A microsatellite-based genetic linkage map of the waterflea, Daphnia pulex: On the prospect of crustacean genomics

We describe the first genetic linkage map for Daphnia pulex using 185 microsatellite markers, including 115 new markers reported in this study. Our approach was to study the segregation of polymorphisms in 129 F2 progeny of one F1 hybrid obtained by crossing two genetically divergent lineages of Daphnia isolated from two Oregon populations. The map spanned 1206 Kosambi cM and had an average intermarker distance of 7 cM. Linkage groups ranged in size from 7 to 185 cM and contained 4 to 27 markers. The map revealed 12 linkage groups corresponding to the expected number of chromosomes and covers approximately 87% of the genome. Tests for random segregation of alleles at individual loci revealed that 21% of the markers showed significant transmission ratio distortion (primarily homozygote deficiency) likely due to markers being linked to deleterious recessive alleles. This map will become the anchor for the physical map of the Daphnia genome and will serve as a starting point for mapping single and quantitative trait loci affecting ecologically important phenotypes. By mapping 342 tentative orthologous gene pairs (Daphnia/Drosophila) into the Daphnia linkage map, we facilitate future comparative projects.     

High rate of recent intron gain and loss in simultaneously duplicated Arabidopsis genes

We examined the gene structure of a set of 2563 Arabidopsis thaliana paralogous pairs that were duplicated simultaneously 20-60 MYA by tetraploidy. Out of a total of 23,164 introns in these genes, we found that 10,004 pairs have been conserved and 578 introns have been inserted or deleted in the time since the duplication event. This intron insertion/deletion rate of 2.7 x 10(-3) to 9.1 x 10(-4) per site per million years is high in comparison to previous studies. At least 56 introns were gained and 39 lost based on parsimony analysis of the phylogenetic distribution of these introns. We found weak evidence that genes undergoing intron gain and loss are biased with respect to gene ontology terms. Gene pairs that experienced at least 2 intron insertions or deletions show evidence of enrichment for membrane location and transport and transporter activity function. We do not find any relationship of intron flux to expression level or G + C content of the gene. Detection of a bias in the location of intron gains and losses within a gene depends on the method of measurement: an intragene method indicates that events (specifically intron losses) are biased toward the 3' end of the gene. Despite the relatively recent acquisition of these introns, we found only one case where we could identify the mechanism of intron origin--the TOUCH3 gene has experienced 2 tandem, partial, internal gene duplications that duplicated a preexisting intron and also created a novel, alternatively spliced intron that makes use of a duplicated pair of cryptic splice sites.     

Formation of new genes explains lower intron density in mammalian Rhodopsin G protein-coupled receptors

Mammalian G protein-coupled receptor (GPCR) genes are characterised by a large proportion of intronless genes or a lower density of introns when compared with GPCRs of invertebrates. It is unclear which mechanisms have influenced intron density in this protein family, which is one of the largest in the mammalian genomes. We used a combination of Hidden Markov Models (HMM) and BLAST searches to establish the comprehensive repertoire of Rhodopsin GPCRs from seven species and performed overall alignments and phylogenetic analysis using the maximum parsimony method for over 1400 receptors in 12 subgroups. We identified 14 different Ancestral Receptor Groups (ARGs) that have members in both vertebrate and invertebrate species. We found that there exists a remarkable difference in the intron density among ancestral and new Rhodopsin GPCRs. The intron density among ARGs members was more than 3.5-fold higher than that within non-ARG members and more than 2-fold higher when considering only the 7TM region. This suggests that the new GPCR genes have been predominantly formed intronless while the ancestral receptors likely accumulated introns during their evolution. Many of the intron positions found in mammalian ARG receptor sequences were found to be present in orthologue invertebrate receptors suggesting that these intron positions are ancient. This analysis also revealed that one intron position is much more frequent than any other position and it is common for a number of phylogenetically different Rhodopsin GPCR groups. This intron position lies within a functionally important, conserved, DRY motif which may form a proto-splice site that could contribute to positional intron insertion. Moreover, we have found that other receptor motifs, similar to DRY, also contain introns between the second and third nucleotide of the arginine codon which also forms a proto-splice site. Our analysis presents compelling evidence that there was not a major loss of introns in mammalian GPCRs and formation of new GPCRs among mammals explains why these have fewer introns compared to invertebrate GPCRs. We also discuss and speculate about the possible role of different RNA- and DNA-based mechanisms of intron insertion and loss.     

