Molecular structure of canine LINE-1 elements in canine transmissible venereal tumor

We determined the 4251-bp sequence of open reading frame 2 (ORF2) of canine LINE-1 retroposon that encodes 1275 amino acids. The truncated LINE-1 inserts associated with transmissible venereal tumor (TVT) of dogs contained the 1378-bp LINE-1 insert (TVT-LINE) flanked by 10-bp direct repeats upstream to c-myc gene. The TVT-LINE elements were composed of 416 bp inverse sequences homologous to the complementary strand of the LINE-1, a 5-bp deletion and 962-bp sequences homologous to the 3' region of the LINE-1.     

Naturally occurring endo-siRNA silences LINE-1 retrotransposons in human cells through DNA methylation

Long interspersed nuclear element 1 (LINE-1) retrotransposons are mutagens that are capable of generating deleterious mutations by inserting themselves into genes and affecting gene function in the human genome. In normal cells, the activity of LINE-1 retrotransposon is mostly repressed, maintaining a stable genome structure. In contrast, cancer cells are characterized by aberrant expression of LINE-1 retrotransposons, which, in principle, have the potential to contribute to genomic instability. The mechanistic pathways that regulate LINE-1 expression remain unclear. Using deep-sequencing small RNA analysis, we identified a subset of differentially expressed endo-siRNAs that directly regulate LINE-1 expression. Detailed analyses suggest that these endo-siRNAs are significantly depleted in human breast cancer cells compared with normal breast cells. The overexpression of these endo-siRNAs in cancer cells markedly silences endogenous LINE-1 expression through increased DNA methylation of the LINE-1 5′-UTR promoter. The finding that endo-siRNAs can silence LINE-1 activity through DNA methylation suggests that a functional link exists between the expression of endo-siRNAs and LINE-1 retrotransposons in human cells.     

Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells.

The LINE-1 repeat family is interspersed throughout mammalian genomes and is thought to be the result of duplicative transposition of LINE-1 sequences via an RNA intermediate. This report describes a ribonucleoprotein particle with LINE-1 RNA in the mouse embryonal carcinoma cell line F9. This ribonucleoprotein particle is a potential intermediate in the transposition of LINE-1 in the mouse genome.     

Systematic identification of LINE-1 repetitive DNA sequence differences having species specificity between Mus spretus and Mus domesticus

LINE-1 is a family of repetitive DNA sequences interspersed among mammalian genes. In the mouse haploid genome there are about 100,000 LINE-1 copies. We asked if the subspecies Mus spretus and Mus domesticus have developed species-specific LINE-1 subfamilies. Sequences from 14 M. spretus LINE-1 elements were obtained and compared to M. domesticus LINE-1 sequences. Using a molecular phylogenetic tree we identified several differences shared among a subset of young repeats in one or the other species as candidates for species-specific LINE-1 variants. Species specificity was tested using oligonucleotide probes complementary to each putative species-specific variant. When hybridized to genomic DNAs, single-variant probes detected an expanded number of elements in the expected mouse. In the other species these probes detected a smaller number of matches consistent with the average rate of random divergence among LINE-1 elements. It was further found that the combination of two species-specific sequence differences in the same probe reduced the detection background in the wrong species below our detection limit.     

Integration of a vector containing a repetitive LINE-1 element in the human genome.

Mammalian cells contain numerous nonallelic repeated sequences, such as multicopy genes, gene families, and repeated elements. One common feature of nonallelic repeated sequences is that they are homeologous (not perfectly identical). Our laboratory has been studying recombination between homeologous sequences by using LINE-1 (L1) elements as substrates. We showed previously that an exogenous L1 element could readily acquire endogenous L1 sequences by nonreciprocal homologous recombination. In the study presented here, we have investigated the propensity of exogenous L1 elements to be involved in a reciprocal process, namely, crossing-overs. This would result in the integration of the exogenous L1 element into an endogenous L1 element. Of over 400 distinct integration events analyzed, only 2% involved homologous recombination between exogenous and endogenous L1 elements. These homologous recombination events were imprecise, with the integrated vector being flanked by one homologous and one illegitimate junction. This type of structure is not consistent with classical crossing-overs that would result in two homologous junctions but rather is consistent with one-sided homologous recombination followed by illegitimate integration. Contrary to what has been found for reciprocal homologous integration, the degree of homology between the exogenous and endogenous L1 elements did not seem to play an important role in the choice of recombination partners. These results suggest that although exogenous and endogenous L1 elements are capable of homologous recombination, this seldom leads to crossing-overs. This observation could have implications for the stability of mammalian genomes.     

