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PubMed1998

Impaired mismatch extension by a herpes simplex DNA polymerase mutant with an editing nuclease defect.

The D368A mutation within the 3'-5'-exonuclease domain of the herpes simplex type 1 DNA polymerase inactivates this nuclease and severely interferes with virus viability. Compared with the wild type enzyme, the D368A mutant exhibits substantially elevated rates of incorrect nucleotide incorporation, as measured in a LacZ reversion assay. This high rate occurs in the presence of high levels of dNTPs, a condition that forces the enzyme to extend mismatched primers. Hence, the mutant fails to correct many misincorporations that are removed in the wild type. In addition, the mutant shows a much reduced ability to replicate DNA templates primed with a 3'-mismatch as compared with wild type. This extension defect also appears more severe than observed for replicases which naturally lack editing nucleases. Based on these findings, we suggest that the inability of the D368A herpes simplex mutant polymerase to replicate beyond a mismatched base pair severely inhibits viral replication.

Amino Acid Sequence↗
PubMed2006

Wikipedias: collaborative web-based encyclopedias as complex networks.

Wikipedia is a popular web-based encyclopedia edited freely and collaboratively by its users. In this paper we present an analysis of Wikipedias in several languages as complex networks. The hyperlinks pointing from one Wikipedia article to another are treated as directed links while the articles represent the nodes of the network. We show that many network characteristics are common to different language versions of Wikipedia, such as their degree distributions, growth, topology, reciprocity, clustering, assortativity, path lengths, and triad significance profiles. These regularities, found in the ensemble of Wikipedias in different languages and of different sizes, point to the existence of a unique growth process. We also compare Wikipedias to other previously studied networks.

Computer Communication Networks↗
PubMed1997

Estimation of Polymer Rigidity in Cell Walls of Growing and Nongrowing Celery Collenchyma by Solid-State Nuclear Magnetic Resonance in Vivo.

When the growth of a plant cell ceases, its walls become more rigid and lose the capacity to extend. Nuclear magnetic resonance relaxation methods were used to determine the molecular mobility of cell wall polymers in growing and nongrowing live celery (Apium graveolens L.) collenchyma. To our knowledge, this is the first time this approach has been used in vivo. Decreased polymer mobility in nongrowing cell walls was detected through the 13C-nuclear magnetic resonance spectrum by decreases in the proton spin-spin relaxation time constant and in the intensity of a sub-spectrum corresponding to highly mobile pectins, which was obtained by a spectral editing technique based on cross-polarization rates. Flexible, highly methyl-esterified pectins decreased in relative quantity when growth ceased. A parallel increase in the net longitudinal orientation of cellulose microfibrils was detected in isolated cell walls by polarized Fourier-transformed infrared spectrometry.

Journal Article↗
PubMed1981

Stop identification from vocalic transition plus vowel segments of CV and VC syllables: a follow-up study.

In a recent article in the Journal [L. C. W. Pols and M. E. H. Schouten, J. Acoust. Soc. Am. 64, 1333-1337 (1978)], it was concluded that perception experiments which present CV syllables with the consonant bursts deleted invoke a "click sensation" and that this sensation has a deleterious affect on the perception of the consonants. In order to directly assess the "click sensation" hypothesis, a computer-based speech editing system was used which smooths onsets and offsets of stimuli and therefore eliminates the possibility of an acoustic click. Subjects identified voiced and voiceless stops from vocalic transition + vowel stimuli edited from CV and VC syllables. The findings were as follows: (1) Consonant identification from vocalic transition + vowel segments in VC syllables was significantly greater than that from vocalic transition + vowel segments in CV syllables; and (2) The superiority of the scores for VC vocalic transition + vowel segment was found for both voiced and voiceless stops, but it was not as great for voiced stops. Although there are differences in magnitude of findings between this study and those which employed other deletion techniques, the reliability and direction of the results were similar. The results do not support the "click sensation"hypothesis and suggest that other mechanisms must account for differences between CV and VC syllables in the perception of sound segments.