Intron mobility in phage T4 is dependent upon a distinctive class of endonucleases and independent of DNA sequences encoding the intron core: mechanistic and evolutionary implications

Although mobility of the phylogenetically widespread group I introns appears to be mechanistically similar, the phage T4 intron-encoded endonucleases that promote mobility of the td and sunY introns are different from their eukaryotic counterparts. Most notably, they cleave at a distance from the intron insertion sites. The td enzyme was shown to cleave 23-26 nt 5' and the sunY endonuclease 13-15 nt 3' to the intron insertion site to generate 3-nt or 2-nt 3'-OH extensions, respectively. The absolute coconversion of exon markers between the distant cleavage and insertion sites is consistent with the double-strand-break repair model for intron mobility. As a further critical test of the model we have demonstrated that the mobility event is independent of DNA sequences that encode the catalytic intron core structure. Thus, in derivatives in which the lacZ or kanR coding sequences replace the intron, these marker genes are efficiently inserted into intron-minus alleles when the cognate endonuclease is provided in trans. The process is therefore endonuclease-dependent, rather than dependent on the intron per se. These findings, which imply that the endonucleases rather than the introns themselves were the primordial mobile elements, are incorporated into a model for the evolution of mobile introns.     

A very high fraction of unique intron positions in the intron-rich diatom Thalassiosira pseudonana indicates widespread intron gain

Although spliceosomal introns are present in all characterized eukaryotes, intron numbers vary dramatically, from only a handful in the entire genomes of some species to nearly 10 introns per gene on average in vertebrates. For all previously studied intron-rich species, significant fractions of intron positions are shared with other widely diverged eukaryotes, indicating that 1) large numbers of the introns date to much earlier stages of eukaryotic evolution and 2) these lineages have not passed through a very intron-poor stage since early eukaryotic evolution. By the same token, among species that have lost nearly all of their ancestral introns, no species is known to harbor large numbers of more recently gained introns. These observations are consistent with the notion that intron-dense genomes have arisen only once over the course of eukaryotic evolution. Here, we report an exception to this pattern, in the intron-rich diatom Thalassiosira pseudonana. Only 8.1% of studied T. pseudonana intron positions are conserved with any of a variety of divergent eukaryotic species. This implies that T. pseudonana has both 1) lost nearly all of the numerous introns present in the diatom-apicomplexan ancestor and 2) gained a large number of new introns since that time. In addition, that so few apparently inserted T. pseudonana introns match the positions of introns in other species implies that insertion of multiple introns into homologous genic sites in eukaryotic evolution is less common than previously estimated. These results suggest the possibility that intron-rich genomes may have arisen multiple times in evolution. These results also provide evidence that multiple intron insertion into the same site is rare, further supporting the notion that early eukaryotic ancestors were very intron rich.     

Heterogeneous yet similar introns reside in identical positions of the rRNA genes in natural isolates of the archaeon Aeropyrum pernix

Some archaeal ribosomal DNA (rDNA) introns carry homing endonuclease-like genes and are therefore assumed to propagate by "intron homing". A previous study demonstrated that three introns are located within the rRNA operon (arnSL) of Aeropyrum pernix strain K1, two of which, Ialpha and Igamma, harbor open reading frames (ORFs) encoding putative LAGLIDADG-type endonucleases. In an effort to understand further the rDNA intron distribution in natural A. pernix populations, 11 A. pernix strains were isolated from marine hydrothermal biotopes, and comparative nucleotide sequence analysis of the arnSL alleles was performed. Of the 11 isolates, eight contained multiple introns, and three patterns of intron insertion were found. Three novel introns, Idelta (62 bp in length), Ivarepsilon (122 bp) and Izeta (57 bp) were identified. They were all ORF-less, but their predicted RNA secondary structure at the exon-intron junctions was consistent with the bulge-helix-bulge motif. The insertion positions and the terminal inverted repeat sequences of Idelta and Izeta were in agreement with those of Ialpha and Igamma, respectively. This suggests that these intron variants were generated by large indels (insertions/deletions) during their evolution.     

