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1.  Methylated DNA is over-represented in whole-genome bisulfite sequencing data 
Frontiers in Genetics  2014;5:341.
The development of whole-genome bisulfite sequencing (WGBS) has resulted in a number of exciting discoveries about the role of DNA methylation leading to a plethora of novel testable hypotheses. Methods for constructing sodium bisulfite-converted and amplified libraries have recently advanced to the point that the bottleneck for experiments that use WGBS has shifted to data analysis and interpretation. Here we present empirical evidence for an over-representation of reads from methylated DNA in WGBS. This enrichment for methylated DNA is exacerbated by higher cycles of PCR and is influenced by the type of uracil-insensitive DNA polymerase used for amplifying the sequencing library. Future efforts to computationally correct for this enrichment bias will be essential to increasing the accuracy of determining methylation levels for individual cytosines. It is especially critical for studies that seek to accurately quantify DNA methylation levels in populations that may segregate for allelic DNA methylation states.
doi:10.3389/fgene.2014.00341
PMCID: PMC4204604  PMID: 25374580
DNA methylation; whole genome bisulfite sequencing; Epigenomics; epigenetics; PCR bias
2.  Genomic Distribution of H3K9me2 and DNA Methylation in a Maize Genome 
PLoS ONE  2014;9(8):e105267.
DNA methylation and dimethylation of lysine 9 of histone H3 (H3K9me2) are two chromatin modifications that can be associated with gene expression or recombination rate. The maize genome provides a complex landscape of interspersed genes and transposons. The genome-wide distribution of DNA methylation and H3K9me2 were investigated in seedling tissue for the maize inbred B73 and compared to patterns of these modifications observed in Arabidopsis thaliana. Most maize transposons are highly enriched for DNA methylation in CG and CHG contexts and for H3K9me2. In contrast to findings in Arabidopsis, maize CHH levels in transposons are generally low but some sub-families of transposons are enriched for CHH methylation and these families exhibit low levels of H3K9me2. The profile of modifications over genes reveals that DNA methylation and H3K9me2 is quite low near the beginning and end of genes. Although elevated CG and CHG methylation are found within gene bodies, CHH and H3K9me2 remain low. Maize has much higher levels of CHG methylation within gene bodies than observed in Arabidopsis and this is partially attributable to the presence of transposons within introns for some maize genes. These transposons are associated with high levels of CHG methylation and H3K9me2 but do not appear to prevent transcriptional elongation. Although the general trend is for a strong depletion of H3K9me2 and CHG near the transcription start site there are some putative genes that have high levels of these chromatin modifications. This study provides a clear view of the relationship between DNA methylation and H3K9me2 in the maize genome and how the distribution of these modifications is shaped by the interplay of genes and transposons.
doi:10.1371/journal.pone.0105267
PMCID: PMC4133378  PMID: 25122127
3.  DNA Topoisomerase 1α Promotes Transcriptional Silencing of Transposable Elements through DNA Methylation and Histone Lysine 9 Dimethylation in Arabidopsis 
PLoS Genetics  2014;10(7):e1004446.
RNA-directed DNA methylation (RdDM) and histone H3 lysine 9 dimethylation (H3K9me2) are related transcriptional silencing mechanisms that target transposable elements (TEs) and repeats to maintain genome stability in plants. RdDM is mediated by small and long noncoding RNAs produced by the plant-specific RNA polymerases Pol IV and Pol V, respectively. Through a chemical genetics screen with a luciferase-based DNA methylation reporter, LUCL, we found that camptothecin, a compound with anti-cancer properties that targets DNA topoisomerase 1α (TOP1α) was able to de-repress LUCL by reducing its DNA methylation and H3K9me2 levels. Further studies with Arabidopsis top1α mutants showed that TOP1α silences endogenous RdDM loci by facilitating the production of Pol V-dependent long non-coding RNAs, AGONAUTE4 recruitment and H3K9me2 deposition at TEs and repeats. This study assigned a new role in epigenetic silencing to an enzyme that affects DNA topology.
