Evolutionary implications of multiple SINE insertions in an intronic region from diverse mammals

An analysis of the nuclear β-fibrinogen intron 7 locus from 30 taxa representing 12 placental orders of mammals reveals the enriched occurrences of short interspersed element (SINE) insertion events. Mammalian-wide interspersed repeats (MIRs) are present at orthologous sites of all examined species except those in the order Rodentia. The higher substitution rate in mouse and a rare MIR deletion from rat account for the absence of MIR in the rodents. A minimum of five lineage-specific SINE sequences are also found to have independently inserted into this intron in Carnivora, Artiodactyla and Lagomorpha. In the case of Carnivora, the unique amplification pattern of order-specific CAN SINE provides important evidence for the “pan-carnivore” hypothesis of this repeat element and reveals that the CAN SINE family may still be active today. Particularly interesting is the finding that all identified lineage-specific SINE elements show a strong tendency to insert within or in very close proximity to the preexisting MIRs for their efficient integrations, suggesting that the MIR element is a hot spot for successive insertions of other SINEs. The unexpected MIR excision as a result of a random deletion in the rat intron locus and the non-random site targeting detected by this study indicate that SINEs actually have a greater insertional flexibility and regional specificity than had previously been recognized. Implications for SINE sequence evolution upon and following integration, as well as the fascinating interactions between retroposons and the host genomes are discussed.     

SINE Retrotransposition During the Evolution of the Pecoran Ruminants

SINE retrotransposition events have proven their value as phylogenetic markers in several eukaryotic taxa at different taxonomic levels. The genomes of ruminants contain three related SINE elements, Bov-tA, Bov-A2, and Bov-B. To estimate the time points of retrotransposition of individual copies of these SINEs, we designed PCR primers on database sequences containing SINE insertions in cattle, sheep, or goat genomes and tested for the presence of these copies in the genomes of other ruminants. It was checked by sequencing whether length variation of the PCR products reflected a SINE retrotransposition. One Bov-B and nine Bov-tA insertions were shared by cattle, sheep, goat, and giraffe, indicating an early retrotransposition event before the radiation of the Pecora, while three other Bov-tA and two Bov-B elements were apparently inserted later. The ruminant α-lactalbumine gene contains a hotspot of early and more recent Bov-tA insertions, a Bov-tA replacement as well as a recent Bov-B insertion. Three Bov-A2 insertions were found to be shared only by the Bovidae, the Bovini, and the Bos and Bison species, respectively, indicating that most Bov-A2 insertions are relatively recent. The time elapsed since the retrotransposition was also reflected in the degeneration of the direct repeats that flank SINE inserts. We suggest that retrotransposition of SINEs may serve as phylogenetic markers in the ruminant families, subfamilies, and even tribes. In addition, sequencing of SINE insertions revealed several other unique deletions/insertions that also may be informative for phylogenetic reconstructions of ruminants. Received: 19 January 2001 / Accepted: 6 June 2001     

A retroposon analysis of Afrotherian phylogeny

Recent comprehensive studies of DNA sequences support the monophyly of Afrotheria, comprising elephants, sirenians (dugongs and manatees), hyraxes, tenrecs, golden moles, aardvarks, and elephant shrews, as well as that of Paenungulata, comprising elephants, sirenians, and hyraxes. However, phylogenetic relationships among paenungulates, as well as among nonpaenungulates, have remained ambiguous. Here we applied an extensive retroposon analysis to these problems to support the monophyly of aardvarks, tenrecs, and golden moles, with elephant shrews as their sister group. Regarding phylogenetic relationships in Paenungulata, we could characterize only one informative locus, although we could isolate many insertions specific to each of three lineages, namely, Proboscidea, Sirenia, and Hyracoidea. These data prompted us to reexamine phylogenetic relationships among Paenungulata using 19 nuclear gene sequences resulting in three different analyses, namely, short interspersed element (SINE) insertions, nuclear sequence analyses, and morphological cladistics, supporting different respective phylogenies. We concluded that these three lineages diverged very rapidly in a very short evolutionary period, with the consequence that ancestral polymorphism present in the last common ancestor of Paenungulata results in such incongruence. Our results suggest the rapid fixation of many large-scale morphological synapomorphies for Tethytheria; implications of this in relation to the morphological evolution in Paenungulata are discussed.     

