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Eline T Luning Prak

Publications and source records attributed to Eline T Luning Prak.

8 recordsLinked to original sources
PubMed2006

Consequences of receptor editing at the lambda locus: multireactivity and light chain secretion.

To investigate the manner in which B cells with lambda light (L) chains undergo receptor editing, we have studied hybridoma panels from 56R/kappa-deleted (kdel) mice. 56R/kdel mice only produce four L chains (lambda1, lambda2, lambda3, and lambdaX). They also have a simplified heavy (H) chain repertoire: All B cells start out with a 56R anti-DNA H chain. A few frankly autoreactive 56R lambda1 cells appear to escape into the periphery, but the majority of the peripheral B cell repertoire in 56R/kdel is made up of B cells expressing the 56R H chain with the lambdaX L chain. Surprisingly, 56R lambdaX B cells are multireactive, binding to a variety of self and nonself antigens, including dsDNA (albeit at reduced affinity compared with the other lambda L chains). Another significant population in the 56R/kdel mouse consists of allelically included B cells that express lambdaX along with another L chain. The multireactivity of both 56R lambdaX and 56R lambdaX/lambda1 receptors could contribute to autoimmunity if these B cells were to become activated. Also found among 56R/kdel hybridomas are clones that have inactivated the H chain and secrete only L chains. These clones may represent products of exhaustive rearrangement. Multireactivity, allelic inclusion, and L chain secretion are three consequences of editing at the lambda locus that may predispose toward the development of autoimmunity.

Alleles↗
PubMed2006

Development and selection of edited B cells in B6.56R mice.

Tolerance to dsDNA is broken in mice with a high-affinity anti-DNA H chain transgene, 56R, on the C57BL/6 background (B6.56R). B6.56R produce more anti-dsDNA Abs than BALBc.56R. To investigate how anti-DNA Abs are regulated on the B6 background, phenotypic and genetic studies were performed. B6.56R have reduced numbers of B cells and phenotypically altered B cell subsets, including relative increases in the proportions of IgM-negative bone marrow B cells, cells with a marginal zone phenotype, and cells with a transitional T3 phenotype. The peripheral B cell repertoire in B6.56R is restricted: most B cells express the 56R H chain and use a similar, limited subset of editor L chains. DNA binding is more common in B6.56R because the repertoire is shifted toward L chains that are more permissive for DNA binding. H chain editing is also observed and is increased in spontaneous as compared with LPS hybridomas. A subset of spontaneous hybridomas appears to lack H chain expression.

Animals↗
PubMed2006

Gamma radiation increases endonuclease-dependent L1 retrotransposition in a cultured cell assay.

Long Interspersed Elements (LINE-1s, L1s) are the most active mobile elements in the human genome and account for a significant fraction of its mass. The propagation of L1 in the human genome requires disruption and repair of DNA at the site of integration. As Barbara McClintock first hypothesized, genotoxic stress may contribute to the mobilization of transposable elements, and conversely, element mobility may contribute to genotoxic stress. We tested the ability of genotoxic agents to increase L1 retrotransposition in a cultured cell assay. We observed that cells exposed to gamma radiation exhibited increased levels of L1 retrotransposition. The L1 retrotransposition frequency was proportional to the number of phosphorylated H2AX foci, an indicator of genotoxic stress. To explore the role of the L1 endonuclease in this context, endonuclease-deficient tagged L1 constructs were produced and tested for their activity in irradiated cells. The activity of the endonuclease-deficient L1 was very low in irradiated cells, suggesting that most L1 insertions in irradiated cells still use the L1 endonuclease. Consistent with this interpretation, DNA sequences that flank L1 insertions in irradiated cells harbored target site duplications. These results suggest that increased L1 retrotransposition in irradiated cells is endonuclease dependent. The mobilization of L1 in irradiated cells potentially contributes to genomic instability and could be a driving force for secondary mutations in patients undergoing radiation therapy.

Aminoglycosides↗
PubMed2006

The potential regulation of L1 mobility by RNA interference.

The hypothesis that RNA interference constrains L1 mobility seems inherently reasonable: L1 mobility can be dangerous and L1 RNA, the presumed target of RNAi, serves as a critical retrotransposition intermediate. Despite its plausibility, proof for this hypothesis has been difficult to obtain. Studies attempting to link the L1 retrotransposition frequency to alterations in RNAi activity have been hampered by the long times required to measure retrotransposition frequency, the pleiotropic and toxic effects of altering RNAi over similar time periods, and the possibility that other cellular machinery may contribute to the regulation of L1s. Another problem is that the commonly used L1 reporter cassette may serve as a substrate for RNAi. Here we review the L1-RNAi hypothesis and describe a genetic assay with a modified reporter cassette that detects approximately 4 times more L1 insertions than the conventional retrotransposition assay.

Journal Article↗
PubMed2006

DNA damage and L1 retrotransposition.

Barbara McClintock was the first to suggest that transposons are a source of genome instability and that genotoxic stress assisted in their mobilization. The generation of double-stranded DNA breaks (DSBs) is a severe form of genotoxic stress that threatens the integrity of the genome, activates cell cycle checkpoints, and, in some cases, causes cell death. Applying McClintock's stress hypothesis to humans, are L1 retrotransposons, the most active autonomous mobile elements in the modern day human genome, mobilized by DSBs? Here, evidence that transposable elements, particularly retrotransposons, are mobilized by genotoxic stress is reviewed. In the setting of DSB formation, L1 mobility may be affected by changes in the substrate for L1 integration, the DNA repair machinery, or the L1 element itself. The review concludes with a discussion of the potential consequences of L1 mobilization in the setting of genotoxic stress.

Journal Article↗
PubMed2004

More active human L1 retrotransposons produce longer insertions.

The vast majority of L1 insertions are 5' truncated and thus inactive. Yet, the mechanism of 5' truncation is unknown. To examine whether the frequency of L1 retrotransposition is directly correlated with the length of genomic L1 insertions, we used a cell culture assay to measure retrotransposition frequency and a PCR-based assay to measure L1 insertion length. We tested five full-length human L1 elements that retrotranspose at different frequencies: LRE3, L1(RP), L1.3, L1.2A and L1.2B. Our data suggest that L1 insertion length correlates with L1 retrotransposition frequency for insertions >1 kb in length. For two elements, L1(RP) and L1.2A, we found that swapping the reverse transcriptase domains had little effect. Instead, we found that genomic insertion length and retrotransposition frequency are substantially affected by amino acid substitutions at positions 363, 1220 and 1259 in ORF2. We suggest that the region containing residues 1220 and 1259 may be important in the binding of ORF2p to L1 RNA to facilitate reverse transcription.

Amino Acid Sequence↗
PubMed2003

Tracking an embryonic L1 retrotransposition event.

Long interspersed nuclear elements 1 (L1) are active retrotransposons that reside in many species, including humans and rodents. L1 elements produce an RNA intermediate that is reverse transcribed to DNA and inserted in a new genomic location. We have tagged an active human L1 element (L1(RP)) with a gene encoding enhanced GFP (EGFP). Expression of GFP occurs only if L1-EGFP has undergone a cycle of transcription, reverse transcription, and integration into a transcriptionally permissive genomic region. We show here that L1-EGFP can undergo retrotransposition in vivo and produce fluorescence in mouse testis. The retrotransposition event characterized here has occurred at a very early stage in the development of an L1-EGFP transgenic founder mouse.

Animals↗