Transposable elements (TEs) shape genome evolution through periodic bursts of amplification. In this study prior knowledge of the mPing/Ping/Pong TE family is exploited to track their copy numbers and distribution in genome sequences from 3,000 accessions of domesticated Oryza sativa (rice) and the wild progenitor Oryza rufipogon. We find that mPing bursts are restricted to recent domestication and is likely due to the accumulation of two TE components, Ping16A and Ping16A_Stow, that appear to be critical for mPing hyperactivity. Ping16A is a variant of the autonomous element with reduced activity as shown in a yeast transposition assay. Transposition of Ping16A into a Stowaway element generated Ping16A_Stow, the only Ping locus shared by all bursting accessions, and shown here to correlate with high mPing copies. Finally, we show that sustained activity of the mPing/Ping family in domesticated rice produced the components necessary for mPing bursts, not the loss of epigenetic regulation.
Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing‐based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.
Cullin-2 (CUL2) based cullin-RING ligases (CRL2s) comprise a family of ubiquitin E3 ligases that exist only in multi-cellular organisms and are crucial for cellular processes such as embryogenesis and viral pathogenesis. CUL2 is the scaffold protein that binds one of the interchangeable substrate receptor modules, which consists of adaptor proteins and the substrate receptor protein. The VHL protein is a substrate receptor known to target hypoxia-inducible factor α (HIF1α) for ubiquitination and degradation. Because of its critical role in the ubiquitination of important cellular factors such as HIF1α, CRL2s have been investigated for their biological functions and the development of novel therapeutics against diseases. Given the importance of CRL2s in biological and biomedical research, methods that efficiently produce functional CUL2 proteins will greatly facilitate studies on the mechanism and regulation of CRL2s. Here, we report two cost-effective systems for the expression and purification of recombinant human CUL2 from E. coli cells. The purified CUL2 proteins were ~ 95% pure, could bind their substrate receptor modules, and were enzymatically active in transferring ubiquitin or ubiquitin-like protein to the corresponding substrate in in vitro assays. The presented methodological advancements will help advance research in CRL2 function and regulation.
Maintenance of protein homeostasis is crucial for virtually every aspect of eukaryotic biology. The ubiquitin-proteasome system (UPS) represents a highly regulated quality control machinery that protects cells from a variety of stress conditions as well as toxic proteins. A large body of evidence has shown that UPS dysfunction contributes to the pathogenesis of cardiovascular diseases. This review highlights the latest findings regarding the physiological and pathological roles of cullin-RING ubiquitin ligases (CRLs), an essential player in the UPS, in the cardiovascular system. To inspire potential therapeutic invention, factors regulating CRL activities are also discussed.
21produced the components necessary for the mPing burst, not the loss of epigenetic 36 regulation. 37 38 39 majority of TE bursts have been inferred after the fact -via computational analysis of 45 whole genome sequence -the stealth features they require for success have remained 46 largely undiscovered. 47Revealing these stealth features requires the identification of a TE in the midst of a 48 burst. This was accomplished for the miniature inverted-repeat transposable element 49 (MITE) mPing from rice 1,2 . MITEs are non-autonomous Class II (DNA) elements that are 50 the most common TE associated with the non-coding regions of plant genes 3 . To 51 understand how MITEs attain high copy numbers despite a preference for insertion into 52 genic regions, a computational approach was used to identify mPing, and its source of 53 transposase, encoded by the related autonomous Ping element ( Fig. 1a) 1 . 54Ongoing bursts of mPing were discovered in four temperate japonica strains: EG4, 55 HEG4, A119, and A123, whose genomes were sequenced and insertion sites and 56 epigenetic landscape determined 2,4,5 . These analyses uncovered two features of 57 successful bursts. First, mPing targets genic regions but avoids exon sequences, thus 58 minimizing harm to the host 2,5 . Second, because mPing does not share coding 59 sequences with Ping (Fig. 1a), increases in its copy number and host recognition of its 60 sequences does not silence Ping genes, thus allowing the continuous production of the 61 proteins necessary to sustain the burst for decades 4 . 62The contributions of two other features to the success of the bursts could not be 63 assessed previously and are the focus of this study. These features are a single SNP at 64 position 16 (+16G/A) that distinguishes mPing and Ping sequences (Fig. 1a), and a 65 single Ping locus (called Ping16A_Stow) that is the only Ping locus shared by all 66 bursting strains 4 . To understand the origin of these features and their possible role in 67 109 Copy number variation of mPing and Ping elements in domesticated and wild rice 110None of the 3,000 rice strains analyzed in this study have more mPing elements than 111 the 231-503 copies found in the four temperate japonica strains (HEG4, EG4, A119, 112 A123) in the midst of mPing bursts 4 . Of the 3,000 rice strains, 2,780 (92.7%) contain 113 (Table 1 and Supplementary Fig. 2). These data suggest that it is likely that Ping was 129 selected against or lost from most strains during the hypothesized two or more 130 domestication events from O. rufipogon populations 6,16 . 131 132 Origin of a Ping variant and its possible significance 133Analysis of the extensive collection of rice genomes revealed that a SNP distinguishing 134Ping and mPing (+16G/A) located adjacent to the 15-bp terminal inverted repeat (TIR) 135 ( Fig. 3a) and may be implicated in mPing bursts. Pings having these SNPs are referred 136
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