The double-strand-break repair model for recombination

We have isolated and characterized several members of the P transposable element family from a Drosophila melanogaster P strain. Large 2.9 kb elements are present as multiple highly conserved copies together with smaller (0.5-1.6 kb), heterogeneous elements. The complete DNA sequences of the 2.9 kb element and four small elements (previously isolated from hybrid-dysgenesis-induced mutations of the white locus) have been determined. Each small element appears to have arisen from the 2.9 kb element by a different internal deletion. P elements have 31 bp perfect inverse terminal repeats and upon insertion duplicate an 8 bp sequence found only once at the site of insertion. Three of the insertions into the white locus occurred at the same nucleotide, indicating a high degree of local site specificity for insertion. The basis of this specificity has been investigated by DNA sequence analysis of the sites where 18 P elements are found. A revertant of one of the white locus mutants has been found to result from precise excision of the P element, restoring the wild-type DNA sequence.     

Evolutionary History of B1 Retroposons in the Genus Mus

Short interspersed DNA elements (SINEs) amplify by retroposition either by (i) successive waves of amplification from one or a few evolving master genes or by (ii) the generation of new master genes that coexist with their progenitors. Individual, highly conserved, elements of the B1 SINE family were identified from the GenBank nucleotide database using various B1 subfamily consensus query sequences to determine their integration times into the mouse genome. A comparison of orthologous loci in various species of the genus Mus demonstrated that four subfamilies of B1 elements have been amplifying within the last 1–3 million years. Therefore, B1 sequences are generated by coexisting source genes. Additionally, three B1 subfamilies have been concurrently propagated during subspecies divergence and strain formation in Mus, indicating very recent activity of this retroposon family. The patterns of intra- and interspecies variations of orthologous loci demonstrate the usefulness of B1 integrations as a phylogenetic tool. A single inconsistency in the phylogenetic trends was depicted by the presence of a B1 insert in an orthologous locus exclusively in M. musculus and M. pahari. However, DNA sequence analysis revealed that these were independent integrations at the same genomic site. One highly conserved B1 element that integrated at least 4–6 million years ago suggests the possibility of occasional function for B1 integrations. Received: 25 February 2000 / Accepted: 5 June 2000     

Retrotransposon insertion polymorphisms in six rice genes and their evolutionary history

Retrotransposons are abundant in higher plant genomes. Although retrotransposons associated with plant genes have been identified, little is known about their evolutionary conservation at the level of species and subspecies. In the present study, we investigated the phylogenetic distribution of long terminal repeat (LTR) retrotransposon, long interspersed nuclear element (LINE) and short interspersed nuclear element (SINE) insertions in six genes in 95 cultivated and wild rice genotypes. These six genes are likely to be functional based on nonsynonymous (Ka) to synonymous (Ks) substitution ratios which were found to be significantly <1. Different conservation patterns of these retrotransposons in genes were observed in cultivated and wild rice species. Four out of seven retrotransposon insertions appear to predate the ancestral Oryza AA genome. Two of these insertions in genes 4 and 5 occurred early in the evolutionary history of Oryza. Two retrotransposon insertions in gene 1 arose after the divergence of Asian cultivated rice from its wild ancestor. Furthermore, the retrotransposon insertion in gene 3 appears to have occurred in the ancestral lineage leading to temperate japonicas. Conservation of retrotransposon insertions in genes in specific groups, species, and lineages might be related to their specific function.     

