Short Interspersed Repetitive Elements Research Articles

Whole-genome identification of transposable elements reveals the equine repetitive element insertion polymorphism in Chinese horses.

Transposable elements (TEs) are diverse, abundant, and complicated in genomes. They not only can drive the genome evolution process but can also act as special resources for adaptation. However, little is known about the evolutionary processes that shaped horses. In this work, 126 horse assemblages involved in most horse breeds in China were used to investigate the patterns of TE variation for the first time. By using RepeatMasker and melt software, we found that the horse-specific short interspersed repetitive elements family, equine repetitive elements (ERE1), exhibited polymorphisms in horse genomes. Phylogenetic analysis based on these ERE1 loci (minor allele frequency ≥0.05) revealed three major horse groups, namely, those in northern China, southern China, and Qinghai-Tibetan, which mirrors the result determined by SNPs to some extent. The present ERE1 family emerged ~0.26 to 1.77 Mya ago, with an activity peak at ~0.49 Mya, which matches the early stage of the horse lineage and decreases after the divergence of Equus caballus and Equus ferus przewalskii. To detect the functional ERE1(s) associated with adaptation, locus-specific branch length, genome-wide association study, and absolute allele frequency difference analyses were conducted and resulted in two common protein-coding genes annotated by candidate ERE1s. They were clustered into the vascular smooth muscle contraction (p=0.01, EDNRA) and apelin signalling pathways (p = 0.02, NRF1). Notably, ERE1 insertion into the EDNRA gene showed a higher association with adaptation among southern China horses and other horses in 15 populations and 451 individuals (p = 4.55 e-8). Our results provide a comprehensive understanding of TE variations to analyse the phylogenetic relationships and traits relevant to adaptive evolution in horses.

Global DNA methylation and chondrogenesis of rat limb buds in a three-dimensional organ culture system.

Although DNA methylation epigenetically regulates development, data on global DNA methylation during the development of limb buds (LBs) are scarce. We aimed to investigate the global DNA methylation developmental dynamics in rat LBs cultivated in a serum supplemented (SS) and in chemically defined serum- and protein-free (SF) three-dimensional organ culture. Fischer rat front- and hind-LBs at the 13th and 14th gestation days (GDs) were cultivated at the air-liquid interface in Eagle’s Minimal Essential Medium (MEM) or MEM with 50% rat serum for 14 days, as SF and SS conditions, respectively. The methylation of repetitive DNA sequences (short interspersed DNA repetitive element [SINE] rat ID elements) was assessed by pyrosequencing. Development was evaluated by light microscopy and extracellular matrix glycosaminoglycans staining by safranin O. On isolation, weak safranin O staining was present only in more developed GD14 front-LBs. Chondrogenesis proceeded well in all cultures toward day 14, except in the SF-cultivated GD13 hind-LBs, where safranin O staining was almost absent on day 3 that was associated with a higher percentage of DNA methylation than in SF-cultivated GD13 front-LBs on day 3. In SF-cultivated front-LBs, a significant methylation increase between the 3rd and 14th days was detected. In SS-cultivated GD13 front-LBs, methylation increased significantly on day 3 and then decreased. In older GD14 SS-cultivated LBs, there was no increase of DNA methylation, but they were significantly hypomethylated relative to the SS-cultivated GD13 at days 3 and 14. We confirmed that the global DNA methylation increase is associated with less developed limb organ primordia that strive toward differentiation in vitro, which is of importance for regenerative medicine strategies.