Epigenetic regulation and role of LINE-1 retrotransposon in embryogenesis

LINE-1 retrotransposon is the most common mobile genetic element in the genomes of various mammals, including humans. Its genes are represented by the greatest number of copies. For a long time, it has been considered that the presence of LINE-1 in genome reflects the limited ability of cells to eliminate it, and the retrotransposon activity is negative owing to the insertional mutagenesis. In recent years, the increased expression of LINE-1 retrotransposon and the activity of their encoded proteins observed in mammalian cells at different stages of development and, first of all, in early embryogenesis have been discussed in the literature. Is early embryogenesis the stage of development when the organism is more susceptible to the activity of retrotransposons, or does LINE-1 play some positive role in early embryonic development? This review is aimed at classifying the available data on the epigenetic regulation and the role of LINE-1 retrotransposon in embryogenesis of mammals. The link between the mechanisms of regulation of LINE-1 expression and the waves of epigenetic reprogramming is tracked in germ cells, during fertilization, and in blastocyst, as well as during the differentiation of embryonic and extraembryonic tissues.     

Isolation and characterization of repetitive DNA sequences from Panax ginseng

Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization and evolution of the genome of interest. We report the isolation and characterization of the major classes of repetitive sequences from the genome of Panax ginseng. The isolation of repetitive DNA from P. ginseng was achieved by the reannealing of chemically hydrolyzed (200 bp-1 kb fragments) and heat-denatured genomic DNA to low C(o)t value. The low C(o)t fraction was cloned, and fifty-five P. ginseng clones were identified that contained repetitive sequences. Sequence analysis revealed that the fraction includes repetitive telomeric sequences, species-specific satellite sequences, chloroplast DNA fragments and sequences that are homologous to retrotransposons. Two of the retrotransposon-like sequences are homologous to Ty1/ copia-type retroelements of Zea mays, and six cloned sequences are homologous to various regions of the del retrotransposon of Lilium henryi. The del retrotransposon-like sequences and several novel repetitive DNA sequences from P. ginseng were used to differentiate P. ginseng from P. quinquefolius, and should be useful for evolutionary studies of these disjunct species.     

Genomic rearrangements by LINE-1 insertion-mediated deletion in the human and chimpanzee lineages

Long INterspersed Elements (LINE-1s or L1s) are abundant non-LTR retrotransposons in mammalian genomes that are capable of insertional mutagenesis. They have been associated with target site deletions upon insertion in cell culture studies of retrotransposition. Here, we report 50 deletion events in the human and chimpanzee genomes directly linked to the insertion of L1 elements, resulting in the loss of ~18 kb of sequence from the human genome and ~15 kb from the chimpanzee genome. Our data suggest that during the primate radiation, L1 insertions may have deleted up to 7.5 Mb of target genomic sequences. While the results of our in vivo analysis differ from those of previous cell culture assays of L1 insertion-mediated deletions in terms of the size and rate of sequence deletion, evolutionary factors can reconcile the differences. We report a pattern of genomic deletion sizes similar to those created during the retrotransposition of Alu elements. Our study provides support for the existence of different mechanisms for small and large L1-mediated deletions, and we present a model for the correlation of L1 element size and the corresponding deletion size. In addition, we show that internal rearrangements can modify L1 structure during retrotransposition events associated with large deletions.     

Structural analysis of repetitive DNA sequences in the bovine corticotropin-beta-lipotropin precursor gene region.

Repetitive DNA sequences in the bovine corticotropin-beta-lipotropin precursor gene region have been mapped and subjected to nucleotide sequence analysis. Two of the four repetitive DNA segments found are located in the 5'-flanking region, and one each within the intervening sequences. Each repetitive DNA segment contains one to three highly homologous unit sequences with an approximate length of 120 base pairs. All the unit sequences are flanked on the 3' side by tandem repeats. There are about 10(5) copies of the repetitive DNA in the bovine genome. Comparison of the bovine repetitive sequences with those of other mammalian species reveals the presence of a homologous segment of approximately 40 base pairs. This segment and the region preceding it in the bovine repetitive DNA exhibit sequence homology with the region encompassing the origin of DNA replication in papovaviruses.     

Loss of LINE-1 activity in the megabats

LINE-1 (L1) retrotransposons are the most abundant type of mammalian retroelement. They have profound effects on genome plasticity and have been proposed to fulfill essential host functions, yet it remains unclear where they lie on the spectrum from parasitism to mutualism. Their ubiquity makes it difficult to determine the extent of their effects on genome evolution and gene expression because of the relative dearth of animal models lacking L1 activity. We have isolated L1 sequences from 11 megabat species by a method that enriches for recently inserted L1s and have done a bioinformatic examination of L1 sequences from a 12th species whose genome was recently shotgun sequenced. An L1 extinction event appears to have occurred at least 24 million years ago (MYA) in an ancestor of the megabats. The ancestor was unusual in having maintained two highly divergent long-term L1 lineages with different levels of activity, which appear, on an evolutionary scale, to have simultaneously lost that activity. These megabat species can serve as new animal models to ask what effect loss of L1 activity has on mammalian genome evolution and gene expression.     

Protein-primed DNA replication: role of inverted terminal repeats in the Escherichia coli bacteriophage PRD1 life cycle.