Author Summary
DNA topoisomerase is an enzyme that releases the torsional stress in DNA generated during DNA replication or transcription. Here, we uncovered an unexpected role of DNA topoisomerase 1α (TOP1α) in the maintenance of genome stability. Eukaryotic genomes are usually littered with transposable elements (TEs) and repeats, which pose threats to genome stability due to their tendency to move or recombine. Mechanisms are in place to silence these elements, such as RNA-directed DNA methylation (RdDM) and histone H3 lysine 9 dimethylation (H3K9me2) in plants. Two plant-specific RNA polymerases, Pol IV and Pol V, generate small and long noncoding RNAs, respectively, from TEs and repeats. These RNAs then recruit protein factors to deposit DNA methylation or H3K9me2 to silence the loci. In this study, we found that treatment of plants with camptothecin, a TOP1α inhibitor, or loss of function in TOP1α, led to the de-repression of RdDM target loci, which was accompanied by loss of H3K9me2 or DNA methylation. The role of TOP1α in RdDM could be attributed to its promotion of Pol V, but not Pol IV, transcription to generate long noncoding RNAs.
doi:10.1371/journal.pgen.1004446
PMCID: PMC4080997  PMID: 24992598
4.  Sigma factor-mediated plastid retrograde signals control nuclear gene expression 
SUMMARY
Retrograde signalling from plastids to the nucleus is necessary to regulate the organelle’s proteome during the establishment of photoautotrophy and fluctuating environmental conditions. Studies that used inhibitors of chloroplast biogenesis have revealed that hundreds of nuclear genes are regulated by retrograde signals emitted from plastids. Plastid gene expression is the source of at least one of these signals, but the number of signals and their mechanisms used to regulate nuclear gene expression are unknown. To further examine the effects of plastid gene expression on nuclear gene expression, we analyzed Arabidopsis mutants that were defective in each of the six sigma factor (SIG) genes that encode proteins utilized by plastid-encoded RNA polymerase to transcribe specific sets of plastid genes. We showed that SIG2 and SIG6 have partially redundant roles in plastid transcription and retrograde signalling to control nuclear gene expression. The loss of GUN1 (a plastid-localized pentatricopeptide repeat protein) is able to restore nuclear (but not plastid) gene expression in both sig2 and sig6, whereas an increase in heme synthesis is able to restore nuclear gene expression in sig2 mutants only. These results demonstrate that sigma factor function is the source of at least two retrograde signals to the nucleus; one likely to involve the transcription of tRNAGlu. A microarray analysis showed that these two signals accounted for at least one subset of the nuclear genes that are regulated by the plastid biogenesis inhibitors norflurazon and lincomycin. Together these data suggest that such inhibitors can induce retrograde signalling by affecting transcription in the plastid.
doi:10.1111/tpj.12011
PMCID: PMC3605210  PMID: 22950756
chloroplast; retrograde signalling; transcription; tetrapyrroles; sigma factors; photosynthesis
5.  Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis 
eLife  2013;2:e00675.
The gaseous plant hormone ethylene regulates a multitude of growth and developmental processes. How the numerous growth control pathways are coordinated by the ethylene transcriptional response remains elusive. We characterized the dynamic ethylene transcriptional response by identifying targets of the master regulator of the ethylene signaling pathway, ETHYLENE INSENSITIVE3 (EIN3), using chromatin immunoprecipitation sequencing and transcript sequencing during a timecourse of ethylene treatment. Ethylene-induced transcription occurs in temporal waves regulated by EIN3, suggesting distinct layers of transcriptional control. EIN3 binding was found to modulate a multitude of downstream transcriptional cascades, including a major feedback regulatory circuitry of the ethylene signaling pathway, as well as integrating numerous connections between most of the hormone mediated growth response pathways. These findings provide direct evidence linking each of the major plant growth and development networks in novel ways.
DOI: http://dx.doi.org/10.7554/eLife.00675.001
eLife digest
All multicellular organisms, including plants, produce hormones—chemical messengers that are released in one part of an organism but act in another. The binding of hormones to receptor proteins on the surface of target cells activates signal transduction cascades, leading ultimately to changes in the transcription and translation of genes.
Ethylene is a gaseous plant hormone that acts at trace levels to stimulate or regulate a variety of processes, including the regulation of plant growth, the ripening of fruit and the shedding of leaves. Plants also produce ethylene in response to wounding, pathogen attack or exposure to environmental stresses, such as extreme temperatures or drought. Although the effects of ethylene on plants are well documented, much less is known about how its functions are controlled and coordinated at the molecular level.