Droplet digital PCR for the quantification of Alu methylation status in hematological malignancies

BackgroundAlu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. Alu sequences lie in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu methylation has been associated with tumor aggressiveness, and also previously discussed in hematological malignancies, by applying different approaches. Moreover, today different techniques designed to measure global DNA methylation are focused on the methylation level of specific repeat elements.In this work we propose a new method of investigating Alu differential methylation, based on droplet digital PCR (ddPCR) technology.MethodsForty-six patients with hematological neoplasms were included in the study: 30 patients affected by chronic lymphocytic leukemia, 7 patients with myelodysplastic syndromes at intermediate/high risk, according with the International Prognostic Scoring System, and 9 patients with myelomonocytic leukemia. Ten healthy donors were included as controls. Acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with decitabine (DEC) hypomethylating agent, was also analyzed.DNA samples were investigated for Alu methylation level by digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation, followed by ddPCR.ResultsUsing ddPCR, a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, was observed. Moreover, comparing the global Alu methylation levels at diagnosis and after azacytidine (AZA) treatment in MDS patients, a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis was evident. We also observed a significant decrease of the Alu methylation level in CLL patients compared to HD, and, finally, for CMML patients, a decrease of Alu sequences methylation was observed in patients harboring the SRSF2 hotspot gene mutation c.284C>D.ConclusionsIn our work, we propose a method to investigate Alu differential methylation based on ddPCR technology. This assay introduces ddPCR as a more sensitive and immediate technique for Alu methylation analysis. To date, this is the first application of ddPCR to study DNA repetitive elements. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose.

Droplet Digital PCR for the Quantification of Alu Methylation Status in Hematological Malignancies

Introduction. Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. They are located in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu repeats methylation has been associated with tumor aggressiveness and investigated in some solid tumors, but the global Alu methylation level has not yet been investigated in hematological malignancies. Moreover, today, some of the techniques designed to measure global DNA methylation are focused on the methylation level of specific genomic compartments, including repeat elements.In this work we propose a new method for investigating Alu differential methylation, employing droplet digital PCR (ddPCR) technology, applied in patients affected by chronic lymphocytic leukemia (CLL), myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML).Methods. The study included a total of 46 patients: 30 CLL patients, 7 patients with MDS at intermediate/high risk, and 9 CMML patients. The study also involved acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with azacytidine (AZA) 0.75 µM or decytabine (DEC) 0.75 µM. Four healthy donors (HD) were also included as controls. For each DNA sample two aliquots of 250ng of gDNA were simultaneously digested (with 1 unit of Alu-in/sensitive isoschizomers either MspI or HpaII) and ligated (to a previously prepared synthetic adaptor) in parallel in two separate tubes.Considering that the genomic DNA amount in a human diploid cell is about 6 pg/cell, for each sample we calculated the percentage of methylated consensus Alu sequences as the ratio between the sum of positive droplets obtained from the three wells of both HpaII (MH) and MspI (MM) final dilutions, according to the following formula: [1-(sumMH/sumMM)]x100. The significance level was set at p<0.05 for all analyses.Results. Using our ddPCR assay, we observed a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, and a minor decrease with AZA (p=0.058). Moreover, comparing the global Alu methylation levels at diagnosis and after AZA treatment in MDS patients, we observed a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis.We also extended the assessment of our assay in CLL patients at diagnosis. We observed a significant decrease of the Alu methylation level in CLL patients compared to HD. CLL patients were also classified in the following three cytogenetic risk groups according to the karyotypic alterations identified by Fluorescent In Situ Hybridization (FISH): low (with isolated 13q deletion), intermediate (without 11q, 13q and 17p deletions or with trisomy 12), and high risk (with 11q or, 17p deletions, or more than two chromosomal aberrations). Alu methylation status of the low and high-risk groups was more significantly reduced compared to HD, whereas considering intermediate-risk patients the difference was less evident.Finally, for CMML patients, a significant decrease of Alu sequences methylation was observed in patients harboring the main SRSF2 gene hotspot. However, these preliminary results should be confirmed by extending the analysis to other CMML patients.Conclusions. In our work, we propose a new method to investigate Alu differential methylation based on ddPCR technology. This assay represents an alternative to conventional quantitative-PCR (qPCR), introducing ddPCR as a more sensitive and immediate technique for Alu methylation analysis. Moreover, compared to qPCR, our ddPCR Alu assay may be carried out using very small amounts of digested gDNA (about 6 pg), and does not require a reference gene for the analysis of ddPCR data.To date, this is the first application of ddPCR to study global DNA methylation by inspecting DNA repeats. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose. DisclosuresNo relevant conflicts of interest to declare.

