We recently characterized HAmo SINE and its partner LINE in silver carp and bighead carp based on hybridization capture of repetitive elements from digested genomic DNA in solution using a bead-probe . To reveal the distribution and evolutionary history of SINEs and LINEs in cyprinid genomes, we performed a multi-species search for HAmo SINE and its partner LINE using the bead-probe capture and internal-primer-SINE polymerase chain reaction (PCR) techniques.
Sixty-seven full-size and 125 internal-SINE sequences (as well as 34 full-size and 9 internal sequences previously reported in bighead carp and silver carp) from 17 species of the family Cyprinidae were aligned as well as 14 new isolated HAmoL2 sequences. Four subfamilies (type I, II, III and IV), which were divided based on diagnostic nucleotides in the tRNA-unrelated region, expanded preferentially within a certain lineage or within the whole family of Cyprinidae as multiple active source genes. The copy numbers of HAmo SINEs were estimated to vary from 104 to 106 in cyprinid genomes by quantitative RT-PCR. Over one hundred type IV members were identified and characterized in the primitive cyprinid Danio rerio genome but only tens of sequences were found to be similar with type I, II and III since the type IV was the oldest subfamily and its members dispersed in almost all investigated cyprinid fishes. For determining the taxonomic distribution of HAmo SINE, inter-primer SINE PCR was conducted in other non-cyprinid fishes, the results shows that HAmo SINE- related sequences may disperse in other families of order Cypriniforms but absent in other orders of bony fishes: Siluriformes, Polypteriformes, Lepidosteiformes, Acipenseriformes and Osteoglossiforms.
Depending on HAmo LINE2, multiple source genes (subfamilies) of HAmo SINE actively expanded and underwent retroposition in a certain lineage or within the whole family of Cyprinidae. From this perspective, HAmo SINE should provide useful phylogenetic makers for future analyses of the evolutionary relationships among species in the family Cyprinidae.
Short interspersed nucleotide elements (SINEs), a type of retrotransposon, are widely distributed in various genomes with multiple copies arranged in different orientations, and cause changes to genes and genomes during evolutionary history. This can provide the basis for determining genome diversity, genetic variation and molecular phylogeny, etc. SINE DNA is transcribed into RNA by polymerase III from an internal promoter, which is composed of two conserved boxes, box A and box B. Here we present an approach to isolate novel SINEs based on these promoter elements. Box A of a SINE is obtained via PCR with only one primer identical to box B (B-PCR). Box B and its downstream sequence are acquired by PCR with one primer corresponding to box A (A-PCR). The SINE clone produced by A-PCR is selected as a template to label a probe with biotin. The full-length SINEs are isolated from the genomic pool through complex capture using the biotinylated probe bound to magnetic particles. Using this approach, a novel SINE family, Cn-SINE, from the genomes of Coilia nasus, was isolated. The members are 180–360 bp long. Sequence homology suggests that Cn-SINEs evolved from a leucine tRNA gene. This is the first report of a tRNALeu-related SINE obtained without the use of a genomic library or inverse PCR. These results provide new insights into the origin of SINEs.
transposable element; SINE; tRNA; Coilia nasus
Short interspersed repetitive elements (SINEs) are a type of retroposon, being members of a class of informational molecules that are amplified via cDNA intermediates and flow back into the host genome. In contrast to retroviruses and retrotransposons, SINEs do not encode the enzymes required for their amplification, such as reverse transcriptases, so they are presumed to borrow these enzymes from other sources. In the present study, we isolated a family of long interspersed repetitive elements (LINEs) from the turtle genome. The sequence of this family was found to be very similar to those of the avian CR1 family. To our surprise, the sequence at the 3' end of the LINE in the turtle genome was nearly identical to that of a family of tortoise SINEs. Since CR1-like LINEs are widespread in birds and in many other reptiles, including the turtle, and since the tortoise SINEs are only found in vertical-necked turtles, it seems possible that the sequence at the 3' end of the tortoise SINEs might have been generated by recombination with the CR1-like LINE in a common ancestor of vertical-necked turtles, after the divergence of side-necked turtles. We extended our observations to show that the 3'-end sequences of families of several tRNA-derived SINEs, such as the salmonid HpaI family, the tobacco TS family, and the salmon SmaI family, might have originated from the respective LINEs. Since it appears reasonable that the recognition sites of LINEs for reverse transcriptase are located within their 3'-end sequences, these results provide the basis for a general scheme for the mechanism by which SINEs might acquire retropositional activity. We propose here that tRNA-derived SINEs might have been generated by a recombination event in which a strong-stop DNA with a primer tRNA, which is an intermediate in the replication of certain retroviruses and long terminal repeat retrotransposons, was directly integrated at the 3' end of a LINE.