Here, Chang et al. reveal how ethylene alters the transcription of DNA into messenger DNA (mRNA) in the plant model organism, Arabidopsis thaliana. Ethylene is known to exert some of its effects via a protein called EIN3, which is a transcription factor that acts as the master regulator of the ethylene signaling pathway. To identify the targets of EIN3, Chang et al. exposed plants to ethylene and then used a technique called ChIP-Seq to identify those regions of the DNA that EIN3 binds to. At the same time, they used genome-wide mRNA sequencing to determine which genes showed altered transcription.
Over the course of 24 hr, ethylene induced four distinct waves of transcription, suggesting that discrete layers of transcriptional control are present. EIN3 binding also controlled a multitude of downstream transcriptional cascades, including a major negative feedback loop. Surprisingly, many of the genes that showed altered expression in response to EIN3 binding were also influenced by hormones other than ethylene.
In addition to extending our knowledge of the role of EIN3 in coordinating the effects of ethylene, the work of Chang et al. reveals the extensive connectivity between pathways regulated by distinct hormones in plants. The results may also make it easier to identify key players involved in hormone signaling pathways in other plant species.
DOI: http://dx.doi.org/10.7554/eLife.00675.002
doi:10.7554/eLife.00675
PMCID: PMC3679525  PMID: 23795294
transcriptional regulation; temporal modulation; network; ethylene; hormone; Physcomitrella patens; Arabidopsis
6.  Circadian oscillations of protein-coding and regulatory RNAs in a highly dynamic mammalian liver epigenome 
Cell metabolism  2012;16(6):833-845.
In the mouse liver, circadian transcriptional rhythms are necessary for metabolic homeostasis. Whether dynamic epigenomic modifications are associated with transcript oscillations has not been systematically investigated. We found in addition to mRNAs, several antisense-, linc- and micro-RNA transcripts showed circadian oscillations in adult mouse livers. Robust transcript oscillations were often accompanied by temporally correlated rhythmic histone modifications in promoters, gene bodies or enhancers, although promoter DNA methylation levels were relatively stable. Such integrative analyses identified oscillating expression of a previously undetected antisense transcript (asPer2) to the gene encoding the circadian oscillator component Per2. Robustness of transcript oscillations often accompanied rhythms in multiple histone modifications and recruitment of multiple chromatin-associated clock components. In summary, coupling of the locations of cycling histone modifications with one or more oscillating transcripts within their proximity enabled establishment of a temporal relationship between enhancers, genes and transcripts on a genome-wide, base-resolution scale in a mammalian liver. The results offer a framework to understand intricate dynamic regulation among metabolism, circadian clock, and chromatin modifications to maintain metabolic homeostasis.
doi:10.1016/j.cmet.2012.11.004
PMCID: PMC3541940  PMID: 23217262
7.  ‘Leveling’ the playing field for analyses of single-base resolution DNA methylomes 
Trends in genetics : TIG  2012;28(12):583-585.
doi:10.1016/j.tig.2012.10.012
PMCID: PMC3523709  PMID: 23131467
DNA methylation; bisulfite sequencing; methylation level; epigenomics
8.  Processing and Subcellular Trafficking of ER-Tethered EIN2 Control Response to Ethylene Gas 
Science (New York, N.Y.)  2012;338(6105):390-393.
Ethylene gas is essential for many developmental processes and stress responses in plants. ETHYLENE INSENSITIVE2 (EIN2), an NRAMP-like integral membrane protein, plays an essential role in ethylene signaling, but its function remains enigmatic. Here we report that phosphorylation-regulated proteolytic processing of EIN2 triggers its endoplasmic reticulum (ER)–to–nucleus translocation. ER-tethered EIN2 shows CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) kinase–dependent phosphorylation. Ethylene triggers dephosphorylation at several sites and proteolytic cleavage at one of these sites, resulting in nuclear translocation of a carboxyl-terminal EIN2 fragment (EIN2-C′). Mutations that mimic EIN2 dephosphorylation, or inactivate CTR1, show constitutive cleavage and nuclear localization of EIN2-C′ and EIN3 and EIN3-LIKE1–dependent activation of ethylene responses. These findings uncover a mechanism of subcellular communication whereby ethylene stimulates phosphorylation-dependent cleavage and nuclear movement of the EIN2-C′ peptide, linking hormone perception and signaling components in the ER with nuclear-localized transcriptional regulators.
doi:10.1126/science.1225974
PMCID: PMC3523706  PMID: 22936567
9.  Patterns of Population Epigenomic Diversity 
Nature  2013;495(7440):193-198.
