Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “TadA”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed2026

Optimized TadA-derived base editors efficiently manipulate mRNA splicing by A-to-G and C-to-K editing in potato.

Pre-messenger RNA (pre-mRNA) splicing is a critical mechanism for post-transcriptional regulation in plants. Through alternative splicing, plants produce diverse transcriptomes and proteomes that finely regulate development as well as responses to biotic and abiotic stresses. However, modulating the generation of specific splicing isoforms for functional characterization remains challenging, particularly in the non-model crop potato. Here, we show that two optimized TadA-derived base editors efficiently induce diverse mRNA splice variants by targeting specific splice sites. By evaluating multiple adenosine deaminases and performing multi-dimensional optimization, we developed an efficient adenine base editor RTF-ABE8e for potato. RTF-ABE8e achieved 100% editing efficiency at two StDL1 target sites in stable transgenic potato, with homozygous editing frequencies as high as 93.3% and 91.1%, respectively. We also developed RTF-TadDE, a dual-base editor based on a TadA-derived dual deaminase, for A-to-G and C-to-K (K = T/G) mutations in potato with an overall editing efficiency comparable to that of RTF-ABE8e. By targeting different splice sites with these base editors, we obtained diverse splicing isoforms carrying premature termination codons (PTCs) at StDL1 and StPDS and robust mutant phenotypes. These base editors enable efficient and precise editing of splice sites to trigger missplicing, making them powerful tools for manipulating splicing in plants.

Solanum tuberosum↗
PubMed2026

EndoV does not measurably affect TadA-dependent A-to-I RNA editing in Escherichia coli under exponential-growth conditions in rich medium.

Adenosine-to-inosine (A-to-I) mRNA editing changes the genetic information post-transcriptionally and was only recently reported to occur in bacteria. Here, we examined whether endonuclease V (EndoV; encoded by nfi) cleaves inosine-containing RNAs in vivo and thereby influences the abundance and fate of A-to-I-edited mRNAs in Escherichia coli. We generated an nfi loss-of-function mutant carrying a premature stop codon and performed RNA sequencing alongside the isogenic wild-type strain. We observed that global and site-specific editing occurrence or levels in both mRNAs and tRNAs were indistinguishable between strains. Moreover, overexpression of EndoV did not affect the number of edited sites, motif enrichment, or editing levels compared with a control strain overexpressing mCherry. Our findings suggest that, in contrast to human EndoV, bacterial EndoV does not regulate the steady-state pool of edited mRNAs in E. coli under nutrient-rich, exponential-growth conditions in vivo.IMPORTANCEAdenosine-to-inosine (A-to-I) mRNA editing is an emerging regulatory layer in bacteria, but the factors that act on edited transcripts are largely unknown. Endonuclease V (EndoV) was a prime candidate because it cleaves inosine-containing nucleic acids and can act on inosine-containing RNA in vitro. By combining loss-of-function and overexpression of EndoV with genome-wide RNA editing measurements, we show that EndoV does not measurably influence TadA-dependent A-to-I mRNA editing in Escherichia coli under standard laboratory conditions. This negative result is important because it rules out a natural effector candidate and redirects attention to other bacterial pathways that may process edited RNAs. Our work, therefore, sharpens mechanistic models for bacterial RNA editing and helps focus future searches for its regulators and physiological roles.

Escherichia coli↗
PubMed2025

A streamlined base editor engineering strategy to reduce bystander editing.

Base editing (BE) can permanently correct over half of known human pathogenic genetic variants without requiring a repair template, thus serving as a promising therapeutic tool to treat a broad spectrum of genetic diseases. However, the broad activity windows of current base editors pose a major challenge to their therapeutic application. Here, we show that integrating a naturally occurring oligonucleotide binding module into the deaminase active center of TadA-8e, a highly active deoxyadenosine deaminase, enhances its editing specificity. When conjugated with a Cas9 nickase or alternative PAM Cas9 variants, the engineered TadA variant-TadA-NW1-consistently achieves robust A-to-G editing efficiencies within an editing window consisting of four nucleotides, substantially narrower than the 10-bp editing window of the TadA-8e-derived ABEs. Moreover, compared to ABE8e, ABE-NW1 shows significantly decreased Cas9-dependent and -independent off-target activity while maintaining similar on-target editing efficiency. Further, TadA-NW1 can be reprogrammed to perform desired cytidine deamination and adenine transversion within a restricted editing window. Finally, in a cystic fibrosis (CF) cell model, ABE-NW1 outperforms existing ABEs in accurately and efficiently correcting the CFTR W1282X variant, one of the most common CF-causing mutations. In all, we engineered a suite of base editors with refined activity windows, enabling more precise base editing. Importantly, this study presents a streamlined genome editor re-engineering strategy to accelerate the development of therapeutic base editing.