Short interspersed elements (SINEs) make up a significant fraction of total DNA in mammalian genomes, providing a rich substrate for chromosomal rearrangements by SINE-SINE recombinations. Proliferation of mammalian SINEs is mediated primarily by LINE1 (L1) non-LTR retrotransposons that preferentially integrate at DNA sequence targets with average length ~15 bp and containing conserved endonucleolytic nicking signals at both ends. We report that sequence variations in the first of the two nicking signals, represented by a 5′TT-AAAA consensus sequence, affect the position of the second signal thus leading to target site duplications (TSDs) of different lengths. The length distribution of TSDs appears to be affected also by L1-encoded enzyme variants, since targets with the same 5′ nicking site can be of different average length in different mammalian species. Taking this into account, we re-analyzed the second nicking site and found that it is larger and includes more conserved sites than previously appreciated, with a consensus of 5′ANTNTN-AA. We also studied potential involvement of the nicking sites in stimulating recombinations between SINE elements. We determined that SINE elements retaining TSDs with perfect 5′TT-AAAA nicking sites appear to be lost relatively rapidly from the human and rat genomes, and less rapidly from dog. We speculate that the introduction of single-strand DNA breaks induced by recurring endonucleolytic attacks at these sites, combined with the ubiquitousness of SINEs, may significantly promote recombination between repetitive elements, leading to the observed losses. At the same time new L1 subfamilies may be selected for “incompatibility” with pre-existing targets. This provides a possible driving force for the continual emergence of new L1 subfamilies which, in turn, may affect selection of L1-dependent SINE subfamilies.
non-LTR retrotransposons; recombination; SINE integration targets
Short Interspersed Nucleotide Elements (SINEs) are highly abundant in mammalian genomes. The term SINE has come to be restricted to short retroposons with internal RNA polymerase III promoter sites in a region derived from a structural RNA (usually a tRNA). Here we describe a novel, 260 bp tRNA-derived SINE, some fragments of which have been noted before to be repetitive in mammalian DNA. Unlike previously reported SINEs, which are restricted to closely related species, copies of this element can be found in all mammalian genomes, including marsupials. It is therefore called MIR for mammalian-wide interspersed repeat. Their high divergence and their presence at orthologous sites in different mammals indicate that MIRs, at least in part, amplified before the mammalian radiation. Next to Alu, MIRs are the most common interspersed repeat in primates with an estimated 300,000 copies still discernible, which account for 1 to 2% of our DNA. Interestingly, a small, central region of MIR appears to be much better conserved in the genomic copies than the rest of the sequence.
Although more than 120 families of short interspersed nuclear elements (SINEs) have been isolated from the eukaryotic genomes, little is known about SINEs in insects. Here, we characterize three novel SINEs from the cotton bollworm, Helicoverpa armigera. Two of them, HaSE1 and HaSE2, share similar 5′ -structure including a tRNA-related region immediately followed by conserved central domain. The 3′ -tail of HaSE1 is significantly similar to that of one LINE retrotransposon element, HaRTE1.1, in H. armigera genome. The 3′ -region of HaSE2 showed high identity with one mariner-like element in H. armigera. The third family, termed HaSE3, is a 5S rRNA-derived SINE and shares both body part and 3′-tail with HaSE1, thus may represent the first example of a chimera generated by recombination between 5S rRNA and tRNA-derived SINE in insect species. Further database searches revealed the presence of these SINEs in several other related insect species, but not in the silkworm, Bombyx mori, indicating a relatively narrow distribution of these SINEs in Lepidopterans. Apart from above, we found a copy of HaSE2 in the GenBank EST entry for the cotton aphid, Aphis gossypii, suggesting the occurrence of horizontal transfer.