Role of the LF-SINE-Derived Distal ISL1 Enhancer in Patients with Classic Bladder Exstrophy.

A genome-wide association study and meta-analysis identified ISL1 as the first genome-wide significant susceptibility gene for classic bladder exstrophy (CBE). A short interspersed repetitive element (SINE), first detected in lobe-finned fishes (LF-SINE), was shown to drive Isl1 expression in embryonic mouse genital eminence. Hence, we assumed this enhancer a conclusive target for mutations associated with CBE formation and analyzed a cohort of 200 CBE patients. Although we identified two enhancer variants in five CBE patients, their clinical significance seems unlikely, implying that sequence variants in the ISL1 LF-SINE enhancer are not frequently associated with CBE.

Comparative Genomic In Situ Hybridization and the Possible Role of Retroelements in the Karyotypic Evolution of Three Akodontini Species.

South American Akodontini rodents are characterized by a large number of chromosome rearrangements. Among them, the genus Akodon has been extensively analyzed with classical and molecular cytogenetics, which allowed the identification of a large number of intra- and interspecific chromosomal variation due to Robertsonian rearrangements, pericentric inversions, and heterochromatin additions/deletions. In order to shed some light on the cause of these rearrangements, we comparatively analyzed the karyotypes of three Akodontini species, Akodon cursor (2n = 14, FN = 19), A. montensis (2n = 24, FN = 42), and Necromys lasiurus (2n = 34, FN = 34), after GTG- and CBG-banding. The karyotypes differed by Robertsonian rearrangements, pericentric inversions, centromere repositioning, and heterochromatin variation. Genome comparisons were performed through interspecific fluorescent in situ hybridization (FISH) with total genomic DNAs of each species as probes (GISH). Our results revealed considerable conservation of the euchromatic portions among the three karyotypes suggesting that they mostly differ in their heterochromatic regions. FISH was also performed to assess the distribution of telomeric sequences, long and short interspersed repetitive elements (LINE-1 and B1 SINE) and of the endogenous retrovirus mysTR in the genomes of the three species. The results led us to infer that transposable elements have played an important role in the enormous chromosome variation found in Akodontini.

Identification of a short interspersed repetitive element insertion polymorphism in the porcine MX1 promoter associated with resistance to porcine reproductive and respiratory syndrome virus infection.

The myxovirus resistance (Mx) proteins belong to the dynamin superfamily and are important for innate host defence against RNA viruses. In this study, we demonstrate that positive elements are present in the two promoter regions of -2713 to -2565 and -688 to -431 in the porcine MX1 gene. Sequencing and alignment of the amplified porcine MX1 gene promoter region identified a short interspersed repetitive element (SINE) insertion of 275 bp at site -547. At this site, allele B (an insertion of 275 bp) is dominant in Chinese indigenous pig breeds but has a workable minor allele frequency in western lean-type pig breeds. Luciferase activity was compared between promoters with and without the insertion of the 275-bp fragment in transiently transfected MARC-145 cells. The insertion of the 275-bp fragment increased the luciferase activity significantly (P < 0.05) both prior to and post-porcine reproductive and respiratory syndrome (PRRS) virus inoculation. These results suggest that the SINE insertion polymorphism at site -547 of the MX1 gene promoter region is a potential DNA marker for PRRS resistance in pigs.

