Mammalian retroelements

The eukaryotic genome has undergone a series of epidemics of amplification of mobile elements that have resulted in most eukaryotic genomes containing much more of this \u27junk\u27 DNA than actual coding DNA. The majority of these elements utilize an RNA intermediate and are termed retroelements. Most of these retroelements appear to amplify in evolutionary waves that insert in the genome and then gradually diverge. In humans, almost half of the genome is recognizably derived from retroelements, with the two elements that are currently actively amplifying, L1 and Alu, making up about 25% of the genome and contributing extensively to disease. The mechanisms of this amplification process are beginning to be understood, although there are still more questions than answers. Insertion of new retroelements may directly damage the genome, and the presence of multiple copies of these elements throughout the genome has longer-term influences on recombination events in the genome and more subtle influences on gene expression

LINE-1 RNA splicing and influences on mammalian gene expression

Long interspersed element-1 elements compose on average one-fifth of mammalian genomes. The expression and retrotransposition of L1 is restricted by a number of cellular mechanisms in order to limit their damage in both germ-line and somatic cells. L1 transcription is largely suppressed in most tissues, but L1 mRNA and/or proteins are still detectable in testes, a number of specific somatic cell types, and malignancies. Down-regulation of L1 expression via premature polyadenylation has been found to be a secondary mechanism of limiting L1 expression. We demonstrate that mammalian L1 elements contain numerous functional splice donor and acceptor sites. Efficient usage of some of these sites results in extensive and complex splicing of L1. Several splice variants of both the human and mouse L1 elements undergo retrotransposition. Some of the spliced L1 mRNAs can potentially contribute to expression ofopen reading frame 2-related products and therefore have implications for the mobility of SINEs even if they are incompetent for L1 retrotransposition. Analysis of the human EST database revealed that L1 elements also participate in splicing events with other genes. Such contribution of functional splice sites by L1 may result in disruption of normal gene expression or formation of alternative mRNA transcripts

Transcription and processing of the rodent ID repeat family in germline and somatic cells

ID elements comprise a rodent SINE (short Interspersed DNA repetitive element) family that has amplified by retroposition of a few master genes. In order to understand the Important factors of SINE amplification, we investigated the transcription of rat ID elements. Three different size classes of ID transcripts, BC1, BC2 and T3, have been detected In various rat tissues, including brain and testes. We have analysed the nucleotlde sequences of testes- and brain-derived ID transcripts isolated by size-fractlonation, C-talling and RACE. Nucleotide sequence variation of testes ID transcripts demonstrated derivation from different loci. However, the transcripts represent a preferred set of ID elements that closely match the subfamily consensus sequences. The small ID transcripts, T3, are not comprised of primary transcripts, but are instead processed polyA- transcripts generated from many different loci. These truncated transcripts would be expected to be retroposition-incompetent forms. Therefore, the amplification of ID elements is likely to be regulated at multiple steps of retroposition, which Include transcription and processing. Although brain ID transcripts showed a similar pattern, with the addition of very high levels of transcription from the BC1 locus, we also found evidence that a single locus dominated the production of brain BC2 RNA species. BC1 RNA is highly stable In both germ line and brain cells, based on the low level of detection of the processing product, T3. This stability of BC1 RNA might have been a contributing factor in its role as a master gene for ID amplification. © 1995 Oxford University Press

LINE-1 and Alu retrotransposition exhibit clonal variation

BACKGROUND: The non-long terminal repeat (non-LTR) retrotransposons, long interspersed element-1 (LINE-1) and Alu are currently active retroelements in humans. We, and others, have observed that different populations of HeLa cells from different laboratories support retrotransposition of LINE-1 and Alu to varying degrees. We therefore tested whether individual cell clones of HeLa and HCT116 cell lines supported different levels of LINE-1 and Alu retrotransposition, and whether these variations were stable upon re-cloning. FINDINGS: Standard retrotransposition tissue culture assays were used to measure a cell’s ability to support LINE-1 and Alu retrotransposition in clonal HeLa and HCT116 cell lines. We observed that both LINE-1 and Alu retrotransposition exhibited clonal variation in HeLa cells, with certain HeLa cell clones supporting high levels of LINE-1 and Alu retrotransposition and other cell clones being essentially retrotransposition-dead. This clonal variation was similarly observed in HCT116 cells, although possibly not to the same extent. These patterns of clonal variation are relatively consistent upon re-cloning. CONCLUSIONS: Observations of the variability of LINE-1 and Alu retrotransposition in different populations of the same cell line are supported by our results that indicate in some cell types, individual cell clones can have dramatically differing capacity for retrotransposition. The mixed populations of cells commonly used in laboratories have often been passaged for many generations and accumulated significant genetic and epigenetic diversity. Our results suggest that the clonal variability observed by our cloning experiments may lead to a homogenization of retrotransposition capacity, with the resulting mixed population of cells being composed of individual variants having either increased or decreased retrotransposition potential compared to the starting population

Feedback inhibition of L1 and alu retrotransposition through altered double strand break repair kinetics

