216 research outputs found
Great exaptations
Long interspersed nuclear elements (LINEs) are among the most successful parasitic genetic sequences in higher organisms. Recent work has discovered many instances of LINE incorporation into exons, reminding us of the hazards they pose to genes in their vicinity as well as their potential to be co-opted for the host's purposes
Effect of reverse transcriptase inhibitors on LINE-1 and Ty1 reverse transcriptase activities and on LINE-1 retrotransposition
<p>Abstract</p> <p>Background</p> <p>LINE-1s (L1, Long Interspersed Element-1) are the most abundant autonomous non-LTR retrotransposons in the human genome and replicate by reverse transcription of an RNA intermediate. Full-length L1 encodes two open reading frames (ORF1, ORF2) and ORF2 has reverse transcriptase activity.</p> <p>Results</p> <p>Here we expressed human L1 RT in <it>E. coli </it>and the purified protein displayed the same RT activity as that of ORF2p expressed in insect cells. We tested the effect of different reverse transcriptase inhibitors on L1 RT and found that all four tested nucleoside inhibitors efficiently inhibited L1 RT activity competitively. The K<sub>i </sub>values of NRTIs were calculated (AZTTP, 16.4 Β± 4.21 nM; d4TTP, 0.73 Β± 0.22 nM; ddCTP, 0.72 Β± 0.16 nM; 3TCTP, 12.9 Β± 2.07 nM). L1 RT was less sensitive to non-nucleoside reverse transcriptase inhibitors, among these nevirapine had no effect, even at concentrations up to 500 ΞΌM. We also examined the effect of RT inhibitors on L1 retrotransposition efficiency <it>in vivo </it>using a cell-based retrotransposition assay. Similarly, all analog inhibitors decreased L1 retrotransposition frequency with different potencies whereas nevirapine had little or no effect on L1 retrotransposition. For comparison, we also tested the same inhibitors to highly purified RT of an LTR-retrotransposon (Ty1) and found it was less sensitive to NRTIs than L1 RT and has the same inhibition profile as L1 RT to NNRTIs.</p> <p>Conclusions</p> <p>These data indicate that bacterially expressed L1 RT is an active reverse transcriptase sensitive to nucleoside RT inhibitors but not to non-nucleoside inhibitors.</p
Identifying related L1 retrotransposons by analyzing 3' transduced sequences
BACKGROUND: A large fraction of the human genome is attributable to L1 retrotransposon sequences. Not only do L1s themselves make up a significant portion of the genome, but L1-encoded proteins are thought to be responsible for the transposition of other repetitive elements and processed pseudogenes. In addition, L1s can mobilize non-L1, 3'-flanking DNA in a process called 3' transduction. Using computational methods, we collected DNA sequences from the human genome for which we have high confidence of their mobilization through L1-mediated 3' transduction. RESULTS: The precursors of L1s with transduced sequence can often be identified, allowing us to reconstruct L1 element families in which a single parent L1 element begot many progeny L1s. Of the L1s exhibiting a sequence structure consistent with 3' transduction (L1 with transduction-derived sequence, L1-TD), the vast majority were located in duplicated regions of the genome and thus did not necessarily represent unique insertion events. Of the remaining L1-TDs, some lack a clear polyadenylation signal, but the alignment between the parent-progeny sequences nevertheless ends in an A-rich tract of DNA. CONCLUSIONS: Sequence data suggest that during the integration into the genome of RNA representing an L1-TD, reverse transcription may be primed internally at A-rich sequences that lie downstream of the L1 3' untranslated region. The occurrence of L1-mediated transduction in the human genome may be less frequent than previously thought, and an accurate estimate is confounded by the frequent occurrence of segmental genomic duplications
Sc3.0: revamping and minimizing the yeast genome
From Springer Nature via Jisc Publications RouterHistory: registration 2020-08-04, pub-electronic 2020-08-13, online 2020-08-13, collection 2020-12Publication status: Publishe
Improved statistical analysis of budding yeast TAG microarrays revealed by defined spike-in pools
Saccharomyces cerevisiae knockout collection TAG microarrays are an emergent platform for rapid, genome-wide functional characterization of yeast genes. TAG arrays report abundance of unique oligonucleotide βTAGβ sequences incorporated into each deletion mutation of the yeast knockout collection, allowing measurement of relative strain representation across experimental conditions for all knockout mutants simultaneously. One application of TAG arrays is to perform genome-wide synthetic lethality screens, known as synthetic lethality analyzed by microarray (SLAM). We designed a fully defined spike-in pool to resemble typical SLAM experiments and performed TAG microarray hybridizations. We describe a method for analyzing two-color array data to efficiently measure the differential knockout strain representation across two experimental conditions, and use the spike-in pool to show that the sensitivity and specificity of this method exceed typical current approaches
Improved microarray methods for profiling the yeast knockout strain collection
A remarkable feature of the Yeast Knockout strain collection is the presence of two unique 20mer TAG sequences in almost every strain. In principle, the relative abundances of strains in a complex mixture can be profiled swiftly and quantitatively by amplifying these sequences and hybridizing them to microarrays, but TAG microarrays have not been widely used. Here, we introduce a TAG microarray design with sophisticated controls and describe a robust method for hybridizing high concentrations of dye-labeled TAGs in single-stranded form. We also highlight the importance of avoiding PCR contamination and provide procedures for detection and eradication. Validation experiments using these methods yielded false positive (FP) and false negative (FN) rates for individual TAG detection of 3β6% and 15β18%, respectively. Analysis demonstrated that cross-hybridization was the chief source of FPs, while TAG amplification defects were the main cause of FNs. The materials, protocols, data and associated software described here comprise a suite of experimental resources that should facilitate the use of TAG microarrays for a wide variety of genetic screens
Characterization of a synthetic human LINE-1 retrotransposon ORFeus-Hs
Long interspersed elements, type 1(LINE-1, L1) are the most abundant and only active autonomous retrotransposons in the human genome. Native L1 elements are inefficiently expressed because of a transcription elongation defect thought to be caused by high adenosine content in L1 sequences. Previously, we constructed a highly active synthetic mouse L1 element (ORFeus-Mm), partially by reducing the nucleotide composition bias. As a result, the transcript abundance of ORFeus-Mm was greatly increased, and its retrotransposition frequency was > 200-fold higher than its native counterpart. In this paper, we report a synthetic human L1 element (ORFeus-Hs) synthesized using a similar strategy. The adenosine content of the L1 open reading frames (ORFs) was reduced from 40% to 27% by changing 25% of the bases in the ORFs, without altering the amino acid sequence. By studying a series of native/synthetic chimeric elements, we observed increased levels of full-length L1 RNA and ORF1 protein and retrotransposition frequency, mostly proportional to increased fraction of synthetic sequence. Overall, the fully synthetic ORFeus-Hs has > 40-fold more RNA but is at most only ~threefold more active than its native counterpart (L1RP); however, its absolute retrotransposition activity is similar to ORFeus-Mm. Owing to the elevated expression of the L1 RNA/protein and its high retrotransposition ability, ORFeus-Hs and its chimeric derivatives will be useful tools for mechanistic L1 studies and mammalian genome manipulation
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