Molecular evidence for independent occurrence of IS6110 insertions at the same sites of the genome of Mycobacterium tuberculosis in different clinical isolates

Several characteristics of Mycobacterium tuberculosis (e.g., conserved genome and low growth rate) have severely restricted the study of the microorganism. The discovery of IS6110 raised hopes of overcoming these obstacles. However, our knowledge of this IS element is relatively limited; even its two basic characteristics (transposition mechanism and target site selection) are far from well understood. In this study, IS6110 insertions in ipl loci (iplA and iplB) in two collections of clinical isolates of M. tuberculosis from different geographic locations, one from Scotland and the other from Thailand, were investigated. Five different IS6110 insertions in the loci were identified: ipl-4::IS6110, ipl-5::IS6110, ipl-11::IS6110, ipl-12::IS6110, and ipl-13::IS6110. An attempt to establish the phylogenetic relationship of the isolates containing these insertions was unsuccessful, suggesting that some of these insertions may have arisen from more than one event. This possibility is further supported by the observation that IS6110 copies existed in the same site but with different orientations in different isolates, and the insertion site of ipl-1::IS6110 harbored IS6110 copies in both iplA and iplB in different strains. All these suggest the independent occurrence of IS6110 insertions at the same sites of the genome of M. tuberculosis in different clinical isolates. The implications of this finding are discussed.     

Pattern of somatic transposition in a high copy Ac tomato line

A transgenic tomato line containing between eight and ten copies per genome of an exceptionally active maize transposable element Ac has previously been described. Southern analyses indicated that these elements are somatically active in these plants. In order to characterize further the pattern of somatic transposition in this line, 24 independent Ac insertion events from a single plant were cloned. In 21 cases, Ac inserted into single copy genomic DNA while in three cases Ac inserted into sequences present at two to four copies per genome; none of the insertions occurred into more highly repetitive DNA. The chromosomal locations of 20 insertion sites were determined by RFLP mapping and a pattern of small dispersed clusters emerged. Thirteen of the 20 insertion sites were linked to at least one other insertion site but these were distributed over nine of the 12 tomato chromosomes. Only one Ac insertion was linked to the T-DNA locus. The structural integrity of these Ac elements was examined and no evidence of deletions or other rearrangements suggestive of Ds elements was found. The implications of these findings with respect to the use of Ac as a transposon tag in heterologous species are discussed.     

Toothed whale monophyly reassessed by SINE insertion analysis: the absence of lineage sorting effects suggests a small population of a common ancestral species

Morphological data have indicated that toothed whales form a monophyletic group. However, research published in the last several years has made the issue of the monophyly or paraphyly of toothed whales a subject of debate. Our group previously characterized three independent loci in which SINE insertions were shared among dolphins and sperm whales, thus supporting the traditional, morphologically based hypothesis of toothed whale monophyly. Although in recent years a few additional molecular works proposed this topology, there is still skepticism over this monophyly from the view point of molecular systematics. When the phylogeny of rapidly radiated taxa is examined using the SINE method, it is important to consider the ascertainment bias that arises when choosing a particular taxon for SINE loci screening. To overcome this methodological problem specific to the SINE method, we examined all possible topologies among sperm whales, dolphins and baleen whales by extensively screening SINE loci from species of all three lineages. We characterized nine independent SINE loci from the genomes of sperm whales and dolphins, all of which cluster sperm whales and dolphins but exclude baleen whales. Furthermore, we characterized ten independent loci from baleen whales, all of which were amplified in a common ancestor of these whales. From these observations, we conclude that toothed whales form a monophyletic group and that no ancestral SINE polymorphisms hinder their phylogenetic assignment despite the short divergence times of the major lineages of extant whales during evolution. These results suggest that a small population of common ancestors of all toothed whales ultimately diverged into the lineages of sperm whales and dolphins.     