Summary
Natural epigenetic variation provides a source for the generation of phenotypic diversity, but to understand its contribution to phenotypic diversity, its interaction with genetic variation requires further investigation. Here, we report population-wide DNA sequencing of genomes, transcriptomes, and methylomes of wild Arabidopsis thaliana accessions. Single cytosine methylation polymorphisms are unlinked to genotype. However, the rate of linkage disequilibrium decay amongst differentially methylated regions targeted by RNA-directed DNA methylation is similar to the rate for single nucleotide polymorphisms. Association analyses of these RNA-directed DNA methylation regions with genetic variants identified thousands of methylQTL, which revealed the first population estimate of genetically dependent methylation variation. Analysis of invariably methylated transposons and genes across this population indicates that loci targeted by RNA-directed DNA methylation are epigenetically activated in pollen and seeds, which facilitates proper development of these structures.
doi:10.1038/nature11968
PMCID: PMC3798000  PMID: 23467092
10.  Response to Perspective  
Plant Signaling & Behavior  2013;8(8):e25037.
Cooper questions one specific technical aspect of our study—the site of cleavage in EIN2—and suggests that cleavage of EIN2 likely occurs elsewhere. Here, we explain how our immunoblotting, mass spectrometry and genetic mutation studies justify our conclusions.
doi:10.4161/psb.25037
PMCID: PMC3999072  PMID: 23733051
11.  High-throughput approaches for plant epigenomic studies 
Current opinion in plant biology  2011;14(2):130-136.
In plant cells, DNA is packaged into chromatin by wrapping around histone octamers. Pathways that lead to cytosine DNA methylation, posttranslational histone modifications and certain components of the RNA interfering (RNAi) pathway are critically important in modulating chromatin structure, thereby affecting many molecular processes that take place in a cell. Recent advances in microarray and high-throughput sequencing technologies have made it possible to study these pathways on a genome-wide scale. Results from such epigenomic studies are broadening our understanding of plant genomes and are also providing important clues regarding the mechanisms and functions of these pathways that can be further tested using genetic and biochemical approaches. This review focuses on the high-throughput approaches that have been successfully applied in plant epigenomic studies.
doi:10.1016/j.pbi.2011.03.010
PMCID: PMC3112054  PMID: 21470901
12.  RF1 Knockout Allows Ribosomal Incorporation of Unnatural Amino Acids at Multiple Sites 
Nature Chemical Biology  2011;7(11):779-786.
Stop codons have been exploited for genetic incorporation of unnatural amino acids (Uaas) in live cells, but the efficiency is low possibly due to competition from release factors, limiting the power and scope of this technology. Here we show that the reportedly essential release factor 1 can be knocked out from Escherichia coli by fixing release factor 2. The resultant strain JX33 is stable and independent, and reassigns UAG from a stop signal to an amino acid when a UAG-decoding tRNA/synthetase pair is introduced. Uaas were efficiently incorporated at multiple UAG sites in the same gene without translational termination in JX33. We also found that amino acid incorporation at endogenous UAG codons is dependent on RF1 and mRNA context, which explains why E. coli tolerates apparent global suppression of UAG. JX33 affords a unique autonomous host for synthesizing and evolving novel protein functions by enabling Uaa incorporation at multiple sites.
doi:10.1038/nchembio.657
PMCID: PMC3201715  PMID: 21926996
13.  Surveillance of 3′ Noncoding Transcripts Requires FIERY1 and XRN3 in Arabidopsis 
G3: Genes|Genomes|Genetics  2012;2(4):487-498.
Eukaryotes possess several RNA surveillance mechanisms that prevent undesirable aberrant RNAs from accumulating. Arabidopsis XRN2, XRN3, and XRN4 are three orthologs of the yeast 5′-to-3′ exoribonuclease, Rat1/Xrn2, that function in multiple RNA decay pathways. XRN activity is maintained by FIERY1 (FRY1), which converts the XRN inhibitor, adenosine 3′, 5′-bisphosphate (PAP), into 5′AMP. To identify the roles of XRNs and FRY1 in suppression of non-coding RNAs, strand-specific genome-wide tiling arrays and deep strand-specific RNA-Seq analyses were carried out in fry1 and xrn single and double mutants. In fry1-6, about 2000 new transcripts were identified that extended the 3′ end of specific mRNAs; many of these were also observed in genotypes that possess the xrn3-3 mutation, a partial loss-of-function allele. Mutations in XRN2 and XRN4 in combination with xrn3-3 revealed only a minor effect on 3′ extensions, indicating that these genes may be partially redundant with XRN3. We also observed the accumulation of 3′ remnants of many DCL1-processed microRNA (miRNA) precursors in fry1-6 and xrn3-3. These findings suggest that XRN3, in combination with FRY1, is required to prevent the accumulation of 3′ extensions that arise from thousands of mRNA and miRNA precursor transcripts.