Follow-Up Studies↗
PubMed2025

In vivo expansion of gene-targeted hepatocytes through transient inhibition of an essential gene.

Homology-directed repair (HDR)-based genome editing is an approach that could permanently correct a broad range of genetic diseases. However, its utility is limited by inefficient and imprecise DNA repair mechanisms in terminally differentiated tissues. Here, we tested Repair Drive, a platform technology for selectively expanding HDR-corrected hepatocytes in adult mice in vivo. Repair Drive involves transient conditioning of the liver by knocking down an essential gene, fumarylacetoacetate hydrolase (Fah), and delivering an untargetable version of the essential gene in cis with a therapeutic transgene. We show that Repair Drive increased the percentage of correctly targeted hepatocytes in healthy wild-type mice up to 25%, which resulted in a fivefold increased expression of a therapeutic transgene, human factor IX (FIX). Repair Drive was well tolerated and did not induce toxicity or tumorigenesis during a 1-year follow-up. This approach may broaden the range of liver diseases that can be treated with somatic genome editing.

Animals↗
PubMed2001

Exact algorithms for computing pairwise alignments and 3-medians from structure-annotated sequences (extended abstract).

Given the problem of mutation saturation in ancient molecular sequences, there is great interest in inferring phylogenies from higher-order types of molecular data that change more slowly, such as genomic organization and the secondary and tertiary structures of ribosomal RNA and proteins. In this paper, we define edit distances based on two representations of RNA secondary structure, arc annotation and hierarchical string annotation, and give algorithms for computing these distances on pairs of annotated sequences, aligning pairs of annotated sequences, and computing 3-median annotated sequences from triples of annotated sequences. The 3-median algorithms can be used as part of a well-known iterative heuristic for inferring phylogenies. All given algorithms are adapted from algorithms for computing longest common annotated subsequences of pairs of annotated sequences.

Algorithms↗
PubMed1988

A computer program for rapid generation of receiver operating characteristic curves and likelihood ratios in the evaluation of diagnostic tests.

We describe a MUMPS program to facilitate the evaluation of the diagnostic effectiveness and efficiency of laboratory tests using receiver operating characteristic (ROC) curves and likelihood ratios (LR). Test results from a population with and without a disease are stored into retrievable, editable data bases. Either single or a combination (less than or equal to 9) of different test data files can be accessed to generate tables of calculated frequency distribution, sensitivity, l-specificity and LR at variable decision thresholds. The program also calculates the area under the ROC curve, confidence intervals and SE. The selection of the optimum cut-off value is assisted by graphical plots of frequency distribution, ROC and decision level curves as illustrated by using some 'cardiac' enzyme tests.

Clinical Enzyme Tests↗
PubMed2026

Epigenetics and childhood obesity: DNA methylation coordinates environment and gene regulation.

Childhood obesity is a complex disorder which results from the combined contribution of genetics, the environment, and development, which is programmed and coordinated by epigenetic mechanisms. Of them, DNA methylation has emerged as an important molecular interface between environmental inputs and changes in gene expression. In this review, we provide an overview of the role of DNA methylation in childhood obesity during the key developmental stages, from prenatal life and childhood to adolescence. We also highlight the available evidence from candidate genes and genome-wide association studies implicating critical loci involved in energy homeostasis and adipogenesis, where DNA methylation is altered. Further, we also provide an overview of how maternal obesity, nutritional status, and bariatric surgery shape offspring's methylation profiles and contribute to the increased risk of programming obesity across generations. Although aberrant methylation patterns are consistently associated with altered metabolic phenotypes, disentangling causality remains a significant challenge. Herein, we highlight emerging approaches, such as rigorous longitudinal cohorts, epigenetic Mendelian randomization, and CRISPR-based epigenome editing, that are beginning to provide the analytical clarity needed to move beyond association. Finally, we examine the potential of DNA methylation signatures to inform early risk stratification and prevention possibilities. Although yet to be clinically validated, whole-genome methylation profiling is increasingly integrated with systems biology and multi-omics frameworks, making the identification of robust, clinically actionable markers more promising. A more precise understanding of how epigenetic processes shape susceptibility to childhood obesity could ultimately support strategies capable of altering lifelong metabolic trajectories.