Genetic and Morphological Assessments of Hybridization Between Two Non-Native Geoemydid Turtles,Mauremys reevesiiandMauremys mutica,in Northcentral Japan

Interspecific hybridization has been occasionally reported for various combinations of geoemydid turtles, but genetic consequences of such hybridization have rarely been investigated. We surveyed the hybrid status of 40 individuals from a seemingly hybridizing turtle assemblage in a natural pond in northcentral Honshu, Japan. Analysis of mtDNA sequences confirmed that the parental species of this assemblage were Mauremys reevesii (Chinese three-keeled pond turtle) and Mauremys mutica (Asian yellow pond turtle), neither of which is native to the main islands of Japan, although M. reevesii was naturalized in the area before the 19th century. Extensive examination of pure strain samples of the 2 species yielded 10 morphological and 10 genetic diagnostic characters (allozyme and short interspersed repetitive elements) that were used to determine the hybrid status of the turtles in the assemblage. Morphological examination showed that 19 out of 40 individuals were not different from pure M. reevesii individuals, but the remaining 21 individuals exhibited M. mutica states at 1 to 6 characters, indicating interspecific crosses at a moderate to large scale. In the genetic characters, however, only 2 of the 21 individuals possessed marker alleles of M. mutica at a few loci and all others possessed alleles of M. reevesii only; thus neither pure M. mutica nor F1 hybrids were included in the assemblage. These results suggest that iterative backcrosses with pure M. reevesii have occurred. In the analysis using the genetic hybrid index (GHI), which was defined as the number of M. mutica type alleles over the 10 examined loci, the 2 individuals scored 5 and 2, respectively, and all others scored 0. This discrete distribution of GHI scores negates an assumption of a random mating hybrid swarm but suggests that these two individuals emerged by a different hybridization event, independent from that leading to the many other putative hybrids detected by morphological characters only. Our results suggest that even though the hybrid genotypes rarely prevailed in the population, once introgressed genes were diluted by successive backcrosses, they could have been retained over many generations.

Altered imprinted gene expression and methylation patterns in mid-gestation aborted cloned porcine fetuses and placentas.

To determine the expression patterns of imprinted genes and their methylation status in aborted cloned porcine fetuses and placentas. RNA and DNA were prepared from fetuses and placentas that were produced by SCNT and controls from artificial insemination. The expression of 18 imprinted genes was determined by quantitative real-time PCR (q-PCR). Bisulfite sequencing PCR (BSP) was conducted to determine the methylation status of PRE-1 short interspersed repetitive element (SINE), satellite DNA and H19 differentially methylated region 3 (DMR3). The weight, imprinted gene expression and genome-wide DNA methylation patterns were compared between the mid-gestation aborted and normal control samples. The results showed hypermethylation of PRE-1 and satellite sequences, the aberrant expression of imprinted genes, and the hypomethylation of H19 DMR3 occurred in mid-gestation aborted fetuses and placentas. Cloned pigs generated by somatic cell nuclear transfer (SCNT) showed a greater ratio of early abortion during mid-gestation than did normal controls because of the incomplete epigenetic reprogramming of the donor cells. Altered expression of imprinted genes and the hypermethylation profile of the repetitive regions (PRE-1 and satellite DNA) may be associated with defective development and early abortion of cloned pigs, emphasizing the importance of epigenetics during pregnancy and implications thereof for patient-specific embryonic stem cells for human therapeutic cloning and improvement of human assisted reproduction.

Variation in the coding and 3′ untranslated regions of the porcine prolactin receptor short form modifies protein expression and function

The actions of prolactin (PRL) are mediated by both long (LF) and short isoforms (SF) of the PRL receptor (PRLR). Here, we report on a genetic and functional analysis of the porcine PRLR (pPRLR) SF. Three single nucleotide polymorphisms (SNPs) within exon 11 of the pPRLR-SF give rise to four amino acid haplotypes of the intracellular domain. We identified the dimorphic insertion of a short interspersed repetitive DNA element (PRE-1) along with 32 SNPs and four other insertion/deletion sites within the 3' untranslated region (UTR) of pPRLR-SF. The PRE-1 element reduced protein translation in vitro by 75%, whereas the combination of 10 SNPs and one insertion/deletion decreased translation by 50%. Full-length cDNAs for all four haplotypes of pPRLR-SF were cloned behind the elongation factor 1-alpha promoter and functionally analyzed in vitro. None of the haplotypes could initiate transcription from the ß-casein promoter, whereas all four were dominant negatives against PRL-activation of the pPRLR-LF. Two of the haplotypes completely inhibited pPRLR-LF activity at a four-fold excess, whereas the others required a six-fold excess to impart the same effect. The ligand binding affinities of the pPRLR-SF haplotypes did not differ. Expression of the pPRLR-SF increased linearly during gestation in the endometrium and was hormonally regulated in a tissue-specific manner in the mammary glands and uterus. In conclusion, we identified a PRE-1 and other SNPs in the pPRLR-SF 3' UTR that reduce protein expression and four haplotypes of the pPRLR-SF that suppress pPRLR-LF signaling and may differentially impact the phenotypic effects of PRL in vivo.