Population differentiation in Daphnia alters community assembly in experimental ponds

Most studies of community assembly ignore how genetic differentiation within species affects their colonization and extinction. However, genetic differentiation in ecologically relevant traits may be substantial enough to alter the colonization and extinction processes that drive community assembly. We measured significant molecular genetic and quantitative trait differentiation among three Daphnia pulex × pulicaria populations in southwestern Michigan ponds and investigated whether this differentiation could alter the assembly of pond zooplankton communities in experimental mesocosms. In this study, we monitored the invasion success of different D. pulex × pulicaria populations after their introduction into an established zooplankton community. We also monitored the invasion success of a diverse array of zooplankton species into different D. pulex × pulicaria populations. Zooplankton community composition depended on the D. pulex × pulicaria source population. Daphnia pulex × pulicaria from one population failed to invade zooplankton communities, while those from other populations successfully invaded similar communities. If population differentiation in other species plays a role in community assembly similar to that demonstrated in our study, assembly may be more sensitive to evolutionary processes than has been previously generally considered.     

Spliceosomal intron insertions in genome compacted ray-finned fishes as evident from phylogeny of MC receptors, also supported by a few other GPCRs

Background

Insertions of spliceosomal introns are very rare events during evolution of vertebrates and the mechanisms governing creation of novel intron(s) remain obscure. Largely, gene structures of melanocortin (MC) receptors are characterized by intron-less architecture. However, recently a few exceptions have been reported in some fishes. This warrants a systematic survey of MC receptors for understanding intron insertion events during vertebrate evolution.

Methodology/Principal Findings

We have compiled an extended list of MC receptors from different vertebrate genomes with variations in fishes. Notably, the closely linked MC2Rs and MC5Rs from a group of ray-finned fishes have three and one intron insertion(s), respectively, with conserved positions and intron phase. In both genes, one novel insertion was in the highly conserved DRY motif at the end of helix TM3. Further, the proto-splice site MAG↑R is maintained at intron insertion sites in these two genes. However, the orthologs of these receptors from zebrafish and tetrapods are intron-less, suggesting these introns are simultaneously created in selected fishes. Surprisingly, these novel introns are traceable only in four fish genomes. We found that these fish genomes are severely compacted after the separation from zebrafish. Furthermore, we also report novel intron insertions in P2Y receptors and in CHRM3. Finally, we report ultrasmall introns in MC2R genes from selected fishes.

Conclusions/Significance

The current repository of MC receptors illustrates that fishes have no MC3R ortholog. MC2R, MC5R, P2Y receptors and CHRM3 have novel intron insertions only in ray-finned fishes that underwent genome compaction. These receptors share one intron at an identical position suggestive of being inserted contemporaneously. In addition to repetitive elements, genome compaction is now believed to be a new hallmark that promotes intron insertions, as it requires rapid DNA breakage and subsequent repair processes to gain back normal functionality.     

Gene invasion in distant eukaryotic lineages: discovery of mutually exclusive genetic elements reveals marine biodiversity