Escherichia coli bacteriophage PRD1 and its relatives contain linear double-stranded DNA genomes, the replication of which proceeds via a protein-primed mechanism. Characteristically, these molecules contain 5'-covalently bound terminal proteins and inverted terminal nucleotide sequences (inverted terminal repeats [ITRs]). The ITRs of each PRD1 phage species have evolved in parallel, suggesting communication between the molecule ends during the life cycle of these viruses. This process was studied by constructing chimeric PRD1 phage DNA molecules with dissimilar end sequences. These molecules were created by combining two closely related phage genomes (i) in vivo by homologous recombination and (ii) in vitro by ligation of appropriate DNA restriction fragments. The fate of the ITRs after propagation of single genomes was monitored by DNA sequence analysis. Recombinants created in vivo showed that phages with nonidentical genome termini are viable and relatively stable, and hybrid phages made in vitro verified this observation. However, genomes in which the dissimilar DNA termini had regained identical sequences were also detected. These observations are explained by a DNA replication model involving two not mutually exclusive pathways. The generality of this model in protein-primed DNA replication is discussed.     

Generation of Adenovirus Vectors Devoid of All Viral Genes by Recombination between Inverted Repeats

Direct or inverse repeated sequences are important functional features of prokaryotic and eukaryotic genomes. Considering the unique mechanism, involving single-stranded genomic intermediates, by which adenovirus (Ad) replicates its genome, we investigated whether repetitive homologous sequences inserted into E1-deleted adenoviral vectors would affect replication of viral DNA. In these studies we found that inverted repeats (IRs) inserted into the E1 region could mediate predictable genomic rearrangements, resulting in vector genomes devoid of all viral genes. These genomes (termed DeltaAd.IR) contained only the transgene cassette flanked on both sides by precisely duplicated IRs, Ad packaging signals, and Ad inverted terminal repeat sequences. Generation of DeltaAd.IR genomes could also be achieved by coinfecting two viruses, each providing one inverse homology element. The formation of DeltaAd.IR genomes required Ad DNA replication and appeared to involve recombination between the homologous inverted sequences. The formation of DeltaAd. IR genomes did not depend on the sequence within or adjacent to the inverted repeat elements. The small DeltaAd.IR vector genomes were efficiently packaged into functional Ad particles. All functions for DeltaAd.IR replication and packaging were provided by the full-length genome amplified in the same cell. DeltaAd.IR vectors were produced at a yield of approximately 10(4) particles per cell, which could be separated from virions with full-length genomes based on their lighter buoyant density. DeltaAd.IR vectors infected cultured cells with the same efficiency as first-generation vectors; however, transgene expression was only transient due to the instability of deleted genomes within transduced cells. The finding that IRs present within Ad vector genomes can mediate precise genetic rearrangements has important implications for the development of new vectors for gene therapy approaches.     

Characterization of full-length LINE-1 insertions in 154 genomes

Long interspersed nuclear elements (LINEs) are retrotransposons that contribute to genetic variation in the human genome. LINE-1 elements in larger-scale studies are challenging to identify using sequencing technologies due to cost and scalability. We developed an approach using optical mapping for detection of full-length LINE-1 insertions and 10× sequencing for confirmation. We found 51 true positive full-length LINE-1 insertions, of which 4 are novel insertions, in NA12878. Repeating our analysis on a larger sample set representing 26 populations, we identified 329 full-length LINE-1 elements, of which 123 are novel. 24.8% of these 329 LINE-1 insertions were shared amongst all 5 superpopulations (AFR, AMR, EUR, EAS, SAS). The African superpopulation has a higher percentage of population-specific LINE-1 insertions than any other superpopulation. These data indicate that our approach can provide high-speed, cost-effective, and increased accuracy for LINE-1 detection. These data also provide an insight into variations of LINE-1 elements between different populations.     

Human DNA sequences isolated with an immunoglobulin switch region probe: sequence,chromosomal localization,and restriction fragment length polymorphisms

Summary We have screened a human genomic DNA library with an immunoglobulin (Ig) derived switch (S) region specific probe for homologous sequences. Five Ig independent phage clones were isolated and characterized. The S sequence homologous DNA fragments are short compared to the S region sequences. Ig independent S sequences are flanked by highly repetitive DNA elements and perfect inverted repeats can be demonstrated in their close vicinity. Using subclones of S homologous sequences restriction fragment length polymorphisms were shown within DNA of different T cell leukemias. Burkitt lyphhomas, lymphoblastoid cell lines, and DNA of healthy individuals. One of the five clones isolated with the S region probe was evidently localized to chromosome 2 and/or 40 and showed a complex hybridisation pattern with several different human DNAs. S homologous sequences of another clone are most likely localized on chromosome 1. It is possible that these Ig indenpendent S sequences have arisen by amplification and transposition and that they are involved in genetic recombination.