<p>Abstract</p> <p>Background</p> <p>Cells adapt to various chronic toxic exposures in a multitude of ways to minimize further damage and maximize their growth potential. Expression of L1 elements in the human genome can be greatly deleterious to cells, generating numerous double strand breaks (DSBs). Cells have been reported to respond to chronic DSBs by altering the repair of these breaks, including increasing the rate of homology independent DSB repair. Retrotransposition is strongly affected by proteins involved in DSB repair. Therefore, L1 expression has the potential to be a source of chronic DSBs and thus bring about the changes in cellular environment that could ultimately restrict its own retrotransposition.</p> <p>Results</p> <p>We demonstrate that constitutive L1 expression leads to quicker DSB repair and decreases in the retrotransposition potential of L1 and other retrotransposons dependent on L1 expression for their mobility. This cellular adaptation results in reduced sensitivity to L1 induced toxicity. These effects can be induced by constitutive expression of the functional L1 ORF2 alone, but not by the constitutive expression of an L1 open reading frame 2 with mutations to its endonuclease and reverse transcriptase domains. This adaptation correlates with the relative activity of the L1 introduced into the cells.</p> <p>Conclusions</p> <p>The increased number of DSBs resulting from constitutive expression of L1 results in a more rapid rate of repair. The cellular response to this L1 expression also results in attenuation of retrotransposition and reduced sensitivity of the cells to negative consequences of L1 ORF2 expression. The influence does not appear to be through RNA interference. We believe that the increased rate of DSB repair is the most likely cause of the attenuation of retrotransposition. These alterations act as a fail safe mechanism that allows cells to escape the toxicity associated with the unchecked L1 expression. This gives cells that overexpress L1, such as tumor cells, the ability to survive the high levels of expression. However, the increased rate of break repair may come at the cost of accuracy of repair of the lesion, resulting in increased genomic instability.</p

Introduction for the Gene special issue dedicated to the meeting Genomic impact of eukaryotic transposable elements at Asilomar

The issues related to \u27Genomic Impact of Eukaryotic Transposable Elements\u27, which took place in Pacific Grove, California between March 31st and April 4th 2006, are discussed. The meeting celebrated the extraordinary contributions of Dr. Carl W. Schmid to the study of repeated DNA sequences and mobile elements. With the advent of recombinant DNA technology, he led the discovery of human Alu elements, and the discovery of their amplification. The idea of the conference was to gather and disseminate information in transposable elements (TEs) on the state-of-the-art tools and approaches. The core sessions from the conference covered research on transposable elements with a strong emphasis on their impact on genomic stability and evolution. The scientific sessions were complemented by after-dinner workshop sessions focusing on Repbase, computer tools used in annotation and analysis of repetitive DNA and open problems related to the field

A droplet digital PCR detection method for rare L1 insertions in tumors

Background: The active human mobile element, long interspersed element 1 (L1) currently populates human genomes in excess of 500,000 copies per haploid genome. Through its mobility via a process called target primed reverse transcription (TPRT), L1 mobilization has resulted in over 100 de novo cases of human disease and has recently been associated with various cancer types. Large advances in high-throughput sequencing (HTS) technology have allowed for an increased understanding of the role of L1 in human cancer; however, researchers are still limited by the ability to validate potentially rare L1 insertion events detected by HTS that may occur in only a small fraction of tumor cells. Additionally, HTS detection of rare events varies greatly as a function of read depth, and new tools for de novo element discovery are needed to fill in gaps created by HTS. Results: We have employed droplet digital PCR (ddPCR) to detect rare L1 loci in mosaic human genomes. Our assay allows for the detection of L1 insertions as rare as one cell in every 10,000. Conclusions: ddPCR represents a robust method to be used alongside HTS techniques for detecting, validating and quantitating rare L1 insertion events in tumors and other tissues. Electronic supplementary material The online version of this article (doi:10.1186/s13100-014-0030-4) contains supplementary material, which is available to authorized users

A new restriction-site polymorphism in exon 18 of the low density lipoprotein receptor (LDLR) gene

Abstract A new restriction fragment length polymorphism (RFLP) in exon 18 of the low density lipoprotein receptor (LDLR) gene is described. It should be a useful marker in linkage to familial hypercholesterolemia. Source~description. Primers, based on the nucleotide sequence of the low density lipoprotein receptor (LDLR) gene exon 18 PCR conditions. Genomic DNA (500 ng) was added to a 50-~tl volume reaction mix containing 1 x Taq buffer (Promega), 3.0 mM MgCI2, 200 gM dNTPs, 250 nM each primer [1019 (5&quot;-ACTTCAAAGCCGTGATCGTGA-3&quot;) and 2029 (5&quot;-TGCAACAGTAACACGGCGATT-3&quot;)] and 1 unit Taq polymerase (Promega) and amplified using Hybaid (Omnigene) thermal cycler (block control) under the following conditions: 94~ 3 min 1 x; 94~ 1:30 min., 52~ 1:30 min, 72~ 3 min, 30 x; 72~ 3 min. An 8-~tl sample of the PCR reaction mix was digested in a 20-~1 reaction using excess enzyme and 1 x buffer (NEB) and examined following electrophoresis on ethidium bromide stained 1.5% agarose gels. Frequency. In 20 unrelated caucasians A1 = 0.70, A2 = 0.30, observed heterozygosity = 0.50; in 22 unrelated African Americans A1 = 0.30, A2 = 0.70, observed heterozygosity = 0.41