Method for efficient soluble expression and purification of recombinant hyperactive Tn5 transposase.

Efficient preparation of libraries is the key step of next-generation sequencing (NGS) methods. Tn5 transposase enables simple, robust and highly efficient tagmentation-based library construction. Here, we report a simple and reliable expression and purification strategy based on fusing Tn5 to the small B1 immunoglobulin binding domain of Streptococcal protein G (GB1) and high affinity 10× His tag. The purified recombinant Tn5 showed high DNA tagmentation activity and ultra-low nucleic acid contamination. This method greatly cuts the costs of Tn5-based NGS library construction and is beneficial to the development of new NGS methods.

Cotranslational control of DNA transposition: a window of opportunity.

Transposable elements are important in genome dynamics and evolution. Bacterial insertion sequences (IS) constitute a major group in number and impact. Understanding their role in shaping genomes requires knowledge of how their transposition activity is regulated and interfaced with the host cell. One IS regulatory phenomenon is a preference of their transposases (Tpases) for action on the element from which they are expressed (cis) rather than on other copies of the same element (trans). Using IS911, we show in vivo that activity in cis was ~200 fold higher than in trans. We also demonstrate that a translational frameshifting pause signal influences cis preference presumably by facilitating sequential folding and cotranslational binding of the Tpase. In vitro, IS911 Tpase bound IS ends during translation but not after complete translation. Cotranslational binding of nascent Tpase permits tight control of IS proliferation providing a mechanistic explanation for cis regulation of transposition involving an unexpected partner, the ribosome.

Kicking against the PRCs - A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2.

The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits PcG silencing by blocking the interaction of the core PRC2 with accessory components that promote its HMTase activity or its role in inhibiting transcription. ALP1 is the first example of a domesticated transposase acquiring a novel function as a PcG component. The antagonistic interaction of a modified transposase with the PcG machinery is novel and may have arisen as a means for the cognate transposon to evade host surveillance or for the host to exploit features of the transposition machinery beneficial for epigenetic regulation of gene activity.

A cis-encoded sRNA, Hfq and mRNA secondary structure act independently to suppress IS200 transposition.

IS200 is found throughout Enterobacteriaceae and transposes at a notoriously low frequency. In addition to the transposase protein (TnpA), IS200 encodes an uncharacterized Hfq-binding sRNA that is encoded opposite to the tnpA 5'UTR. In the current work we asked if this sRNA represses tnpA expression. We show here that the IS200 sRNA (named art200 for antisense regulator of transposase IS200) basepairs with tnpA to inhibit translation initiation. Unexpectedly, art200-tnpA pairing is limited to 40 bp, despite 90 nt of perfect complementarity. Additionally, we show that Hfq and RNA secondary structure in the tnpA 5'UTR each repress tnpA expression in an art200-independent manner. Finally, we show that disrupting translational control of tnpA expression leads to increased IS200 transposition in E. coli. The current work provides new mechanistic insight into why IS200 transposition is so strongly suppressed. The possibility of art200 acting in trans to regulate a yet-unidentified target is discussed as well as potential applications of the IS200 system for designing novel riboregulators.

Hfq binds directly to the ribosome-binding site of IS10 transposase mRNA to inhibit translation.

Hfq is a critical component of post-transcriptional regulatory networks in most bacteria. It usually functions as a chaperone for base-pairing small RNAs, although non-canonical regulatory roles are continually emerging. We have previously shown that Hfq represses IS10/Tn10 transposase expression through both antisense RNA-dependent and independent mechanisms. In the current work, we set out to define the regulatory role of Hfq in the absence of the IS10 antisense RNA. We show here that an interaction between the distal surface of Hfq and the ribosome-binding site of transposase mRNA (RNA-IN) is required for repressing translation initiation. Additionally, this interaction was critical for the in vivo association of Hfq and RNA-IN. Finally, we present evidence that the small RNA ChiX activates transposase expression by titrating Hfq away from RNA-IN. The current results are considered in the broader context of Hfq biology and implications for Hfq titration by ChiX are discussed.