Humans↗
PubMedDate not supplied

[ATPase subunit 6 gene of Leptomonas seymouri (Trypanosomatidae) is transcribed and edited as a polycistronic transcript].

The maturation of mitochondrial RNA transcripts proceeds through several steps. Here we use insects trypanosomatid Leptomonas seymouri as a model organism for analysis of transcription and posttranscriptional processing of mitochondria encoded gene for the subunit 6 of ATPase (A6). It was shown that Cyt b (cytochrome b) and A6 genes were transcribed and edited as a polycistronic template. Analysis of twelve RT-PCR products of both genes led to identification of four types of differently and/or partially edited cDNA molecules. Based on the analysis of two fully edited A6 transcripts we propose the existence of two alternatively edited products.

Adenosine Triphosphatases↗
PubMed1996

Protein synthesis editing by a DNA aptamer.

Potential errors in decoding genetic information are corrected by tRNA-dependent amino acid recognition processes manifested through editing reactions. One example is the rejection of difficult-to-discriminate misactivated amino acids by tRNA synthetases through hydrolytic reactions. Although several crystal structures of tRNA synthetases and synthetase-tRNA complexes exist, none of them have provided insight into the editing reactions. Other work suggested that editing required active amino acid acceptor hydroxyl groups at the 3' end of a tRNA effector. We describe here the isolation of a DNA aptamer that specifically induced hydrolysis of a misactivated amino acid bound to a tRNA synthetase. The aptamer had no effect on the stability of the correctly activated amino acid and was almost as efficient as the tRNA for inducing editing activity. The aptamer has no sequence similarity to that of the tRNA effector and cannot be folded into a tRNA-like structure. These and additional data show that active acceptor hydroxyl groups in a tRNA effector and a tRNA-like structure are not essential for editing. Thus, specific bases in a nucleic acid effector trigger the editing response.

Base Sequence↗
PubMed1993

On the evolution of RNA editing.

The term 'RNA editing' encompasses a variety of processes that change the primary nucleotide sequence of an RNA transcript from that of its encoding DNA. As in the case of certain other molecular genetic phenomena, for example RNA splicing, the discovery of RNA editing presented molecular biologists with an evolutionary puzzle, since the existence of RNA editing offers no obvious selective advantage. A three-step model for the evolution of RNA editing is proposed, based on the co-evolution of editing activity and editing sites, with genetic drift as an important component. The implications of this model for the known forms of RNA editing are discussed.

Animals↗
PubMed1995

The phosphoprotein gene of a dolphin morbillivirus isolate exhibits genomic variation at the editing site.

The nucleotide sequence of the phosphoprotein (P) gene of a dolphin morbillivirus (DMV) isolate was determined. Like those of other morbilliviruses the DMV P gene encoded P and C proteins in overlapping open reading frames and V protein by editing the P gene transcript. Among P mRNA based clones the editing site variants GGGC, GGGG, GAGC and GGGGGGC predicting a P protein, and the variants GGGGC and GGGGGG predicting a V protein, were found. Surprisingly, the three variants GGGC, GGGG and GAGC were also found among clones generated from genomic RNA of the DMV isolate. Thus, more than one viral genome type appeared to be present in cells infected with the DMV isolate. By a similar analysis of the virus genomes in the tissue from which the DMV isolate was obtained, only the GGGC type was found, indicating that the GGGG and GAGC types arose during adaptation of the virus to growth in cell cultures. No editing site variants likely to have arisen by editing the GAGC type were encountered, and it remains ot be determined whether mRNA encoding V protein can be transcribed from genomes with this editing site. Using antisera raised against the common N terminus and unique C termini of the predicted P and V proteins, the in vivo expression of these proteins was demonstrated.