Swine liver methylomes of Berkshire, Duroc and Landrace breeds by MeDIPS

Using a methyl-DNA immunoprecipitation technique in combination with next-generation deep sequencing, we conducted comprehensive DNA methylation profiling of liver genomes from three pig breeds: Berkshire, Duroc and Landrace. The profiles revealed that the distribution patterns of methylation signals along the genome are conserved among the three pig breeds. Specifically, many signals in coding genes were found in introns, and most signals in the repetitive elements were identified in non-long terminal repeat (LTR) retrotransposons such as long and short interspersed repetitive elements, implying a significant association with alternative splicing and expression of retrotransposable elements respectively. Differentially methylated regions among the three pig breeds were identified in the non-LTR retrotransposons, suggesting that they may lead to differential retrotransposable element activity. Altogether, this study provides advanced swine methylome data and valuable resources for understanding the function of DNA methylation in the evolutionary divergence of different pig breeds.

A SINE-Derived Element Constitutes a Unique Modular Enhancer for Mammalian Diencephalic Fgf8

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.

Molecular characterization of the short interspersed repetitive element SIRE in the six discrete typing units (DTUs) of Trypanosoma cruzi

Repetitive sequences constitute an important proportion of the Trypanosoma cruzi genome; hence, they have been used as molecular markers and as amplification targets to identify the parasite presence via PCR. In this study, a molecular characterization of the SIRE repetitive element was performed in the six discrete typing units (DTUs) of T. cruzi. The results evidenced that this element, located in multiple chromosomes, was interspersed in the genome of all DTUs of the parasite. The presence of several motifs implicated in element insertion, duplication, and functionality suggests that SIRE could be an active element in the parasite genome. Of interest, there were SIRE specific Alu I fragments that allowed to discriminate DTU I from the others DTUs. Moreover, an UPGMA phenetic tree constructed from fragment sharing Southern blot data showed that T. cruzi I isolates conform a cluster separated from the T. cruzi II–VI isolates. When the relative number of SIRE copies was determined, a variation from 105 to 2,000 copies per haploid genome was observed among the different isolates without kept a DTU-relationship. In all, these findings suggest that SIRE sequence is a good target for parasite DNA amplification.

Transcriptional Regulation of Translocator Protein (Tspo) via a SINE B2-Mediated Natural Antisense Transcript in MA-10 Leydig Cells1

Translocator protein (18 kDa; TSPO) is a mitochondrial cholesterol- and drug-binding protein involved in cholesterol import into mitochondria, the rate-limiting step in steroidogenesis. TSPO is expressed at high levels in Leydig cells of the testis, and its expression levels dictate the ability of the cells to form androgen. In search of mechanisms that regulate Tspo expression, a number of transcription factors acting on its promoter region have been identified. We report herein the presence of a mechanism of regulation of Tspo expression via complementation with a natural antisense transcript (NAT). At the Tspo locus, a short interspersed repetitive element (SINE) of the SINE B2 family has the potential for high transcriptional activity. The extension of the SINE B2 element-mediated transcript overlapped with exon 3 of the Tspo gene and formed a NAT specific for Tspo (Tspo-NAT) in MA-10 mouse tumor Leydig cells. The identified Tspo-NAT was also found in testis and kidney tissues. Overexpression of the Tspo-NAT regulated Tspo gene expression and its function in steroid formation in MA-10 cells. Time-course studies have indicated that Tspo-NAT expression is regulated by cAMP and could regulate TSPO levels to maintain optimal steroid production by MA-10 Leydig cells. Taken together, these results suggest a new micro-transcriptional mechanism that regulates Tspo expression and thus steroidogenesis via an intron-based SINE B2-driven NAT specific for the Tspo gene.