The periodontal pathogen Porphyromonas gingivalis induces expression of transposases and cell death of Streptococcus mitis in a biofilm model.

Oral microbial communities are extremely complex biofilms with high numbers of bacterial species interacting with each other (and the host) to maintain homeostasis of the system. Disturbance in the oral microbiome homeostasis can lead to either caries or periodontitis, two of the most common human diseases. Periodontitis is a polymicrobial disease caused by the coordinated action of a complex microbial community, which results in inflammation of tissues that support the teeth. It is the most common cause of tooth loss among adults in the United States, and recent studies have suggested that it may increase the risk for systemic conditions such as cardiovascular diseases. In a recent series of papers, Hajishengallis and coworkers proposed the idea of the "keystone-pathogen" where low-abundance microbial pathogens (Porphyromonas gingivalis) can orchestrate inflammatory disease by turning a benign microbial community into a dysbiotic one. The exact mechanisms by which these pathogens reorganize the healthy oral microbiome are still unknown. In the present manuscript, we present results demonstrating that P. gingivalis induces S. mitis death and DNA fragmentation in an in vitro biofilm system. Moreover, we report here the induction of expression of multiple transposases in a Streptococcus mitis biofilm when the periodontopathogen P. gingivalis is present. Based on these results, we hypothesize that P. gingivalis induces S. mitis cell death by an unknown mechanism, shaping the oral microbiome to its advantage.

The interplay of mRNA stimulatory signals required for AUU-mediated initiation and programmed -1 ribosomal frameshifting in decoding of transposable element IS911.

The IS911 bacterial transposable element uses -1 programmed translational frameshifting to generate the protein required for its mobility: translation initiated in one gene (orfA) shifts to the -1 frame and continues in a second overlapping gene (orfB), thus generating the OrfAB transposase. The A-AAA-AAG frameshift site of IS911 is flanked by two stimulatory elements, an upstream Shine-Dalgarno sequence and a downstream stem-loop. We show here that, while they can act independently, these stimulators have a synergistic effect when combined. Mutagenic analyses revealed features of the complex stem-loop that make it a low-efficiency stimulator. They also revealed the dual role of the upstream Shine-Dalgarno sequence as (i) a stimulator of frameshifting, by itself more potent than the stem-loop, and (ii) a mandatory determinant of initiation of OrfB protein synthesis on an AUU codon directly preceding the A6G motif. Both roles rely on transient base pairing of the Shine-Dalgarno sequence with the 3' end of 16S rRNA. Because of its effect on frameshifting, the Shine-Dalgarno sequence is an important determinant of the level of transposase in IS911-containing cells, and hence of the frequency of transposition.

High-level genomic integration, epigenetic changes, and expression of sleeping beauty transgene.

Sleeping Beauty transposon (SB-Tn) has emerged as an important nonviral vector for integrating transgenes into mammalian genomes. We report here a novel dual fluorescent reporter cis SB-Tn system that permitted nonselective fluorescent-activated cell sorting for SB-Tn-transduced K562 erythroid cells. Using an internal ribosome entry site element, the green fluorescent protein (eGFP) was linked to the SB10 transposase gene as an indirect marker for the robust expression of SB10 transposase. Flourescence-activated cell sorting (FACS) by eGFP resulted in significant enrichment (>60%) of cells exhibiting SB-Tn-mediated genomic insertions and long-term expression of a DsRed transgene. The hybrid erythroid-specific promoter of DsRed transgene was verified in erythroid or megakaryocyte differentiation of K562 cells. Bisulfite-mediated genomic analyses identified different DNA methylation patterns between DsRed(+) and DsRed(-) cell clones, suggesting a critical role in transgene expression. Moreover, although the host genomic copy of the promoter element showed no CpG methylation, the same sequence carried by the transgene was markedly hypermethylated. Additional evidence also suggested a role for histone deacetylation in the regulation of DsRed transgene. The presence of SB transgene affected the expression of neighboring host genes at distances >45 kb. Our data suggested that a fluorescent reporter cis SB-Tn system can be used to enrich mammalian cells harboring SB-mediated transgene insertions. The observed epigenetic changes also demonstrated that transgenes inserted by SB could be selectively modified by endogenous factors. In addition, long-range activation of host genes must now be recognized as a potential consequence of an inserted transgene cassette containing enhancer elements.