Gene Editing↗
PubMed1975

Phosphorylation of a 22,000-dalton component of the cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase.

Cardiac microsomes were incubated with [gamma-32P]ATP and a cardiac adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase in the presence of ethylene glycol bis(bets-aminoethyl ether)-N,N'-tetraacetic acid. After solubilization in sodium dodecyl sulfate and fractionation by polyacrylamide gel electrophoresis, a single microsomal protein component of approximately 22,000 daltons was found to bind most of the 32P label. The 32P labeling of this component increased several fold when NaF was included in the incubation medium. No other component of cardiac microsomes, including sarcoplasmic reticulum ATPase protein, contained significant amounts of 32P label. This 22,000-dalton phosphoprotein formed by cyclic AMP-dependent protein kinase had stability characteristics of a phosphoester rather than an acyl phosphate. Washing of microsomes with buffered KCl did not decrease the amount of 32P labeling to the 22,000-dalton protein, suggesting that this protein is associated with the membranes of sarcoplasmic reticulum rather than being a contaminant from other soluble proteins. The 22,000-dalton protein was susceptible to trypsin. Brief digestion with trypsin in the presence of 1 M sucrose did not significantly affect microsomal calcium transport activity, but prevented both subsequent phosphorylation of the 22,000-dalton protein and stimulation of calcium uptake by cyclic AMP-dependent protein kinase, suggesting that this protein is a modulator of the calcium pump. These results are consistent with previous findings (Kirchberger, M.A., Tada, M., and Katz, A.M. (1974) J. Biol. Chem. 249, 6166-6173; Tada, M., Kirchberger, M.A., Repke, D.I., and Katz, A.M. (1974) J. Biol. Chem. 249, 6174-6180) that cyclic AMP-dependent protein kinase-catalyzed phosphorylation is associated with stimulation of calcium transport in the cardiac sarcoplasmic reticulum, and further indicate that this phosphorylation occurs at a component of low mass (22,000 daltons) of the cardiac sarcoplasmic reticulum which, while separable from the calcium transport ATPase protein (100,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has the ability to regulate calcium transport by the cardiac sarcoplasmic reticulum.

Animals↗
PubMed1977

Immunosuppressive factor(s) specific for L-glutamic acid50-L-tyrosine50 (GT) II. Presence of I-J determinants on the GT-suppressive factor.

The responses to the synthetic antigens, L-glutamic acid(60)-L- alanine(30)-L-tyrosine(10) (GAT) and L-glutamic acid(50)-L-tyrosine(50) (GT) are controlled by genes in the I region of the mouse H-2 complex (1-3). Preimmunization of the mice bearing the H-2(p,q,s) nonresponder haplotypes with GAT stimulates the development of suppressor T cells that inhibit in vivo or in vitro antibody responses to GAT complexed to the immunogenic carrier, methylated bovine serum albumin (GAT-MBSA) (4). The copolymer GT is not immunogenic in any inbred mouse strain tested, and has a suppressive effect on the antibody responses to GT-MBSA in mouse strains bearing the H-2(d,f,k,s) haplotypes; suppressor T cells have been demonstrated to be responsible for specific GT suppression (3). We have obtained specific suppressive extracts from thymus and spleen cells of GAT-or GT-primed suppressor strains (5,6). The specific suppressive T-cell factors in the active extracts have been characterized (6,7) and appear similar to the carrier-specific suppressor factor described by Tada and Taniguchi (8). These products belong to a family of newly identified molecules coded for by the I region of the H-2 complex with affinity for antigen and helper (9,10) or suppressive (5-8) regulatory activity on the immune response. Recently, Tada et al. have reported that the keyhole limpet hemocyanin (KLH)-specific suppressor factor is coded for by the I-J subregion of the H-2 complex (11). We now demonstrate also that a GT-specific suppressor factor extracted from the spleens and thymuses of B10.BR (H-2(k)) mice bears determinants controlled by the I-J subregion of the H-2 complex.