Inteins are rare, translated genetic parasites mainly found in bacteria and archaea, while spliceosomal introns are distinctly eukaryotic features abundant in most nuclear genomes. Using targeted metagenomics, we discovered an intein in an Atlantic population of the photosynthetic eukaryote, Bathycoccus, harbored by the essential spliceosomal protein PRP8 (processing factor 8 protein). Although previously thought exclusive to fungi, we also identified PRP8 inteins in parasitic (Capsaspora) and predatory (Salpingoeca) protists. Most new PRP8 inteins were at novel insertion sites that, surprisingly, were not in the most conserved regions of the gene. Evolutionarily, Dikarya fungal inteins at PRP8 insertion site a appeared more related to the Bathycoccus intein at a unique insertion site, than to other fungal and opisthokont inteins. Strikingly, independent analyses of Pacific and Atlantic samples revealed an intron at the same codon as the Bathycoccus PRP8 intein. The two elements are mutually exclusive and neither was found in cultured Bathycoccus or other picoprasinophyte genomes. Thus, wild Bathycoccus contain one of few non-fungal eukaryotic inteins known and a rare polymorphic intron. Our data indicate at least two Bathycoccus ecotypes exist, associated respectively with oceanic or mesotrophic environments. We hypothesize that intein propagation is facilitated by marine viruses; and, while intron gain is still poorly understood, presence of a spliceosomal intron where a locus lacks an intein raises the possibility of new, intein-primed mechanisms for intron gain. The discovery of nucleus-encoded inteins and associated sequence polymorphisms in uncultivated marine eukaryotes highlights their diversity and reveals potential sexual boundaries between populations indistinguishable by common marker genes.     

Novel Group I Introns Encoding a Putative Homing Endonuclease in the Mitochondrial <Emphasis Type="Italic">cox1</Emphasis> Gene of Scleractinian Corals

Analyses of mitochondrial sequences revealed the existence of a group I intron in the cytochrome oxidase subunit 1 (cox1) gene in 13 of 41 genera (20 out of 73 species) of corals conventionally assigned to the suborder Faviina. With one exception, phylogenies of the coral cox1 gene and its intron were concordant, suggesting at most two insertions and many subsequent losses. The coral introns were inferred to encode a putative homing endonuclease with a LAGLI-DADG motif as reported for the cox1 group I intron in the sea anemone Metridium senile. However, the coral and sea anemone cox1 group I introns differed in several aspects, such as the intron insertion site and sequence length. The coral cox1 introns most closely resemble the mitochondrial cox1 group I introns of a sponge species, which also has the same insertion site. The coral introns are also more similar to the introns of several fungal species than to that of the sea anemone (although the insertion site differs in the fungi). This suggests either a horizontal transfer between a sponge and a coral or independent transfers from a similar fungal donor (perhaps one with an identical insertion site that has not yet been discovered). The common occurrence of this intron in corals strengthens the evidence for an elevated abundance of group I introns in the mitochondria of anthozoans. [Reviewing Editor: Dr. Niles Lehman]     

Coevolution of a homing endonuclease and its host target sequence

We have determined the specificity profile of the homing endonuclease I-AniI and compared it to the conservation of its host gene. Homing endonucleases are encoded within intervening sequences such as group I introns. They initiate the transfer of such elements by cleaving cognate alleles lacking the intron, leading to their transfer via homologous recombination. Each structural homing endonuclease family has arrived at an appropriate balance of specificity and fidelity that avoids toxicity while maximizing target recognition and invasiveness. I-AniI recognizes a strongly conserved target sequence in a host gene encoding apocytochrome B and has fine-tuned its specificity to correlate with wobble versus nonwobble positions across that sequence and to the amount of degeneracy inherent in individual codons. The physiological target site in the host gene is not the optimal substrate for recognition and cleavage: at least one target variant identified during a screen is bound more tightly and cleaved more rapidly. This is a result of the periodic cycle of intron homing, which at any time can present nonoptimal combinations of endonuclease specificity and insertion site sequences in a biological host.     

The I-CeuI endonuclease recognizes a sequence of 19 base pairs and preferentially cleaves the coding strand of the Chlamydomonas moewusii chloroplast large subunit rRNA gene.