Transposable elements, including short interspersed repetitive elements (SINEs), comprise nearly half the mammalian genome. Moreover, they are a major source of conserved non-coding elements (CNEs), which play important functional roles in regulating development-related genes, such as enhancing and silencing, serving for the diversification of morphological and physiological features among species. We previously reported a novel SINE family, AmnSINE1, as part of mammalian-specific CNEs. One AmnSINE1 locus, named AS071, showed an enhancer property in the developing mouse diencephalon. Indeed, AS071 appears to recapitulate the expression of diencephalic fibroblast growth factor 8 (Fgf8). Here we established three independent lines of AS071-transgenic mice and performed detailed expression profiling of AS071-enhanced lacZ in comparison with that of Fgf8 across embryonic stages. We demonstrate that AS071 is a distal enhancer that directs Fgf8 expression in the developing diencephalon. Furthermore, enhancer assays with constructs encoding partially deleted AS071 sequence revealed a unique modular organization in which AS071 contains at least three functionally distinct sub-elements that cooperatively direct the enhancer activity in three diencephalic domains, namely the dorsal midline and the lateral wall of the diencephalon, and the ventral midline of the hypothalamus. Interestingly, the AmnSINE1-derived sub-element was found to specify the enhancer activity to the ventral midline of the hypothalamus. To our knowledge, this is the first discovery of an enhancer element that could be separated into respective sub-elements that determine regional specificity and/or the core enhancing activity. These results potentiate our understanding of the evolution of retroposon-derived cis-regulatory elements as well as the basis for future studies of the molecular mechanism underlying the determination of domain-specificity of an enhancer.
Short interspersed nuclear elements (SINEs) are a type of class 1 transposable element (retrotransposon) with features that allow investigators to resolve evolutionary relationships between populations and species while providing insight into genome composition and function. Characterization of a Carnivora-specific SINE family, Can-SINEs, has, has aided comparative genomic studies by providing rare genomic changes, and neutral sequence variants often needed to resolve difficult evolutionary questions. In addition, Can-SINEs constitute a significant source of functional diversity with Carnivora. Publication of the whole-genome sequence of domestic dog, domestic cat, and giant panda serves as a valuable resource in comparative genomic inferences gleaned from Can-SINEs. In anticipation of forthcoming studies bolstered by new genomic data, this review describes the discovery and characterization of Can-SINE motifs as well as describes composition, distribution, and effect on genome function. As the contribution of noncoding sequences to genomic diversity becomes more apparent, SINEs and other transposable elements will play an increasingly large role in mammalian comparative genomics.
carnivore; genome; SINE
The genome of the carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii, Order: Dasyuromorphia), was sequenced in the hopes of finding a cure for or gaining a better understanding of the contagious devil facial tumor disease that is threatening the species’ survival. To better understand the Tasmanian devil genome, we screened it for transposable elements and investigated the dynamics of short interspersed element (SINE) retroposons.
The temporal history of Tasmanian devil SINEs, elucidated using a transposition in transposition analysis, indicates that WSINE1, a CORE-SINE present in around 200,000 copies, is the most recently active element. Moreover, we discovered a new subtype of WSINE1 (WSINE1b) that comprises at least 90% of all Tasmanian devil WSINE1s. The frequencies of WSINE1 subtypes differ in the genomes of two of the other Australian marsupial orders. A co-segregation analysis indicated that at least 66 subfamilies of WSINE1 evolved during the evolution of Dasyuromorphia. Using a substitution rate derived from WSINE1 insertions, the ages of the subfamilies were estimated and correlated with a newly established phylogeny of Dasyuromorphia. Phylogenetic analyses and divergence time estimates of mitochondrial genome data indicate a rapid radiation of the Tasmanian devil and the closest relative the quolls (Dasyurus) around 14 million years ago.
