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At least 199 records · Page 11Linked to original sources
PubMed1999

Uridine insertion/deletion RNA editing in trypanosome mitochondria--a review.

The uridine insertion/deletion RNA editing in trypanosome mitochondria is a unique posttranscriptional RNA maturation process that involves the addition or removal of uridine residues at precise sites usually within the coding regions of mitochondrial transcripts. This process creates initiation and termination codons, corrects frameshifts and even builds entire open-reading frames from nonsense sequences. The development of several in-vitro editing assays has provided much insight into the molecular mechanism of RNA editing, which appears to involve cleavage, U addition, exonuclease trimming and ligation, essentially as proposed in the original 'enzyme cascade' model (Blum, B., Bakalara, N., Simpson, L., 1990. A model for RNA editing in kinetoplastid mitochondria: 'Guide' RNA molecules transcribed from maxicircle DNA provide the edited information. Cell 60, 189-198). However, little is known about the biochemical properties of the proteins involved and the significance and role of this process. This article is a review of recent findings on uridine-insertion/deletion editing in trypanosome mitochondria, with an emphasis on the proteins isolated and characterized that may have a role in this process.

Animals↗
PubMed1999

In vitro uridine insertion RNA editing mediated by cis-acting guide RNAs.

Uridine (U) insertion/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa is a posttranscriptional process mediated by guide RNAs (gRNAs). The gRNAs direct the precise insertion and deletion of Us by a cleavage-ligation mechanism involving base pairing. We show that a cognate gRNA in cis at the 3' end of a preedited NADH dehydrogenase 7 (ND7) mRNA substrate can direct U insertions at editing site 1 when incubated with a mitochondrial lysate from Leishmania tarentolae. The efficiency of gRNA-dependent U insertion mediated by a cis-acting gRNA is greater on a molar basis than that for a trans-acting gRNA, as expected for a unimolecular gRNA:mRNA interaction. Blocking the 3' end of a cis-acting gRNA lacking a 3' oligo[U] tail has no effect on gRNA-dependent U insertions, nor does providing the gRNA in cis upstream of the mRNA, confirming the previous observation that the terminal 2'- and 3'-hydroxyls of the gRNA are not involved in U insertion activity. These results also establish that the oligo[U] tail is not required for U insertion in vitro. Increasing the extent of base pairing between the 3' end of the gRNA and the 5' end of the mRNA significantly increases in vitro gRNA-dependent U insertion at site 1, presumably by maintaining the mRNA 5' cleavage fragment within the editing complex. We speculate that, in vivo, protein:RNA and/or protein:protein interactions may be responsible for maintaining the mRNA 5' cleavage fragment in close proximity to the mRNA 3' cleavage fragment, and that such interactions may be rate limiting in vitro.

Animals↗
PubMed2026

Chiron3D: an interpretable deep learning framework for understanding the DNA code of chromatin looping.

MOTIVATION: Three-dimensional folding of the genome into structures such as chromatin loops is essential for gene regulation. Current experimental methods for mapping these structures, like Hi-C and HiChIP, are labor-intensive and require repeated assays to test hypothesized mutation effects. This motivates the need for predictive approaches that reveal the sequence determinants of chromatin loops. RESULTS: In this work, we present a novel and interpretable computational pipeline for predicting CTCF-mediated chromatin loops. We propose Chiron3D, a DNA-only model trained in a cell-type specific manner to predict CTCF HiChIP contact maps. By leveraging pre-trained embeddings from a foundation model, our approach is competitive with baselines that take CTCF ChIP-seq as additional input, while enabling nucleotide-level attribution to the input DNA sequence. Using our framework, we provide likely mechanistic insights into the physical control of loop dynamics. Specifically, we find that the strength of the loop extrusion anchorage site is largely governed by the amount and binding affinity of CTCF sites at the boundaries. Furthermore, we reveal that loop stability is regulated by the amount of intra-loop CTCF binding sites, where fewer intra-loop sites are associated with greater loop stability. Using targeted, single-nucleotide edit simulations with Chiron3D, we show that both loop strength and stability can be precisely controlled. Together, these results provide novel mechanistic insights into the physical control of genome organization and highlight the potential of decoding the DNA sequence logic in silico. AVAILABILITY: The Chiron3D pipeline is made available at https://github.com/BoevaLab/Chiron3D.