The I-CeuI endonuclease is a member of the growing family of homing endonucleases that catalyse mobility of group I introns by making a double-strand break at the homing site of these introns in cognate intronless alleles during genetic crosses. In a previous study, we have shown that a short DNA fragment of 26 bp, encompassing the homing site of the fifth intron in the Chlamydomonas eugametos chloroplast large subunit rRNA gene (Ce LSU.5), was sufficient for I-CeuI recognition and cleavage. Here, we report the recognition sequence of the I-CeuI endonuclease, as determined by random mutagenesis of nucleotide positions adjacent to the I-CeuI cleavage site. Single-base substitutions that completely abolish endonuclease activity delimit a 15-bp sequence whereas those that reduce the cleavage rate define a 19-bp sequence that extends from position -7 to position +12 with respect to the Ce LSU.5 intron insertion site. As the other homing endonucleases that have been studied so far, the I-CeuI endonuclease recognizes a non-symmetric degenerate sequence. The top strand of the recognition sequence is preferred for I-CeuI cleavage and the bottom strand most likely determines the rate of double-strand breaks.     

Distribution of rRNA introns in the three-dimensional structure of the ribosome

More than 1200 introns have been documented at over 150 unique sites in the small and large subunit ribosomal RNA genes (as of February 2002). Nearly all of these introns are assigned to one of four main types: group I, group II, archaeal and spliceosomal. This sequence information has been organized into a relational database that is accessible through the Comparative RNA Web Site (http://www.rna.icmb.utexas.edu/) While the rRNA introns are distributed across the entire tree of life, the majority of introns occur within a few phylogenetic groups. We analyzed the distributions of rRNA introns within the three-dimensional structures of the 30S and 50S ribosomes. Most sites in rRNA genes that contain introns contain only one type of intron. While the intron insertion sites occur at many different coordinates, the majority are clustered near conserved residues that form tRNA binding sites and the subunit interface. Contrary to our expectations, many of these positions are not accessible to solvent in the mature ribosome. The correlation between the frequency of intron insertions and proximity of the insertion site to functionally important residues suggests an association between intron evolution and rRNA function.     

A site-specific endonuclease and co-conversion of flanking exons associated with the mobile td intron of phage T4

The product of the td intron open reading frame (ORF) of phage T4 is required for high-frequency transfer of the intervening sequence from intron-plus (In+) to intron-minus (In-) alleles. In vivo studies have demonstrated that the td ORF product targets cleavage of td In- DNA, and that cleavage is correlated with intron inheritance [Quirk et al., Cell 56 (1989) 455-465]. In the present study we show by in vitro synthesis of the td intron ORF product, that the protein possesses endonuclease activity and efficiently cleaves double-stranded DNA at or near the site of intron integration. In addition, we demonstrate that intron insertion is accompanied by co-conversion of the flanking exon sequences. Co-conversion of markers within 50 nt surrounding the site of intron insertion occurred at a high frequency (80-100%), and decreased at greater distance from the intervening sequence. Co-conversion may provide a mechanism for maintaining exon-intron RNA contacts required for accurate splicing of the relocated intron. Cleavage of target DNA by an intron endonuclease and co-conversion of flanking exon sequences are both features associated with mobile introns of eukaryotes, indicating a common mechanism for intron transfer in the eukaryotic and prokaryotic kingdoms.     

New maximum likelihood estimators for eukaryotic intron evolution

The evolution of spliceosomal introns remains poorly understood. Although many approaches have been used to infer intron evolution from the patterns of intron position conservation, the results to date have been contradictory. In this paper, we address the problem using a novel maximum likelihood method, which allows estimation of the frequency of intron insertion target sites, together with the rates of intron gain and loss. We analyzed the pattern of 10,044 introns (7,221 intron positions) in the conserved regions of 684 sets of orthologs from seven eukaryotes. We determined that there is an average of one target site per 11.86 base pairs (bp) (95% confidence interval, 9.27 to 14.39 bp). In addition, our results showed that: (i) overall intron gains are ~25% greater than intron losses, although specific patterns vary with time and lineage; (ii) parallel gains account for ~18.5% of shared intron positions; and (iii) reacquisition following loss accounts for ~0.5% of all intron positions. Our results should assist in resolving the long-standing problem of inferring the evolution of spliceosomal introns.