Amino Acid Sequence↗
PubMed2005

Selective homonuclear Hartmann-Hahn transfer method for in vivo spectral editing in the human brain.

A novel selective homonuclear Hartmann-Hahn transfer method for in vivo spectral editing is proposed and applied to measurements of gamma-aminobutyric acid (GABA) in the human brain at 3 T. The proposed method utilizes a new concept for in vivo spectral editing, the spectral selectivity of which is not based on a conventional editing pulse but based on the stringent requirement of the doubly selective Hartmann-Hahn match. The sensitivity and spectral selectivity of GABA detection achieved by this doubly selective Hartmann-Hahn match scheme was superior to that achievable by conventional in vivo spectral editing techniques providing both sensitivity enhancement and excellent suppression of overlapping resonances in a single shot. Since double-quantum filtering gradients were not employed, singlets such as the NAA methyl group at 2.02 ppm and the creatine methylene group at 3.92 ppm were detected simultaneously. These singlets may serve as navigators for the spectral phase of GABA and for frequency shifts during measurements. The estimated concentration of GABA in the frontoparietal region of the human brain in vivo was 0.7 +/- 0.2 mumol/g (mean +/- SD, n = 12).

Brain Chemistry↗
PubMed1996

Control of gene expression by base deamination: the case of RNA editing in wheat mitochondria.

The term 'RNA editing' was used for the first time in 1986 to describe the process of uridylate insertion into trypanosomal mitochondrial transcripts. Since then, the term has been used more generally to describe a large variety of processes involving base insertions, deletions and conversions that generate RNAs with a primary sequence different to those encoded by the gene. RNA editing has been observed in the mitochondrial fraction of trypanosomes, plants and other organisms, in the animal nuclear fraction in the case of the apolipoprotein B and glutamate brain receptors mRNAs as well as in viruses like paramyxovirus, hepatitis delta and probably HIV. The role of cytidine and adenine deamination leading to C to U and A to I transitions has became pivotal to explain this process by base conversion. In this review we will focus mainly on the work performed in our group on plant mitochondria and more specifically on the mechanism and the functional significance of RNA editing in wheat organelles. The original contributions of our laboratory in this field are: i) showing that RNA editing is reflected at the protein level; ii) settling three in vitro systems to assay C to U conversion using a wheat mitochondrial lysate as source of enzymes and factors, and unedited mRNA from the same source, as substrate; iii) determination by double labelling of the unedited substrate that RNA editing in wheat mitochondria occurs via a deamination step; and iv) that introducing unedited proteins in the mitochondria of transgenic plants leads to the emergence of cytoplasmic male sterility supporting the idea that the role of this process is to produce functional proteins. Using the antisense approach in transgenic plants we were able to obtain a significant male fertility restoration.

Cytosine Deaminase↗
PubMed1999

Phylogenetic analysis of the apolipoprotein B mRNA-editing region. Evidence for a secondary structure between the mooring sequence and the 3' efficiency element.

Apolipoprotein (apo) B mRNA editing is the deamination of C(6666) to uridine, which changes the codon at position 2153 from a genomically encoded glutamine (CAA) to an in-frame stop codon (UAA). The apoB mRNA-editing enzyme complex recognizes the editing region of the apoB pre-mRNA with exquisite precision. Four sequence elements spanning 139 nucleotides (nt) on the apoB mRNA have been identified that specify this precision. In cooperation with the indispensable mooring sequence and spacer element, a 5' efficiency element and a 3' efficiency element enhance editing in vitro. A phylogenetic comparison of 32 species showed minor differences in the apoB mRNA sequence, and the apoB mRNA from 31 species was robustly edited in vitro. However, guinea pig mRNA was poorly edited. Compared with the consensus sequences of these 31 species, guinea pig apoB mRNA has three variations in the 3' efficiency element, and the conversion of these to the consensus sequence increased editing to the levels in the other species. From this information, a model for the secondary structure was formulated in which the mooring sequence and the 3' efficiency element form a double-stranded stem. Thirty-one mammalian apoB mRNA sequences are predicted to form this stem positioning C(6666) two nucleotides upstream of the stem. However, the guinea pig apoB mRNA has a mutation in the 3' efficiency element (C(6743) to U) that predicts an extension of the stem and hence the lower editing efficiency. A test of this model demonstrated that a single substitution at 6743 (U to C) in the guinea pig apoB mRNA, that should reduce the stem, enhanced editing, and mutations in the 3' efficiency element that extended the stem for three base pairs dramatically reduced editing. Furthermore, the addition of a 20-nucleotide 3' efficiency element RNA, to a 58-nucleotide guinea pig apoB mRNA lacking the 3' efficiency element more than doubled the in vitro editing activity. Based on these results, a model is proposed in which the mooring sequence and the 3' efficiency element form a double-stranded stem, thus suggesting a mechanism of how the 3' efficiency element enhances editing.