Generation of an inducible and optimized piggyBac transposon system.

Genomic studies in the mouse have been slowed by the lack of transposon-mediated mutagenesis. However, since the resurrection of Sleeping Beauty (SB), the possibility of performing forward genetics in mice has been reinforced. Recently, piggyBac (PB), a functional transposon from insects, was also described to work in mammals. As the activity of PB is higher than that of SB11 and SB12, two hyperactive SB transposases, we have characterized and improved the PB system in mouse ES cells. We have generated a mouse codon-optimized version of the PB transposase coding sequence (CDS) which provides transposition levels greater than the original. We have also found that the promoter sequence predicted in the 5'-terminal repeat of the PB transposon is active in the mammalian context. Finally, we have engineered inducible versions of the optimized piggyBac transposase fused with ERT2. One of them, when induced, provides higher levels of transposition than the native piggyBac CDS, whereas in the absence of induction its activity is indistinguishable from background. We expect that these tools, adaptable to perform mouse-germline mutagenesis, will facilitate the identification of genes involved in pathological and physiological processes, such as cancer or ES cell differentiation.

Benomyl induction of brain aromatase and toxic effects in the zebrafish embryo.

Benomyl is a benzimidazole fungicide that has been widely used on a variety of food crops and ornamental plants. It is known to cause adverse effects on reproductive systems, including decreased testicular and epididymal weights and reduced epididymal sperm counts and fertility. The brain aromatase gene is up-regulated by estrogens and estrogen mimics and considered a target gene to screen estrogen mimics. This study was designed to test the estrogenic potential and toxic effects of benomyl in the zebrafish system, and validated this system as a model that may correspond to the effect of benomyl in rodents. Concentrations of 20 x 10(-6), 40 x 10(-6) and 80 x 10(-6) M of benomyl-treated embryos showed decreased survival, hatching and heart rates, and increased incidence of malformations, such as pericardial edema, spinal lordosis, elongated heart, head edema, eye lens protrusion and caudal fin disappearance. Benomyl induced enhanced green fluorescent protein (EGFP) expression in the mediobasal hypothalamus (MBH) in transient zebrafish embryos with a brain aromatase-based reporter gene. In this study, we determined that benomyl has estrogenic potential based on zebrafish brain aromatase gene induction, and that benomyl is toxic at 20 x 10(-6) M concentration and higher. These results demonstrate the usefulness of zebrafish embryos as an in vivo system to examine the estrogenic and developmental toxic potential of unknown compounds.

A meta-analysis of multiple matched aCGH/expression cancer datasets reveals regulatory relationships and pathway enrichment of potential oncogenes.

The copy numbers of genes in cancer samples are often highly disrupted and form a natural amplification/deletion experiment encompassing multiple genes. Matched array comparative genomics and transcriptomics datasets from such samples can be used to predict inter-chromosomal gene regulatory relationships. Previously we published the database METAMATCHED, comprising the results from such an analysis of a large number of publically available cancer datasets. Here we investigate genes in the database which are unusual in that their copy number exhibits consistent heterogeneous disruption in a high proportion of the cancer datasets. We assess the potential relevance of these genes to the pathology of the cancer samples, in light of their predicted regulatory relationships and enriched biological pathways. A network-based method was used to identify enriched pathways from the genes' inferred targets. The analysis predicts both known and new regulator-target interactions and pathway memberships. We examine examples in detail, in particular the gene POGZ, which is disrupted in many of the cancer datasets and has an unusually large number of predicted targets, from which the network analysis predicts membership of cancer related pathways. The results suggest close involvement in known cancer pathways of genes exhibiting consistent heterogeneous copy number disruption. Further experimental work would clarify their relevance to tumor biology. The results of the analysis presented in the database METAMATCHED, and included here as an R archive file, constitute a large number of predicted regulatory relationships and pathway memberships which we anticipate will be useful in informing such experiments.