First application of the SINE (short interspersed repetitive element) method to infer phylogenetic relationships in reptiles: an example from the turtle superfamily Testudinoidea

Although turtles (order Testudines) constitute one of the major reptile groups, their phylogenetic relationships remain largely unresolved. Hence, we attempted to elucidate their phylogeny using the SINE (short interspersed repetitive element) method, in which the sharing of a SINE at orthologous loci is indicative of synapomorphy. First, a detailed characterization of the tortoise polIII/SINE was conducted using 10 species from eight families of hidden-necked turtles (suborder Cryptodira). Our analysis of 382 SINE sequences newly isolated in the present study revealed two subgroups, namely Cry I and Cry II, which were distinguishable according to diagnostic nucleotides in the 3' region. Furthermore, four (IA-ID) and five (IIA-IIE) different SINE types were identified within Cry I and Cry II subgroups, respectively, based on features of insertions/deletions located in the middle of the SINE sequences. The relative frequency of occurrence of the subgroups and the types of SINEs in this family were highly variable among different lineages of turtles, suggesting active differential retroposition in each lineage. Further application of the SINE method using the most retrotranspositionally active types, namely IB and IC, challenged the established phylogenetic relationships of Bataguridae and its related families. The data for 11 orthologous loci demonstrated a close relationship between Bataguridae and Testudinidae, as well as the presence of the three clades within Bataguridae. Although the SINE method has been used to infer the phylogenies of a number of vertebrate groups, it has never been applied to reptiles. The present study represents the first application of this method to a phylogenetic analysis of this class of vertebrates, and it provides detailed information on the SINE subgroups and types. This information may be applied to the phylogenetic resolution of relevant turtle lineages.     

Retroposition of the AFC Family of SINEs (Short Interspersed Repetitive Elements) Before and During the Adaptive Radiation of Cichlid Fishes in Lake Malawi and Related Inferences About Phylogeny

Lake Malawi is home to more than 450 species of endemic cichlids, which provide a spectacular example of adaptive radiation. To clarify the phylogenetic relationships among these fish, we examined the presence and absence of SINEs (short interspersed repetitive elements) at orthologous loci. We identified six loci at which a SINE sequence had apparently been specifically inserted by retroposition in the common ancestor of all the investigated species of endemic cichlids in Lake Malawi. At another locus, unique sharing of a SINE sequence was evident among all the investigated species of endemic non-Mbuna cichlids with the exception of Rhamphochromis sp. The relationships were in good agreement with those deduced in previous studies with various different markers, demonstrating that the SINE method is useful for the elucidation of phylogenetic relationships among cichlids in Lake Malawi. We also characterized a locus that exhibited transspecies polymorphism with respect to the presence or absence of the SINE sequence among non-Mbuna species. This result suggests that incomplete lineage sorting and/or interspecific hybridization might have occurred or be occurring among the species in this group, which might potentially cause misinterpretation of phylogenetic data, in particular when a single-locus marker, such as a sequence in the mitochondrial DNA, is used for analysis. Received: 15 December 2000 / Accepted: 30 January 2001     

Consistency of SINE insertion topology and flanking sequence tree: quantifying relationships among cetartiodactyls

Short interspersed nuclear elements (SINEs) have been used to generate unambiguous phylogenetic topologies relating eukaryotic taxa. The irreversible nature of SINE retroposition is supported by a large body of comparative genome data and is a fundamental assumption inherent in the value of this qualitative method of inference. Here, we assess the key assumption of unidirectional SINE insertion by comparing the SINE insertion-derived topology and the phylogenetic tree based on seven independent loci of five taxa in the order Cetartiodactyla (Cetacea + Artiodactyla). The data sets and analyses were largely independent, but the loci were, by definition, linked, and thus their consistency supported an irreversible pattern of SINE retroposition. Moreover, our analyses of the flanking sequences provided estimates of divergence times among cetartiodactyl lineages unavailable from SINE insertion analysis alone. Unexpected rate heterogeneity among sites of SINE-flanking sequences and other noncoding DNA sequences were observed. Sequence simulations suggest that this rate heterogeneity may be an artifact resulting from the inaccuracies of the substitution model used.     