The radiation and abundance of CORE-SINEs in marsupial genomes indicates that they may be a major player in the evolution of marsupials. It is evident that the early phases of evolution of the carnivorous marsupial order Dasyuromorphia was characterized by a burst of SINE activity. A correlation between a speciation event and a major burst of retroposon activity is for the first time shown in a marsupial genome.
SINE; WSINE1; Retroposon; Tasmanian devil; Sarcophilus; Genome; Marsupials
The C family of short, interspersed repeats (SINES) is highly repeated in the rabbit genome, and most members have a structure suggestive of a model for their dispersal via reinsertion of a double-stranded copy of an RNA polymerase III transcribed RNA. We have determined the nucleotide sequence of additional members of the repeat family and have compiled them to obtain an improved consensus sequence. This compilation shows that although most regions of the repeat are well conserved, two regions show high variability. Some individual repeats are truncated, and one truncated repeat retains the characteristic structures of a retroposon. The consensus sequence for C repeats does not match the sequence of any other sequenced mammalian SINE over large regions, but short imperfect matches to several primate and rodent SINES are observed. A sequence similar to the 27 nucleotide consensus sequence TCCCAGCAACCACATGGGAGGCAGAGA was found in all mammalian SINES examined. The 3' portion of this sequence matches a DNA segment found at the replication origins of papovaviruses.
SINEBase (http://sines.eimb.ru) integrates the revisited body of knowledge about short interspersed elements (SINEs). A set of formal definitions concerning SINEs was introduced. All available sequence data were screened through these definitions and the genetic elements misidentified as SINEs were discarded. As a result, 175 SINE families have been recognized in animals, flowering plants and green algae. These families were classified by the modular structure of their nucleotide sequences and the frequencies of different patterns were evaluated. These data formed the basis for the database of SINEs. The SINEBase website can be used in two ways: first, to explore the database of SINE families, and second, to analyse candidate SINE sequences using specifically developed tools. This article presents an overview of the database and the process of SINE identification and analysis.
Short interspersed elements (SINEs) are one of the two most prolific mobile genomic elements in most of the higher eukaryotes. Although their biology is still not thoroughly understood, unusual life cycle of these simple elements amplified as genomic parasites makes their evolution unique in many ways. In contrast to most genetic elements including other transposons, SINEs emerged de novo many times in evolution from available molecules (for example, tRNA). The involvement of reverse transcription in their amplification cycle, huge number of genomic copies and modular structure allow variation mechanisms in SINEs uncommon or rare in other genetic elements (module exchange between SINE families, dimerization, and so on.). Overall, SINE evolution includes their emergence, progressive optimization and counteraction to the cell's defense against mobile genetic elements.
repetitive elements; mobile elements; transposons; retrotransposons; SINEs; evolution
The evolution, mobility and deleterious genetic effects of human Alus are fairly well understood. The complexity of regulated transcriptional expression of Alus is becoming apparent and insight into the mechanism of retrotransposition is emerging. Unresolved questions concern why mobile, highly repetitive short interspersed elements (SINEs) have been tolerated throughout evolution and why and how families of such sequences are periodically replaced. Either certain SINEs are more successful genomic parasites or positive selection drives their relative success and genomic maintenance. A complete understanding of the evolutionary dynamics and significance of SINEs requires determining whether or not they have a function(s). Recent evidence suggests two possibilities, one concerning DNA and the other RNA. Dispersed Alus exhibit remarkable tissue-specific differences in the level of their 5-methylcytosine content. Differences in Alu methylation in the male and female germlines suggest that Alu DNA may be involved in either the unique chromatin organization of sperm or signaling events in the early embryo. Alu RNA is increased by cellular insults and stimulates protein synthesis by inhibiting PKR, the eIF2 kinase that is regulated by double-stranded RNA. PKR serves other roles potentially linking Alu RNA to a variety of vital cell functions. Since Alus have appeared only recently within the primate lineage, this proposal provokes the challenging question of how Alu RNA could have possibly assumed a significant role in cell physiology.