Stress-induced mobility of OPHIO1 and OPHIO2, DNA transposons of the Dutch elm disease fungi.

The mobility of transposable elements (TEs) can contribute to genome plasticity, under- or over-expression of genes and ectopic recombination. The data collected in this study provide evidence of stress-induced mobility of OPHIO1 and OPHIO2 transposons, recently detected in Ophiostoma ulmi and O. novo-ulmi, the causal agents of Dutch elm disease (DED). The analyses of OPHIO UTRs and TIRs indicated the presence of two potential binding site motifs and a heat shock protein (hsp) promoter which could be involved in the mobility of OPHIO1 following a heat shock stress. The exact position of the hsp promoter was determined by 5' RACE PCR. After confirmation of the expression by RT-PCR of both OPHIO1 and OPHIO2 transposases in the absence of stress factors, we tested two experimental procedures to induce mobility of OPHIO TEs: (1) an exogenous (cloned) copy of OPHIO1 was introduced into the O. novo-ulmi subsp. americana strain W2 (OPHIO1 free strain) to give mutant strain W2:OPHIO1. After exposure of W2:OPHIO1 to a 55 degrees C heat shock treatment, some of the survivors showed signs of incomplete transposition (excision without reinsertion) of OPHIO1. (2) The O. novo-ulmi subsp. novo-ulmi strain AST27, introgressed from O. ulmi and carrying a distinct endogenous copy of OPHIO2 (OPHIO2-int.), was subjected to a series of abiotic stress treatments. Although a promoter sequence could not be identified, both exposures to UV light and to a 4 degrees C cold treatment caused perfect excision of OPHIO2-int. In contrast to OPHIO1, heat shock stress did not induce OPHIO2-int. mobility. Taken together, these results allow us to hypothesize a potential interspecific invasion of OPHIO transposons due to their mobility in Ophiostoma spp.

Efficient and stable transgene expression in human embryonic stem cells using transposon-mediated gene transfer.

Efficient and stable genetic modification of human embryonic stem (ES) cells is required to realize the full scientific and potential therapeutic use of these cells. Currently, only limited success toward this goal has been achieved without using a viral vector. The Sleeping Beauty (SB) transposon system mediates nonviral gene insertion and stable expression in target cells and tissues. Here, we demonstrate use of the nonviral SB transposon system to effectively mediate stable gene transfer in human ES cells. Transposons encoding (a) green fluorescent protein coupled to the zeocin gene or (b) the firefly luciferase (luc) gene were effectively delivered to undifferentiated human ES cells with either a DNA or RNA source of transposase. Only human ES cells cotransfected with transposon- and transposase-encoding sequences exhibited transgene expression after 1 week in culture. Molecular analysis of transposon integrants indicated that 98% of stable gene transfer resulted from transposition. Stable luc expression was observed up to 5 months in human ES cells cotransfected with a transposon along with either DNA or RNA encoding SB transposase. Genetically engineered human ES cells demonstrated the ability to differentiate into teratomas in vivo and mature hematopoietic cells in vitro while maintaining stable transgene expression. We conclude that the SB transposon system provides an effective approach with several advantages for genetic manipulation and durable gene expression in human ES cells.

[Molecular characterization and expression profile of a novel porcine gene differentially expressed in the muscle and backfat tissues from Chinese Meishan and Russian Large White pigs].

