate evolution. Intron loss has been more extensive in the invertebrate genes, and obvious patterns have yet to emerge in this more limited data set. Received: 5 March 2001 / Accepted: 17 May 2001     

Multiple origins of fungal group I introns located in the same position of nuclear SSU rRNA gene

The archiascomycetous fungus Protomyces pachydermus has two group I introns within the nuclear small subunit (nSSU) rRNA gene. One of these introns has an internal open reading frame (ORF) that encodes a predicted protein of 228 amino acid residues. On the other hand, Protomyces macrosporus has two group I introns that insert at the same positions as P. pachydermus, which have no ORF. Each alignment was constructed with Protomyces group I introns located in the same position and other introns retrieved by the BLAST Search. Each phylogenetic tree based on the alignment shows that Protomyces introns are monophyletic but the relationships among fungal introns do not reflect on the fungal phylogeny. Therefore, it is suggested that two different horizontal transfers of group I introns occurred at the early stage of Protomyces species diversification. Received: 11 June 1997 / Accepted: 2 September 1997     

Common features of analogous replacement histone H3 genes in animals and plants

Phylogenetic analysis of histone H3 protein sequences demonstrates the independent origin of the replacement histone H3 genes in animals and in plants. Multiple introns in the replacement histone H3 genes of animals in a pattern distinct from that in plant replacement H3 genes supports this conclusion. It is suggested that replacement H3 genes arose at the same time that, independently, multicellular forms of animals and of plants evolved. Judged by the degree of invariant and functionally constrained amino acid positions, histones H3 and H4, which form together the tetramer kernel of the nucleosome, have co-evolved with equal rates of sequence divergence. Residues 31 and 87 in histone H3 are the only residues that consistently changed across each gene duplication event that created functional replacement histone H3 variant forms. Once changed, these residues have remained invariant across divergent speciation. This suggests that they are required to allow replacement histone H3 to participate in the assembly of nucleosomes in non–S-phase cells. The abundant occurrence of polypyrimidine sequences in the introns of all replacement H3 genes, and the replacement of an intron by a polypyrimidine motif upstream of the alfalfa replacement H3 gene, suggests a function. It is speculated that they may contribute to the characteristic cell-cycle-independent pattern of replacement histone H3 genes by binding nucleosome-excluding proteins.     

A Group I Intron in the Nuclear Small Subunit rRNA Gene of Cryptendoxyla hypophloia, an Ascomycetous Fungus: Evidence for a New Major Class of Group I Introns

The ascomycetous fungus Cryptendoxyla hypophloia contains an insertion of 433 base pairs in the genes encoding nuclear small subunit ribosomal RNA. Secondary structure analyses of the insert reveal characteristics indicative of a Group I intron, including elements P, Q, R, and S; however, the sequences of these conserved regions deviate significantly from recognized consensus sequences for Group I introns. Principal-components analysis, based on 79 nucleotide positions from the conserved core sequences of 93 Group I introns, identified 17 introns similar to that of C. hypophloia. This grouping, which includes inserts from phylogenetically diverse organisms, cannot readily be classified in any previously recognized major group of Group I introns. We propose the creation of a new group, IE, to accommodate these sequences, and discuss the evolutionary relationships between group IE and other major groups of Group I introns. Received: 11 January 1998 / Accepted: 12 October 1998     

Evolutionary Comparisons of RecA-Like Proteins Across All Major Kingdoms of Living Organisms

Protein sequences with similarities to Escherichia coli RecA were compared across the major kingdoms of eubacteria, archaebacteria, and eukaryotes. The archaeal sequences branch monophyletically and are most closely related to the eukaryotic paralogous Rad51 and Dmc1 groups. A multiple alignment of the sequences suggests a modular structure of RecA-like proteins consisting of distinct segments, some of which are conserved only within subgroups of sequences. The eukaryotic and archaeal sequences share an N-terminal domain which may play a role in interactions with other factors and nucleic acids. Several positions in the alignment blocks are highly conserved within the eubacteria as one group and within the eukaryotes and archaebacteria as a second group, but compared between the groups these positions display nonconservative amino acid substitutions. Conservation within the RecA-like core domain identifies possible key residues involved in ATP-induced conformational changes. We propose that RecA-like proteins derive evolutionarily from an assortment of independent domains and that the functional homologs of RecA in noneubacteria comprise an array of RecA-like proteins acting in series or cooperatively. Received: 25 October 1996 / Accepted: 31 December 1996     