In addition to the Alu family of short interspersed repetitive DNA elements (SINEs), we have previously characterized one other repetitive DNA family (Type II) in the prosimian, Galago crassicaudatus. We present here a detailed analysis of seventeen members of a third galago SINE family designated as the Monomer family. Both the Monomer and Type II families are shown to be specific for the galago genome as compared to other primates, including another prosimian, the lemur. Moreover, in vitro transcription of galago SINEs suggests that the Monomer and Type II families have appreciably stronger RNA polymerase III promoters than does the Alu family. This agrees with the promoter sequence for each of these SINE families, in that the Monomer and Type II family promoters are more closely related to the RNA polymerase III promoter consensus sequence than is the Alu family promoter. These promoter strength analyses also correlate with copy number and sequence divergence analyses, which suggests that the SINE families with the strongest promoters have been amplified most recently in the galago genome.
L1 and Alu elements are long and short interspersed
retrotransposable elements (LINEs and SINEs) in humans,
respectively. Proteins encoded in the autonomous L1 mediate
retrotransposition of the nonautonomous Alu and cellular
mRNAs. Alu is the only active SINE in the human genome
and is derived from 7SL RNA of signal recognition particle. In the
other eukaryotic genomes, various tRNA- and 5S rRNA-derived SINEs
are found. Some of the tRNA- and 5S rRNA-derived SINEs have
partner LINEs of which 3′ sequences are similar to those of the
SINEs. One of the tRNA-derived SINEs is shown to be mobilized by
its partner LINE. Many copies of tRNA and 5S rRNA pseudogenes are
present in the human genome. These pseudogenes may have been
generated via the retrotransposition process using L1 proteins.
Although there are no sequence similarities between L1 and
Alu, L1 functionally links with Alu and even
cellular genes, impacting on our genome shaping.
Lemurs (infraorder: Lemuriformes) are a radiation of strepsirrhine primates endemic to the island of Madagascar. As of 2012, 101 lemur species, divided among five families, have been described. Genetic and morphological evidence indicates all species are descended from a common ancestor that arrived in Madagascar ∼55–60 million years ago (mya). Phylogenetic relationships in this species-rich infraorder have been the subject of debate. Here we use Alu elements, a family of primate-specific Short INterspersed Elements (SINEs), to construct a phylogeny of infraorder Lemuriformes. Alu elements are particularly useful SINEs for the purpose of phylogeny reconstruction because they are identical by descent and confounding events between loci are easily resolved by sequencing. The genome of the grey mouse lemur (Microcebus murinus) was computationally assayed for synapomorphic Alu elements. Those that were identified as Lemuriformes-specific were analyzed against other available primate genomes for orthologous sequence in which to design primers for PCR (polymerase chain reaction) verification. A primate phylogenetic panel of 24 species, including 22 lemur species from all five families, was examined for the presence/absence of 138 Alu elements via PCR to establish relationships among species. Of these, 111 were phylogenetically informative. A phylogenetic tree was generated based on the results of this analysis. We demonstrate strong support for the monophyly of Lemuriformes to the exclusion of other primates, with Daubentoniidae, the aye-aye, as the basal lineage within the infraorder. Our results also suggest Lepilemuridae as a sister lineage to Cheirogaleidae, and Indriidae as sister to Lemuridae. Among the Cheirogaleidae, we show strong support for Microcebus and Mirza as sister genera, with Cheirogaleus the sister lineage to both. Our results also support the monophyly of the Lemuridae. Within Lemuridae we place Lemur and Hapalemur together to the exclusion of Eulemur and Varecia, with Varecia the sister lineage to the other three genera.