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus dorsi muscle and backfat tissues from Chinese Meishan and Russian Large White pigs. One novel gene that was differentially expressed was identified through semi-quantitative RT-PCR and the cDNA complete sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. The cDNA sequence of this gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 402 amino acids that contains the putative conserved transposase DDE domain and further Blast analysis revealed that this protein has 100% homology with the Tn10 transposase from Oryza sativa, Serratia marcescens, and Salmonella, and therefore, this gene can be defined as the swine Tn10 transposase gene. This novel porcine gene was finally assigned to Gene ID: 100049649. The RT-PCR analysis of the tissue expression profile was carried out using the tissue cDNAs of one Meishan pig as the templates, and the result indicated that this novel swine gene is moderately expressed in fat, and weakly expressed in small intestine, liver, kidney, and spleen but almost not expressed in heart, ovary, muscle, and lung. Our experiment established the primary foundation for further research into the biological significance of swine Tn10 transposase gene.

Recombinant Tol2 transposase with activity in Xenopus embryos.

The Tol2 transposon system is a useful gene transduction technique, but the injection of mRNA is not sufficiently effective in Xenopus embryos to express Tol2 transposase (Tol2TP). To overcome this, we bacterially synthesized recombinant Tol2TP (rTol2TP) protein and showed that rTol2TP efficiently excised the Tol2 element from an injected donor plasmid in Xenopus embryos. Furthermore, injected embryos exhibited uniform and ubiquitous expression of an EGFP reporter gene placed within the Tol2 element. Importantly, size-exclusion chromatography suggests that rTol2TP forms a tetramer, which differs from the reported hexamer formed by Hermes transposase, although both belong to the same hAT family. The use of rTol2TP may facilitate efficient gene transduction in Xenopus, and the biochemical characterization of Tol2TP.

Stable gene transfer to human CD34(+) hematopoietic cells using the Sleeping Beauty transposon.

OBJECTIVE: Methods of gene transfer to hematopoietic stem cells that result in stable integration may provide treatments for many inherited and acquired blood diseases. It has been demonstrated previously that a gene delivery system based on the Sleeping Beauty (SB) transposon can be derived where a plasmid transiently expressing the SB transposase can mediate the stable chromosomal integration of a codelivered second plasmid containing a gene expression unit flanked by the inverted repeats derived from the transposon. METHODS: Plasmid DNA containing the elements required for SB transposition was delivered to hematopoietic cells via electroporation. Integrated transgene (enhanced green fluorescent protein [eGFP]) expression was assessed in vitro and in vivo. RESULTS: In the K562 human hematopoietic cell line, we observed stable expression of eGFP in >60% of cells for over 2 months after electroporation of the two plasmids; in contrast, in control cells either not treated with transposase or exposed to a defective mutant transposase, the level of gene expression had fallen to near background (<0.1%) by 2 weeks. In purified human cord blood CD34(+) progenitor cells, the transposase led to stable gene transfer at levels up to 6% for over 4 weeks, but gene transfer to more primitive nonobese diabetic/severe combined immunodeficient repopulating cells or CD34(+)/CD38(-) in long-term culture was low and electroporation of the cells with plasmid DNA caused significant cell death. CONCLUSION: The long-term stable expression highlights the potential of this transposase-based gene delivery method for ameliorating diseases affecting the hematopoietic system, although further improvements in gene transfer efficacy are needed.

High frequencies of Minos transposon mobilization are obtained in insects by using in vitro synthesized mRNA as a source of transposase.

One of the most frequently encountered problems in transposon-mediated transgenesis is low transformation frequency, often resulting from difficulty in expressing from injected plasmid DNA constructs adequate levels of transposase in embryos. Capped RNA corresponding to the spliced transcript of the Minos transposable element has been synthesized in vitro and shown to be an effective source of transposase protein for Minos transposon mobilization. Transposase produced by this mRNA is shown to catalyze excision of a Minos transposon from plasmid DNA in Medfly embryos. When injected into Drosophila or Medfly embryos, transposase mRNA leads to a several-fold increase in transformation efficiencies compared with injected plasmids expressing transposase. Also, frequent mobilization of a Minos transposon from the X chromosome into autosomes was demonstrated after injections of Minos transposase mRNA into pre-blastoderm Drosophila embryos. The high rates of transposition achieved with transposase mRNA suggest that this is a powerful system for genetic applications in Drosophila and other insects.