Following the LINEs: an analysis of primate genomic variation at human-specific LINE-1 insertion sites

The L1 Ta subfamily of long interspersed elements (LINEs) consists exclusively of human-specific L1 elements. Polymerase chain reaction-based screening in nonhuman primate genomes of the orthologous sites for 249 human L1 Ta elements resulted in the recovery of various types of sequence variants for approximately 12% of these loci. Sequence analysis was employed to capture the nature of the observed variation and to determine the levels of gene conversion and insertion site homoplasy associated with LINE elements. Half of the orthologous loci differed from the predicted sizes due to localized sequence variants that occurred as a result of common mutational processes in ancestral sequences, often including regions containing simple sequence repeats. Additional sequence variation included genomic deletions that occurred upon L1 insertion, as well as successive mobile element insertions that accumulated within a single locus over evolutionary time. Parallel independent mobile element insertions at orthologous loci in distinct species may introduce homoplasy into retroelement-based phylogenetic and population genetic data. We estimate the overall frequency of parallel independent insertion events at L1 insertion sites in seven different primate species to be very low (0.52%). In addition, no cases of insertion site homoplasy involved the integration of a second L1 element at any of the loci, but rather largely involved secondary insertions of Alu elements. No independent mobile element insertion events were found at orthologous loci in the human and chimpanzee genomes. Therefore, L1 insertion polymorphisms appear to be essentially homoplasy free characters well suited for the study of population genetics and phylogenetic relationships within closely related species.     

BAC TG-EMBED: one-step method for high-level,copy-number-dependent,position-independent transgene expression

Chromosome position effects combined with transgene silencing of multi-copy plasmid insertions lead to highly variable and usually quite low expression levels of mini-genes integrated into mammalian chromosomes. Together, these effects greatly complicate obtaining high-level expression of therapeutic proteins in mammalian cells or reproducible expression of individual or multiple transgenes. Here, we report a simple, one-step procedure for obtaining high-level, reproducible mini-gene expression in mammalian cells. By inserting mini-genes at different locations within a BAC containing the DHFR housekeeping gene locus, we obtain copy-number-dependent, position-independent expression with chromosomal insertions of one to several hundred BAC copies. These multi-copy DHFR BAC insertions adopt similar large-scale chromatin conformations independent of their chromosome integration site, including insertions within centromeric heterochromatin. Prevention of chromosome position effects, therefore, may be the result of embedding the mini-gene within the BAC-specific large-scale chromatin structure. The expression of reporter mini-genes can be stably maintained during continuous, long-term culture in the presence of drug selection. Finally, we show that this method is extendable to reproducible, high-level expression of multiple mini-genes, providing improved expression of both single and multiple transgenes.     

SINEs of a nearly perfect character

Mobile elements have been recognized as powerful tools for phylogenetic and population-level analyses. However, issues regarding potential sources of homoplasy and other misleading events have been raised. We have collected available data for all phylogenetic and population level studies of primates utilizing Alu insertion data and examined them for potentially homoplasious and other misleading events. Very low levels of each potential confounding factor in a phylogenetic or population analysis (i.e., lineage sorting, parallel insertions, and precise excision) were found. Although taxa known to be subject to high levels of these types of events may indeed be subject to problems when using SINE analysis, we propose that most taxa will respond as the order Primates has--by the resolution of several long-standing problems observed using sequence-based methods.     

Construction and characterization of a Lipotes vexillifer genomic DNA BAC library

We constructed a genomic DNA library for Lipotes vexillifer (L. vexillifer), the Baiji or Yangtze River dolphin, one of the most endangered mammals in the world. The library consists of 149,000 BAC clones, with an average insert size of 83 kb, representing approximately 3.4 haploid genome equivalents. PCR amplification of four known L. vexillifer genes yielded two to four positive clones each. To demonstrate the utility of this library, we isolated and sequenced the L. vexillifer alpha lactalbumin gene, which is a gene specific to mammals and one which has been widely used as molecular tool in phylogenetic analysis. We also end-sequenced 20 randomly selected clones, resulting in the identification of at least five new L. vexillifer genes, five SSR loci, and one SINE locus. These results suggest that this library is a valuable resource for candidate gene cloning, physical mapping, and genome sequencing of this important and threatened species.