Heterogeneity in Codon Usage in the Flatworm Schistosoma mansoni

Synonymous codon choices vary considerably among Schistosoma mansoni genes. Principal components analysis detects a single major trend among genes, which highly correlates with GC content in third codon positions and exons, but does not discriminate among putatively highly and lowly expressed genes. The effective number of codons used in each gene, and its distribution when plotted against GC₃, suggests that codon usage is shaped mainly by mutational biases. The GC content of exons, GC₃, 5′, 3′, and flanking (5′+ 3′+ introns) regions are all correlated among them, suggesting that variations in GC content may exist among different regions of the S. mansoni genome. We propose that this genome structure might be among the most important factors shaping codon usage in this species, although the action of selection on certain sequences cannot be excluded. Received: 10 March 1997 / Accepted: 27 June 1997     

The evolution of gymnosperms redrawn by phytochrome genes: the Gnetatae appear at the base of the gymnosperms

Gymnosperms possess two to four phytochrome types which apparently are the result of successive gene duplications in the genomes of their common ancestors. Phytochromes are nuclear-encoded proteins whose genes, contrary to chloroplast, mitochondrion, and rRNA genes, have hitherto rarely been used to examine gymnosperm phylogenies. Since the individual phytochrome gene types implied phylogenies that were not completely congruent to one another, conflicting branching orders were sorted by the number of gene lineages present in a taxon. The Gnetatae (two gene types) branched at the base of all gymnosperms, a position supported by bootstrap sampling (distance and character state trees, maximum likelihood). The Gnetatae were followed by Ginkgo, Cycadatae, and Pinaceae (three gene types) and the remaining conifers (four gene types). Therefore, in phytochrome trees, the most ancient branch of the conifers (Pinatae) seems to be the Pinaceae. The next split appears to have separated Araucariaceae plus Podocarpaceae from the Taxaceae/Taxodiaceae/Cupressaceae group. Structural arrangements in the plastid genomes (Raubeson and Jansen 1992) corroborate the finding that there is no close connection between Pinaceae and Gnetatae as suggested by some publications. The analyses are based on 60 phytochrome genes (579 positions in an alignment of PCR fragments) from 28 species. According to rough divergence time estimates, the last common ancestor of gymnosperms and angiosperms is likely to have existed in the Carboniferous.     

Detection of Genes with Atypical Nucleotide Sequence in Microbial Genomes

Along the gene, nucleotides in various codon positions tend to exert a slight but observable influence on the nucleotide choice at neighboring positions. Such context biases are different in different organisms and can be used as genomic signatures. In this paper, we will focus specifically on the dinucleotide composed of a third codon position nucleotide and its succeeding first position nucleotide. Using the 16 possible dinucleotide combinations, we calculate how well individual genes conform to the observed mean dinucleotide frequencies of an entire genome, forming a distance measure for each gene. It is found that genes from different genomes can be separated with a high degree of accuracy, according to these distance values. In particular, we address the problem of recent horizontal gene transfer, and how imported genes may be evaluated by their poor assimilation to the host's context biases. By concentrating on the third- and succeeding first position nucleotides, we eliminate most spurious contributions from codon usage and amino-acid requirements, focusing mainly on mutational effects. Since imported genes are expected to converge only gradually to genomic signatures, it is possible to question whether a gene present in only one of two closely related organisms has been imported into one organism or deleted in the other. Striking correlations between the proposed distance measure and poor homology are observed when Escherichia coli genes are compared to Salmonella typhi, indicating that sets of outlier genes in E. coli may contain a high number of genes that have been imported into E. coli, and not deleted in S. typhi. Received: 16 January 2001 / Accepted: 30 August 2001