Alanine↗
PubMed2026

damidBind: an R/bioconductor package for differential DamID analysis and data exploration.

SUMMARY: DamID, and its cell-type specific adaptations, including Targeted DamID (TaDa) and Chromatin Accessibility TaDa (CATaDa), are now widely-adopted as techniques for the genome-wide profiling of DNA binding proteins. Despite this popularity, no dedicated software solution exists for identifying differentially bound or accessible loci, or differentially transcribed genes, between cell types using DamID. The R/Bioconductor package damidBind provides these functions, allowing an end-user to move from processed binding profiles to identifying differentially-bound loci in a reproducible, statistically appropriate and straightforward workflow. AVAILABILITY AND IMPLEMENTATION: damidBind is an open-source R/Bioconductor package and freely available from Bioconductor at https://bioconductor.org/packages/damidBind/, and from GitHub at https://github.com/marshall-lab/damidBind. It is released under the GPLv3 licence.

Software↗
PubMed1975

Phosphoprotein phosphatase-catalyzed dephosphorylation of the 22,000 dalton phosphoprotein of cardiac sarcoplasmic reticulum.

The present study demonstrated the presence within the myocardium of phosphoprotein phosphatase activity which can account for dephosphorylation of a 22,000 dalton phosphoprotein of cardiac sarcoplasmic reticulum that has been associated with the stimulatory effects of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase on calcium transport (Tada, M., Kirchberger, M. A., and Katz, A. M. (1975) J. Biol. Chem. 250:2640-2647). Dog cardiac microsomes, consisting mainly of fragmented sarcomplasmic reticulum, were phosphorylated by incubation with cyclic AMP-dependent protein kinase and [gamma-32P]ATP, and subsequently washed with trichloroacetic acid or buffered KCl. Phosphorylated microsomes contained approximately 1 nmole of 32P bound per mg of microsomal protein, 32P labeling occurring almost exclusively at the 22,000 dalton component. Soluble phosphoprotein phosphatases, isolated from the cytosol, catalyzed dephosphorylation of 32P-labeled microsomes. The existence of a phosphoprotein phosphatase that is associated with the microsomes was demonstrated by the ability of the microsomes to dephosphorylate 32P-histone. This membrane-associated phosphatase activity can also account for a rapid decrease in the amount of 32P-labeling of the 22,000 dalton protein. The dephosphorylation of the phosphorylated 22,000 dalton protein by phosphoprotein phosphatase satisfies an important requirement for the phosphorylation of the 22,000 dalton protein to serve a physiological role, namely, its reversibility.

Animals↗
PubMed2025

Preparation of high-purity RNPs of CRISPR-based DNA base editors.

Since their introduction, CRISPR-based DNA base editors (BEs) have become essential in the field of precision genome editing, revolutionizing the correction of pathogenic SNPs for both basic research and therapeutic applications. As this technology advances, more laboratories are implementing these tools into their workflow. The delivery of BEs as BE-guide RNA complexes (RNPs), rather than as mRNA or plasmids, has been shown to exhibit lower off-target effects, establishing it as the preferred method of delivery. However, there are no protocols describing in detail how to obtain high-purity and highly active BE RNPs. Here, we offer a comprehensive guide for the expression, purification, RNP reconstitution, and in vitro activity assessment of TadA-based BEs. The protocol includes guidance on performing activity assays using commercial denaturing gels, which is convenient and uses standard molecular biology equipment. This allows for rapid quality control testing of reconstituted BE RNPs prior to more expensive and time-consuming in vivo genome editing experiments. Overall, this protocol aims to empower more laboratories to generate tailored BE RNPs for diverse in vitro and in vivo applications.

Gene Editing↗
PubMed2025

Charting the development and engineering of CRISPR base editors: lessons and inspirations.

CRISPR base editors (BEs) have introduced a new chapter in precise genome editing. The brief but fruitful history of BE development documents many case studies that not only lay the foundation of base-editing technology but are also instrumental to future protein engineering efforts. In this review, we summarize the development and engineering of various BEs with a focus on recent progress. These include traditional cytosine and adenine base editors (CBEs and ABEs), novel TadA-derived CBEs, transversion BEs, dual BEs, and CRISPR-free BEs. We discuss each aspect of the workflow and highlight the successes and challenges encountered in the engineering process.

Gene Editing↗
PubMed1979

Surface markers on natural killer cells of the mouse.