doi:10.1534/g3.111.001362
PMCID: PMC3337477  PMID: 22540040
FRY1; XRN3; genome-wide; Arabidopsis; methylation
14.  Transgenerational Epigenetic Instability Is a Source of Novel Methylation Variants 
Science (New York, N.Y.)  2011;334(6054):369-373.
Epigenetic information, which may affect an organisms’ phenotype, can be stored and stably inherited in the form of cytosine DNA methylation. Changes in DNA methylation can produce meiotically stable epialleles that affect transcription and morphology, but the rates of spontaneous gain or loss of DNA methylation are unknown. We examined spontaneously occurring variation in DNA methylation in Arabidopsis thaliana plants propagated by single-seed descent for 30 generations. 114,287 CG single methylation polymorphisms (SMPs) and 2485 CG differentially methylated regions (DMRs) were identified, both of which show patterns of divergence compared to the ancestral state. Thus, transgenerational epigenetic variation in DNA methylation may generate new allelic states that alter transcription providing a mechanism for phenotypic diversity in the absence of genetic mutation.
doi:10.1126/science.1212959
PMCID: PMC3210014  PMID: 21921155
15.  Unexpected Diversity of Chloroplast Noncoding RNAs as Revealed by Deep Sequencing of the Arabidopsis Transcriptome 
G3: Genes|Genomes|Genetics  2011;1(7):559-570.
Noncoding RNAs (ncRNA) are widely expressed in both prokaryotes and eukaryotes. Eukaryotic ncRNAs are commonly micro- and small-interfering RNAs (18–25 nt) involved in posttranscriptional gene silencing, whereas prokaryotic ncRNAs vary in size and are involved in various aspects of gene regulation. Given the prokaryotic origin of organelles, the presence of ncRNAs might be expected; however, the full spectrum of organellar ncRNAs has not been determined systematically. Here, strand-specific RNA-Seq analysis was used to identify 107 candidate ncRNAs from Arabidopsis thaliana chloroplasts, primarily encoded opposite protein-coding and tRNA genes. Forty-eight ncRNAs were shown to accumulate by RNA gel blot as discrete transcripts in wild-type (WT) plants and/or the pnp1-1 mutant, which lacks the chloroplast ribonuclease polynucleotide phosphorylase (cpPNPase). Ninety-eight percent of the ncRNAs detected by RNA gel blot had different transcript patterns between WT and pnp1-1, suggesting cpPNPase has a significant role in chloroplast ncRNA biogenesis and accumulation. Analysis of materials deficient for other major chloroplast ribonucleases, RNase R, RNase E, and RNase J, showed differential effects on ncRNA accumulation and/or form, suggesting specificity in RNase-ncRNA interactions. 5′ end mapping demonstrates that some ncRNAs are transcribed from dedicated promoters, whereas others result from transcriptional read-through. Finally, correlations between accumulation of some ncRNAs and the symmetrically transcribed sense RNA are consistent with a role in RNA stability. Overall, our data suggest that this extensive population of ncRNAs has the potential to underpin a previously underappreciated regulatory mode in the chloroplast.
doi:10.1534/g3.111.000752
PMCID: PMC3276175  PMID: 22384367
RNA-Seq; posttranscriptional regulation; transcription; organelle; plastid
16.  Brahma Is Required for Proper Expression of the Floral Repressor FLC in Arabidopsis 
PLoS ONE  2011;6(3):e17997.
Background
BRAHMA (BRM) is a member of a family of ATPases of the SWI/SNF chromatin remodeling complexes from Arabidopsis. BRM has been previously shown to be crucial for vegetative and reproductive development.