A Profile of Native Integration Sites Used by φC31 Integrase in the Bovine Genome

The Streptomyces phage φC31 integrase can efficiently target attB-bearing transgenes to endogenous pseudo attP sites within mammalian genomes. To better understand the activity of φC31 integrase in the bovine genome, DNA sequences of 44 integration events were analyzed, and 32 pseudo attP sites were identified. The majority of these sites share a sequence motif that contains inverted repeats and has similarities to wild-type attP site. Genomic DNA flanking these sites typically contained repetitive sequence elements, such as short and long interspersed repetitive elements. These sequence features indicate that DNA sequence recognition plays an important role in guiding φC31-mediated site-specific integration. In addition, BF27 integration hotspot sites were identified in the bovine genome, which accounted for 13.6% of all isolated integration events and mapped to an intron of the deleted in liver cancer 1 (DLC1) gene. Also we found that the pseudo attP sites in the bovine genome had other features in common with those in the human genome. This study represents the first time that the sequence features of pseudo attP sites in the bovine genome were analyzed. We conclude that this site-specific integrase system has great potential for applied modifications of the bovine genome.

A Mammalian Conserved Element Derived from SINE Displays Enhancer Properties Recapitulating Satb2 Expression in Early-Born Callosal Projection Neurons

Short interspersed repetitive elements (SINEs) are highly repeated sequences that account for a significant proportion of many eukaryotic genomes and are usually considered “junk DNA”. However, we previously discovered that many AmnSINE1 loci are evolutionarily conserved across mammalian genomes, suggesting that they may have acquired significant functions involved in controlling mammalian-specific traits. Notably, we identified the AS021 SINE locus, located 390 kbp upstream of Satb2. Using transgenic mice, we showed that this SINE displays specific enhancer activity in the developing cerebral cortex. The transcription factor Satb2 is expressed by cortical neurons extending axons through the corpus callosum and is a determinant of callosal versus subcortical projection. Mouse mutants reveal a crucial function for Sabt2 in corpus callosum formation. In this study, we compared the enhancer activity of the AS021 locus with Satb2 expression during telencephalic development in the mouse. First, we showed that the AS021 enhancer is specifically activated in early-born Satb2+ neurons. Second, we demonstrated that the activity of the AS021 enhancer recapitulates the expression of Satb2 at later embryonic and postnatal stages in deep-layer but not superficial-layer neurons, suggesting the possibility that the expression of Satb2 in these two subpopulations of cortical neurons is under genetically distinct transcriptional control. Third, we showed that the AS021 enhancer is activated in neurons projecting through the corpus callosum, as described for Satb2+ neurons. Notably, AS021 drives specific expression in axons crossing through the ventral (TAG1−/NPY+) portion of the corpus callosum, confirming that it is active in a subpopulation of callosal neurons. These data suggest that exaptation of the AS021 SINE locus might be involved in enhancement of Satb2 expression, leading to the establishment of interhemispheric communication via the corpus callosum, a eutherian-specific brain structure.

Mapping and analysis of the LINE and SINE type of repetitive elements in rice.

Non-LTR retrotransposons comprise significant portion of the plants genome. Their complete characterization is thus necessary if the sequenced genome is to be annotated correctly. The long and short interspersed nucleotide repetitive elements (LINE and SINE) may be responsible for alteration in the expression mechanism of neighboring genes, the complete identification of these elements in the rice genome is essential in order studying their putative functional interactions with the plant genes and its role in genome composition. The main emphasis of this work is to assemble a comprehensive dataset of nonLTR (LINEs and SINEs) and the map of completely inserted LINEs and SINE type of retroelement by both intact ends (3' and 5' ends). The assembled information and work may help for further research in this direction.