Repression of IS200 transposase synthesis by RNA secondary structures.

The IS 200 transposase, a 16 kDa polypeptide encoded by the single open reading frame (ORF) of the insertion element, has been identified using an expression system based on T7 RNA polymerase. In wild-type IS 200, two sets of internal inverted repeats that generate RNA secondary structures provide two independent mechanisms for repression of transposase synthesis. The inverted repeat located near the left end of IS 200 is a transcriptional terminator that terminates read-through transcripts before they reach the IS 200 ORF. The terminator is functional in both directions and may terminate >80% of transcripts. Another control operates at the translational level: transposase synthesis is inhibited by occlusion of the ribosome-binding site (RBS) of the IS 200 ORF. The RBS (5'-AGGGG-3') is occluded by formation of a mRNA stem-loop structure whose 3' end is located only 3 nt upstream of the start codon. This mechanism reduces transposase synthesis approximately 10-fold. Primer extension experiments with AMV reverse transcriptase have provided evidence that this stem-loop RNA structure is actually formed. Tight repression of transposase synthesis, achieved through synergistic mechanisms of negative control, may explain the unusually low transposition frequency of IS 200.

Functional zinc finger/sleeping beauty transposase chimeras exhibit attenuated overproduction inhibition.

The sleeping beauty (SB) transposon system has potential utility in gene transfer applications but lacks specificity for genomic integration and exhibits overproduction inhibition which limits in vivo activity. Targeting transposition may be possible by coupling a specific DNA binding domain to the SB transposase, but it is not known if this strategy will preserve or disrupt activity of the system. We engineered and tested chimeric SB transposases with two different human zinc finger DNA binding domain elements, Sp1 and zinc finger 202 (ZNF202). Addition of Sp1 to the C-terminus abolished transposase activity whereas N-terminal addition of either Sp1 or ZNF202 did not. Transposition activity exhibited by N-terminal chimeras was increased to levels similar to native SB through the use of a hyperactive transposase (SB12) and activating transposon mutations. Importantly, addition of DNA binding domains to the transposase N-terminus resulted in attenuation of overproduction inhibition, a major limitation of this system. These findings suggest that SB transposase chimeras may have specific advantages over the native enzyme.

Characterization of the putative transposase mRNA of Tag1, which is ubiquitously expressed in Arabidopsis and can be induced by Agrobacterium-mediated transformation with dTag1 DNA.

Tag1 is an autonomous transposable element of Arabidopsis thaliana. Tag1 expression was examined in two ecotypes of Arabidopsis (Columbia and No-0) that were transformed with CaMV 35S-Tag1-GUS DNA. These ecotypes contain no endogenous Tag1 elements. A major 2.3-kb and several minor transcripts were detected in all major organs of the plants. The major transcript encoded a putative transposase of 84.2 kD with two nuclear localization signal sequences and a region conserved among transposases of the Ac or hAT family of elements. The abundance of Tag1 transcripts varied among transgenic lines and did not correlate with somatic excision frequency or germinal reversion rates, suggesting that factors other than transcript levels control Tag1 excision activity. In untransformed plants of the Landsberg ecotype, which contain two endogenous Tag1 elements, no Tag1 transcripts were detected. Agrobacterium-mediated transformation of these Landsberg plants with a defective 1.4-kb Tag1 element resulted in the appearance of full-length Tag1 transcripts from the endogenous elements. Transformation with control DNA containing no Tag1 sequences did not activate endogenous Tag1 expression. These results indicate that Agrobacterium-mediated transformation with dTag1 can activate the expression of Tag1.