Methodology/Principal Findings
Here we carry out a detailed analysis of the flowering phenotype of brm mutant plants which reveals that, in addition to repressing the flowering promoting genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), BRM also represses expression of the general flowering repressor FLOWERING LOCUS C (FLC). Thus, in brm mutant plants FLC expression is elevated, and FLC chromatin exhibits increased levels of histone H3 lysine 4 tri-methylation and decreased levels of H3 lysine 27 tri-methylation, indicating that BRM imposes a repressive chromatin configuration at the FLC locus. However, brm mutants display a normal vernalization response, indicating that BRM is not involved in vernalization-mediated FLC repression. Analysis of double mutants suggests that BRM is partially redundant with the autonomous pathway. Analysis of genetic interactions between BRM and the histone H2A.Z deposition machinery demonstrates that brm mutations overcome a requirement of H2A.Z for FLC activation suggesting that in the absence of BRM, a constitutively open chromatin conformation renders H2A.Z dispensable.
Conclusions/Significance
BRM is critical for phase transition in Arabidopsis. Thus, BRM represses expression of the flowering promoting genes CO, FT and SOC1 and of the flowering repressor FLC. Our results indicate that BRM controls expression of FLC by creating a repressive chromatin configuration of the locus.
doi:10.1371/journal.pone.0017997
PMCID: PMC3061888  PMID: 21445315
17.  The Role of VIN3-LIKE Genes in Environmentally Induced Epigenetic Regulation of Flowering 
Plant Signaling & Behavior  2007;2(2):127-128.
Given their sessile nature, it is critical for the survival of plants to adapt to their environment. Accordingly, plants have evolved the ability to sense seasonal changes to govern developmental fates such as the floral transition. Temperature and day length are among the seasonal cues that plants sense. We recently reported that VIN3-LIKE 1 (VIL1) is involved in mediating the flowering response to both cold and day length via regulation of two related genes, FLOWERING LOCUS C (FLC) and FLOWERING LOCUS M (FLM), respectively.
PMCID: PMC2633917  PMID: 19704758
flowering; vernalization; photoperiod; chromatin; histone; gene expression
18.  Voluntary Partial Capitation: The Community Nursing Organization Medicare Demonstration 
Health Care Financing Review  2005;26(4):21-37.
In a recently concluded Medicare demonstration, Community Nursing Organizations (CNOs) received capitated payment to provide a subset of Medicare services through a nursing case management delivery system. Demonstration participation was voluntary, both for CNOs and recruited beneficiaries, raising several challenging issues associated with selection. We investigate provider and beneficiary selection, as well as Medicare costs, using multiple evaluation methodologies. We find that CNO enrollment is associated with increased payment by Medicare for CNO-covered services. Results showing CNO enrollees to be more costly to Medicare for non-CNO services are consistent with cost shifting, but could also be accounted for by biased provider selection into the demonstration.
PMCID: PMC4194914  PMID: 17288066
19.  Contemplating Home Health PPS: Current Patterns of Medicare Service Use 
Health Care Financing Review  1994;16(1):109-130.
Implementing a per-episode prospective payment system (PPS) for home health services is one option for Medicare policy makers facing rapid increases in service use and expenditures. Analysis of data on recent episodes of Medicare home health care identified systematic differences in service patterns across provider types; these indicate potential differences in the capacity of agencies of different types to adjust to PPS. The second phase of a national demonstration, which is about to be implemented, will provide information on the extent to which the agency practices that generate much of the observed variation (such as the number of visits provided per episode) are susceptible to management decisions; and whether managers can and do respond to the incentives of per-episode prospective payment.
PMCID: PMC4193476  PMID: 10140150
20.  Release Factor One Is Nonessential in Escherichia coli 
ACS Chemical Biology  2012;7(8):1337-1344.
Recoding a stop codon to an amino acid may afford orthogonal genetic systems for biosynthesizing new protein and organism properties. Although reassignment of stop codons has been found in extant organisms, a model organism is lacking to investigate the reassignment process and to direct code evolution. Complete reassignment of a stop codon is precluded by release factors (RFs), which recognize stop codons to terminate translation. Here we discovered that RF1 could be unconditionally knocked out from various Escherichia coli stains, demonstrating that the reportedly essential RF1 is generally dispensable for the E. coli species. The apparent essentiality of RF1 was found to be caused by the inefficiency of a mutant RF2 in terminating all UAA stop codons; a wild type RF2 was sufficient for RF1 knockout. The RF1-knockout strains were autonomous and unambiguously reassigned UAG to encode natural or unnatural amino acids (Uaas) at multiple sites, affording a previously unavailable model for studying code evolution and a unique host for exploiting Uaas to evolve new biological functions.
doi:10.1021/cb300229q
PMCID: PMC3423824  PMID: 22662873

Results 1-20 (20)