Novel CHR-2 SINE subfamilies and t-SINEs identified in cetaceans using nonradioactive Southern blotting

The SINEs are short interspersed repetitive elements that propagate in eukaryotic genomes via transcription followed by reverse transcription. Recent genomic surveys have demonstrated that SINEs are active in shaping the architecture of genomes and therefore are powerful markers for studies of phylogeny and population biology. In the present study, a new protocol was developed to apply nonradioactive digoxigenin-labeling system into isolation of SINEs rapidly, safely, and efficiently. The application of this approach is illustrated with discovery of thirteen informative SINEs and a novel member of the t-SINE family in the Omura’s whale (Balaenoptera omurai) and sperm whale (Physeter macrocephalus), which is the first record of t-SINE in cetacean. Analysis of subfamily-specific genetic variability and insertions/deletions strongly suggested the existence of some novel CHR-2 SINE subfamilies in the cetacean genomes.

Origin and evolution of LINE-1 derived “half-L1” retrotransposons (HAL1)

LINE-1 (L1) retrotransposons represent the most abundant family of non-LTR retrotransposons in virtually all mammals. The only currently known exception is Platypus, where it is found only in low copy numbers. Autonomous L1s encode two proteins, ORF1p and ORF2p, both of which are required for the transposition of L1s. L1 replicative machinery is also involved in the trans-mobilization of non-autonomous retrotransposons, such as diverse short interspersed repetitive elements (SINEs) and processed pseudogenes. Here, we focus on a unique category of “half -L1” elements (HAL1s), which encode ORF1p but not ORF2p. HAL1s are present both in placental mammals and marsupials. We demonstrate that HAL1s originated independently several times during the evolution of mammals. The youngest mammalian HAL1 elements analyzed in this paper were identified in the guinea pig genome. Our analysis strongly suggests that HAL1-encoded ORF1p is essential for the transposition of HAL1s and indicates that the evolution of ORF1p in HAL1s is faster than in L1s. The implications of HAL1 for the evolution of L1 elements and the host genomes are discussed.

Characterization of a novel SINE superfamily from invertebrates: “Ceph-SINEs” from the genomes of squids and cuttlefish

Five tRNA-derived short interspersed repetitive elements (SINEs), named SepiaSINE, Sepioth-SINE1, Sepioth-SINE2A, Sepioth-SINE2B and OegopSINE, were isolated from the genomes of three decabrachian species [ Sepia officinalis (order Sepiida), Sepiotheuthis lessoniana (suborder Myopsida), and Mastigoteuthis cordiformes (suborder Oegopsida)], by random sequencing and genome screening. In addition, two tRNA-derived SINEs, named IdioSINE1 and IdioSINE2, were further detected from EST (expressed sequence tag) data of Idiosepius paradoxus (order Idiosepiida), using a GenBank FASTA search with a conserved sequence of the SepiaSINE as the query. All the isolated SINEs had a common and unique highly conserved 149-bp sequence in their central structures (Sepioth-SINE2B and IdioSINEs, however, had a continuous 73-bp deletion in the conserved region.), and are therefore grouped as the fourth SINE superfamily “Ceph-SINEs”, following the CORE-SINE, V-SINE, and DeuSINE superfamilies. Our analysis suggested that the central conserved region called the “Ceph-domain” might have originated before the diversification of cephalopods (505 myr ago). A sequence alignment of Sepioth-SINE1, Sepioth-SINE2A, and Sepioth-SINE2B demonstrated that Sepioth-SINE2A has a chimeric structure shared with two other SINEs. The above relationship suggests possible template switching in the central conserved domain during reverse transcription for the birth of Sepioth-SINE2A, providing the possibility that the presence of the conserved domain contributed to yield a variety of SINEs during evolution. Furthermore, the distributions of the isolated SINEs showed that order Sepiida, suborders Oegopsida and Myopsida, and order Idiosepiida have their own independent SINE(s), and suggest that order Sepiida can be largely separated into two groups, with clarification of the phylogenetic relatedness between subfamily Sepioteuthinae and the other loliginid squids.