Rabbit antiserum against mouse brain tissue (anti-brain-associated T cell antigen, anti-BAT) was capable of killing splenic natural killer (NK) cells of CBA/J, BALB/c, C 57 Bl/6J, C 3 H/He and nude mice, which were detected with Molony virus-induced lymphoma (YAC-1) and radiation-induced leukemia (RL male 1) cells as targets. The same antiserum abolished T cell functions, e.g. carrier-specific helper function and the responsiveness to concanavalin A, but not B cell functions, e.g. immunological memory for the secondary antibody response and the responsiveness to lipopolysaccharide. After absorption of the anti-BAT with thymocytes, the ability to kill T cells was completely abrogated, leaving the activity to kill NK cells intact. No other heterologous and isologous antisera, i.e. rabbit anti-mouse thymocyte antiserum, goat antiserum against antigens shared by thymus and B cells, anti-Thy-1.2 and anti-Ia antisera, could eliminate NK function regardless of their definite reactivity against T or B cells. The results indicate that the absorbed anti-BAT can distinguish NK cells from other known subsets of T and B cells.

Animals↗
PubMed1978

Neuroradiology of ossification of the posterior longitudinal spinal ligament. Comparative studies with computer tomography.

Comparative neuroradiologic studies of the posterior longitudinal spinal ligament were performed in 15 cases showing myelopathy. On visualizing the ossified foci CT scan was found to be superior to the conventional roentgenograms, and detailed evaluation of the constricted spinal canal with related neurologic deficits became possible. CT analysis must be performed to differentiate spondylotic myelopathy, which is essential when considering operative intervention.

Humans↗
PubMed1978

Congenital lactic acidosis due to pyruvate carboxylase deficiency: absence of an inhibitor of TPP-ATP phosphoryl transferase.

Two children are described who suffered from episodes of metabolic acidosis and progressive mental and motor deterioration. The patients showed periodic elevation of blood lactate, pyruvate and alanine, which was accompanied by vomiting, hypotonia or convulsions. The concentrations of lactate and pyruvate in cerebrospinal fluid were found to be increased. Liver biopsies revealed a decrease in pyruvate carboxylase activity and normal pyruvate decarboxylase activity. No inhibitor of TPP-ATP phosphoryl transferase was detected in urine from the patients. These findings suggest that congenital lactic acidosis due to pyruvate carboxylase deficiency is probably a different disease entity from Leigh's encephalomyelopathy. A possible mechanism of brain damage caused by a defect in pyruvate carboxylase is postulated.

Acidosis↗
PubMed2026

Quality-of-life assessment in the randomized JBCRG-M06/EMERALD study of eribulin plus dual HER2 blockade in HER2-positive locally advanced or metastatic breast cancer.

BACKGROUND: Although taxanes are a mainstay treatment for locally advanced or metastatic breast cancer (LABC/MBC), they often impair quality of life (QoL). Treatments that avoid taxane-related QoL deteriorations would be valuable. METHODS: The JBCRG-M06/EMERALD trial (NCT03264547, UMIN000027938) compared eribulin with a taxane, each combined with trastuzumab and pertuzumab, in patients with human epidermal growth factor receptor type 2 (HER2)-positive LABC/MBC. QoL was assessed using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Module C30 (EORTC QLQ-C30) version 3.0. QoL deterioration was defined as a decrease in the Global Health Status (GHS) score by ≥ 10 points (minimum clinically important difference), disease progression, or death. RESULTS: QoL data were available for 210 (of 224 randomized) and 205 (of 222 randomized) patients in the eribulin and taxane groups, respectively. The median (95% confidence interval) time to QoL deterioration was 7.16 (6.28-8.34) months in the eribulin group versus 4.57 (4.17-6.14) months in the taxane group, with a hazard ratio of 0.80 (95% confidence interval 0.65-0.98; log-rank P = 0.08). QoL was maintained at 6 and 12 months in greater proportions of the eribulin group (62.7% and 30.5%) compared with the taxane group (43.5% and 25.5%). GHS scores remained stable over time in the eribulin group. GHS deteriorated between weeks 9 and 27 in the taxane group (i.e. during treatment) with subsequent recovery toward baseline. CONCLUSIONS: Eribulin could help avoid the early deteriorations in QoL that occur during taxane therapy and maintain QoL for longer in patents with HER2-positive LABC/MBC receiving trastuzumab and pertuzumab.

Adult↗