The popularity of microsatellites has greatly increased in the last decade on account of their many applications. However, little is currently understood about the factors that influence their genesis and distribution among and within species genomes. In this work, we analyzed carnivore microsatellite clones from GenBank to study their association with interspersed repeats and elucidate the role of the latter in microsatellite genesis and distribution.
We constructed a comprehensive carnivore microsatellite database comprising 1236 clones from GenBank. Thirty-three species of 11 out of 12 carnivore families were represented, although two distantly related species, the domestic dog and cat, were clearly overrepresented. Of these clones, 330 contained tRNALys-derived SINEs and 357 contained other interspersed repeats. Our rough estimates of tRNA SINE copies per haploid genome were much higher than published ones. Our results also revealed a distinct juxtaposition of AG and A-rich repeats and tRNALys-derived SINEs suggesting their coevolution. Both microsatellites arose repeatedly in two regions of the insterspersed repeat. Moreover, microsatellites associated with tRNALys-derived SINEs showed the highest complexity and less potential instability.
Our results suggest that tRNALys-derived SINEs are a significant source for microsatellite generation in carnivores, especially for AG and A-rich repeat motifs. These observations indicate two modes of microsatellite generation: the expansion and variation of pre-existing tandem repeats and the conversion of sequences with high cryptic simplicity into a repeat array; mechanisms which are not specific to tRNALys-derived SINEs. Microsatellite and interspersed repeat coevolution could also explain different distribution of repeat types among and within species genomes.
Finally, due to their higher complexity and lower potential informative content of microsatellites associated with tRNALys-derived SINEs, we recommend avoiding their use as genetic markers.
Woolly mammoths were a species of elephant that populated much of Eurasia and North America until about 10,000 years ago. Using the next-generation sequencing technologies, we generated nearly one-fold nuclear genomic sequences, and investigated their transposable element amplification dynamics. We found that the mammoth genome contains a larger proportion of interspersed repeats than any other mammalian genome reported so far, in which the proliferation of the RTE family of retrotransposons (covering 12% of the genome) may be the main reason for an increased genome size. Phylogenetic analysis showed that RTEs in mammoth are closely related to the family BovB/RTE. The incongruence of the reconstructed RTE phylogeny indicates that RTEs in mammoth may be acquired through an ancient lateral gene transfer event. A recent proliferation of SINEs was also found in the probocidean lineage, whereas the Afrotherian-wide SINEs in mammoth have undergone a rather flat and stepwise expansion. Comparisons of the transposable elements (TEs) between mammoth and other mammals may shed light on the evolutionary history of TEs in various mammalian lineages.
A substantial number of “retrogenes” that are derived from the mRNA of various intron-containing genes have been reported. A class of mammalian retroposons, long interspersed element-1 (LINE1, L1), has been shown to be involved in the reverse transcription of retrogenes (or processed pseudogenes) and non-autonomous short interspersed elements (SINEs). The 3′-end sequences of various SINEs originated from a corresponding LINE. As the 3′-untranslated regions of several LINEs are essential for retroposition, these LINEs presumably require “stringent” recognition of the 3′-end sequence of the RNA template. However, the 3′-ends of mammalian L1s do not exhibit any similarity to SINEs, except for the presence of 3′-poly(A) repeats. Since the 3′-poly(A) repeats of L1 and Alu SINE are critical for their retroposition, L1 probably recognizes the poly(A) repeats, thereby mobilizing not only Alu SINE but also cytosolic mRNA. Many flowering plants only harbor L1-clade LINEs and a significant number of SINEs with poly(A) repeats, but no homology to the LINEs. Moreover, processed pseudogenes have also been found in flowering plants. I propose that the ancestral L1-clade LINE in the common ancestor of green plants may have recognized a specific RNA template, with stringent recognition then becoming relaxed during the course of plant evolution.
Entamoeba histolytica and Entamoeba dispar are closely related protistan parasites but while E. histolytica can be invasive, E. dispar is completely non pathogenic. Transposable elements constitute a significant portion of the genome in these species; there being three families of LINEs and SINEs. These elements can profoundly influence the expression of neighboring genes. Thus their genomic location can have important phenotypic consequences. A genome-wide comparison of the location of these elements in the E. histolytica and E. dispar genomes has not been carried out. It is also not known whether the retrotransposition machinery works similarly in both species. The present study was undertaken to address these issues.