Animals↗
PubMed1995

Development of an interactive data base management system for capturing large volumes of data.

Accurate collection and successful management of data are problems common to all scientific studies. For studies in which large quantities of data are collected by means of questionnaires and/or forms, data base management becomes quite laborious and time consuming. Data base management comprises data collection, data entry, data editing, and data base maintenance. In this article, the authors describe the development of an interactive data base management (IDM) system for the collection of more than 1,400 variables from a targeted population of 6,000 patients undergoing heart surgery requiring cardiopulmonary bypass. The goals of the IDM system are to increase the accuracy and efficiency with which this large amount of data is collected and processed, to reduce research nurse work load through automation of certain administrative and clerical activities, and to improve the process for implementing a uniform study protocol, standardized forms, and definitions across sites.

Cardiac Surgical Procedures↗
PubMed2025

Correction of pathogenic mitochondrial DNA in patient-derived disease models using mitochondrial base editors.

Mutations in the mitochondrial genome can cause maternally inherited diseases, cancer, and aging-related conditions. Recent technological progress now enables the creation and correction of mutations in the mitochondrial genome, but it remains relatively unknown how patients with primary mitochondrial disease can benefit from this technology. Here, we demonstrate the potential of the double-stranded DNA deaminase toxin A-derived cytosine base editor (DdCBE) to develop disease models and therapeutic strategies for mitochondrial disease in primary human cells. Introduction of the m.15150G > A mutation in liver organoids resulted in organoid lines with varying degrees of heteroplasmy and correspondingly reduced ATP production, providing a unique model to study functional consequences of different levels of heteroplasmy of this mutation. Correction of the m.4291T > C mutation in patient-derived fibroblasts restored mitochondrial membrane potential. DdCBE generated sustainable edits with high specificity and product purity. To prepare for clinical application, we found that mRNA-mediated mitochondrial base editing resulted in increased efficiency and cellular viability compared to DNA-mediated editing. Moreover, we showed efficient delivery of the mRNA mitochondrial base editors using lipid nanoparticles, which is currently the most advanced non-viral in vivo delivery system for gene products. Our study thus demonstrates the potential of mitochondrial base editing to not only generate unique in vitro models to study these diseases, but also to functionally correct mitochondrial mutations in patient-derived cells for future therapeutic purposes.

Humans↗
PubMed1990

Kinetoplast DNA minicircles encode guide RNAs for editing of cytochrome oxidase subunit III mRNA.

Guide RNAs (gRNAs) for the editing of sites 1-8 of COIII mRNA and an "unexpected" partially edited COIII mRNA are encoded in the variable regions of specific kinetoplast DNA minicircles. The gRNAs can form 37 and 44 nucleotide perfect hybrids (allowing for G-U base pairs) with edited mRNAs. The gRNAs were detected on Northern blots and shown to have unique 5' ends situated close to the beginning of the potential base pairing with the edited mRNAs. We suggest that kinetoplast DNA minicircle molecules in general may encode gRNAs for editing of cryptogene mRNAs by a mechanism similar to that previously proposed for editing by maxicircle-encoded gRNAs.

Animals↗