Here we extracted all genomic occurrences of full-length copies of EhSINE1 in the E. histolytica genome and matched them with the homologous regions in E. dispar, and vice versa, wherever it was possible to establish synteny. We found that only about 20% of syntenic sites were occupied by SINE1 in both species. We checked whether the different genomic location in the two species was due to differences in the activity of the LINE-encoded endonuclease which is required for nicking the target site. We found that the endonucleases of both species were essentially very similar, both in their kinetic properties and in their substrate sequence specificity. Hence the differential distribution of SINEs in these species is not likely to be influenced by the endonuclease. Further we found that the physical properties of the DNA sequences adjoining the insertion sites were similar in both species.
Our data shows that the basic retrotransposition machinery is conserved in these sibling species. SINEs may indeed have occupied all of the insertion sites in the genome of the common ancestor of E. histolytica and E. dispar but these may have been subsequently lost from some locations. Alternatively, SINE expansion took place after the divergence of the two species. The absence of SINE1 in 80% of syntenic loci could affect the phenotype of the two species, including their pathogenic properties, which needs to be explored.
The major clinical manifestations of Entamoeba histolytica infection include amebic colitis and liver abscess. However the majority of infections remain asymptomatic. Earlier reports have shown that some E. histolytica isolates are more virulent than others, suggesting that virulence may be linked to genotype. Here we have looked at the genomic distribution of the retrotransposable short interspersed nuclear elements EhSINE1 and EhSINE2. Due to their mobile nature, some EhSINE copies may occupy different genomic locations among isolates of E. histolytica possibly affecting adjacent gene expression; this variability in location can be exploited to differentiate strains.
We have looked for EhSINE1- and EhSINE2-occupied loci in the genome sequence of Entamoeba histolytica HM-1:IMSS and searched for homologous loci in other strains to determine the insertion status of these elements. A total of 393 EhSINE1 and 119 EhSINE2 loci were analyzed in the available sequenced strains (Rahman, DS4-868, HM1:CA, KU48, KU50, KU27 and MS96-3382. Seventeen loci (13 EhSINE1 and 4 EhSINE2) were identified where a EhSINE1/EhSINE2 sequence was missing from the corresponding locus of other strains. Most of these loci were unoccupied in more than one strain. Some of the loci were analyzed experimentally for SINE occupancy using DNA from strain Rahman. These data helped to correctly assemble the nucleotide sequence at three loci in Rahman. SINE occupancy was also checked at these three loci in 7 other axenically cultivated E. histolytica strains and 16 clinical isolates. Each locus gave a single, specific amplicon with the primer sets used, making this a suitable method for strain typing. Based on presence/absence of SINE and amplification with locus-specific primers, the 23 strains could be divided into eleven genotypes. The results obtained by our method correlated with the data from other typing methods. We also report a bioinformatic analysis of EhSINE2 copies.
Our results reveal several loci with extensive polymorphism of SINE occupancy among different strains of E. histolytica and prove the principle that the genomic distribution of SINEs is a valid method for typing of E. histolytica strains.
Entamoeba histolytica; Genotype; EhSINE1; SINE occupancy; Polymorphism; Strain typing
In a human genome, we found dispersed repetitive sequences homologous to part of a human endogenous retrovirus termed HERV-K which resembled mouse mammary tumor virus. For elucidation of their structure and organization, we cloned some of these sequences from a human gene library. The sequence common to the cloned DNA was ca. 630 base-pairs (bp) in length with an A-rich tail at the 3' end and was found to be a SINE (short interspersed repeated sequence) type nonviral retroposon. In this retroposon, the 5' end had multiple copies of a 40 bp direct repeat very rich in GC content and about the next 510 nucleotides were homologous to the 3' long terminal repeat and its upstream flanking region of the HERV-K genome. This retroposon was thus given the name, SINE-R element since most of it derived from a retrovirus. SINE-R elements were present at 4,000 to 5,000 copies per haploid human genome. The nucleotide sequence was ca. 90% homologous among the cloned elements.
As bighead carp Hypophthalmichthysnobilis and silver carp H. molitrix (the bigheaded carps) are poised to enter the Laurentian Great Lakes and potentially damage the region’s economically important fishery, information on developmental rates and behaviors of carps is critical to assessing their ability to establish sustainable populations within the Great Lakes basin. In laboratory experiments, the embryonic and larval developmental rates, size, and behaviors of bigheaded carp were tracked at two temperature treatments, one “cold” and one “warm”. Developmental rates were computed using previously described stages of development and the cumulative thermal unit method. Both species have similar thermal requirements, with a minimum developmental temperature for embryonic stages of 12.1° C for silver carp and 12.9° C for bighead carp, and 13.3° C for silver carp larval stages and 13.4° C for bighead carp larval stages. Egg size differed among species and temperature treatments, as egg size was larger in bighead carp, and “warm" temperature treatments. The larvae started robust upwards vertical swimming immediately after hatching, interspersed with intervals of sinking. Vertical swimming tubes were used to measure water column distribution, and ascent and descent rates of vertically swimming fish. Water column distribution and ascent and descent rates changed with ontogeny. Water column distribution also showed some diel periodicity. Developmental rates, size, and behaviors contribute to the drift distance needed to fulfill the early life history requirements of bigheaded carps and can be used in conjunction with transport information to assess invasibility of a river.
Two percentage of the cat genome is a repetitive, feline-specific satellite sequence (FA-SAT) of 483 bp and 65% guanine-cytosine content. Previous chromosomal localization of the satellite has demonstrated the satellite’s presence on several discrete regions of the telomeres of chromosomes, predominately on the D, E, and F chromosome groups. The recent assembly of the 1.9× whole-genome shotgun (WGS) sequence of cat illustrates the challenge of the assembly of these large numbers of relatively short, similar sequences. Clones with paired end reads that include FA-SAT sequence have a high level of assembly discrepancies compared with clones with other types of repetitive elements, such as short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs). The influence of the presence of FA-SAT but not SINEs and LINEs on genome assembly may likely reflect the evolutionary emergence of FA-SAT, which has lead to an excess of FA-SAT copies with identical sequence, which is less an issue with older, more diverse SINE and LINE sequences. The FA-SATs are restricted to a few hundred discrete regions of the cat genome, and associated errors in the assembly seem to be restricted to these loci. The findings regarding the feline-specific sequence should be considered in the pending 8x assembly of the cat genome.
artifacts; FA-SAT; genome assembly; repetitive elements; satellite; whole-genome shotgun
Among the cellular responses observed following treatment with DNA-damaging agents is the activation of Short Interspersed Elements (SINEs; retrotransposable genetic elements that comprise over 10% of the human genome). By placing a human SINE (the Alu element) into murine cells, we have previously shown that DNA-damaging agents such as etoposide can induce both upregulation of SINE transcript levels and SINE retrotransposition. A similarly cytotoxic (but not genotoxic) exposure to vincristine was not associated with SINE activation. Here we demonstrate that multiple other genotoxic exposures are associated with upregulation of SINE transcript levels. By comparing the effects of similarly cytotoxic doses of the topoisomerase II inhibitors etoposide and merbarone, we confirm that DNA strand breakage is specifically associated with SINE induction. By evaluating transcription rate and RNA stability, we demonstrate that SINE induction by genotoxic exposure is associated with transcriptional induction and not with transcript stabilization. Finally we demonstrate that SINE induction by genotoxic stress is mediated by a Trp53-independent pathway, and in fact that Trp53 plays an inhibitory role in attenuating the transcriptional induction of SINE elements following exposure to a genotoxic agent. Together these data support a model in which initial DNA damage can trigger genomic instability due to SINE activation, a response which may be amplified in cancer cells lacking functional TP53.