Chromatin↗
PubMed1999

Genetic analysis of B cell antigen receptor signaling.

In B lymphocytes, a signaling complex that contributes to cell fate decisions is the B cell antigen receptor (BCR). Data from knockout experiments in cell lines and mice have revealed distinct functions for the intracellular protein tyrosine kinases (Lyn, Syk, Btk) in BCR signaling and B cell development. Combinations of intracellular signaling pathways downstream of these PTKs determine the quality and quantity of BCR signaling. For example, concerted actions of the PLC-gamma 2 and PI3-K pathways are required for proper calcium responses. Similarly, the regulation of ERK and JNK responses involves both PLC-gamma 2 and GTPases pathways. Since the immune response in vivo is regulated by alteration of these signaling outcomes, achieving a precise understanding of intracellular molecular events leading to B lymphocyte proliferation, deletion, anergy, receptor editing, and survival still remains a challenge for the future.

Animals↗
PubMed2026

An efficient woody plant protoplast platform enables transgene-free multiplex genome editing and rapid trait validation in pear.

Multiplex editing is crucial for analysing complex multiple-gene traits in woody plants, yet its application remains limited because of low transformation efficiency and lengthy regeneration cycles. To overcome these barriers, in this study we establish an efficient protoplast isolation protocol for pear (Pyrus) that employs 1.0% cellulase R10 and 0.4% macerozyme R10 with an 8.5 h digestion. Its broad applicability using different digestion times across seven other economically important woody plants is demonstrated. Coupling a 40% PEG-4000-mediated transfection regimen with DNA-free CRISPR/Cas9 ribonucleoprotein (RNP) delivery enables multiplex genome editing in isolated protoplasts. Using this platform, we simultaneously disrupted the key components of the chloroplast division apparatus ARC3, PARC6, and FtsZ2-1a in Pyrus bretschneideri and found that it consistently reproduced macro-chloroplast abnormalities, confirming effective multigene perturbation within a single cellular context. Notably, failure of chloroplast division activated chloroplast-to-nucleus retrograde signaling, as evidenced by the induction of the nuclear stress-response genes RBOHD and ZAT12, a concomitant surge in reactive oxygen species, and progression to severe cellular deformation. Thus, our study establishes a rapid, cross-genus protoplast-RNP workflow that enables DNA-free multiplex editing and accelerates genotype-to-phenotype analyses in woody perennials. The approach provides a practical foundation for functional genomics and supports advances in non-transgenic precision breeding of tree crops.

Protoplasts↗
PubMed1997

Refined structure, DNA binding studies, and dynamics of the bacteriophage Pf3 encoded single-stranded DNA binding protein.

The solution structure of the 18-kDa single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been refined using 40 ms 15N- and 13C-edited NOESY spectra and many homo- and heteronuclear J-couplings. The structures are highly precise, but some variation was found in the orientation of the beta-hairpin denoted the DNA binding wing with respect to the core of the protein. Backbone dynamics of the protein was investigated in the presence and absence of DNA by measuring the R1 and R2 relaxation rates of the 15N nuclei and the 15N-1H NOE. It was found that the DNA binding wing is much more flexible than the rest of the protein, but its mobility is largely arrested upon binding of the protein to d(A)6. This confirms earlier hypotheses on the role of this hairpin in the function of the protein, as will be discussed. Furthermore, the complete DNA binding domain of the protein has been mapped by recording two-dimensional TOCSY spectra of the protein in the presence and absence of a small amount of spin-labeled oligonucleotide. The roles of specific residues in DNA binding were assessed by stoichiometric titration of d(A)6, which indicated for instance that Phe43 forms base stacking interactions with the single-stranded DNA. Finally, all results were combined to form a set of experimental restraints, which were subsequently used in restrained molecular dynamics calculations aimed at building a model for the Pf3 nucleoprotein complex. Implying in addition some similarities to the well-studied M13 complex, a plausible model could be constructed that is in accordance with the experimental data.

Crystallography, X-Ray↗
PubMed2001

A comparison of quantitative coronary angiography systems using a unique set of in vivo coronary stenosis images.

OBJECTIVE: To compare the accuracy and precision of measurements of minimum lumen diameter (MLD) among two existing, and one new, quantitative coronary angiography systems. MATERIALS AND METHODS: The analysis was performed using in vivo cinearteriograms of precisely drilled, radiolucent plastic beads that were inserted percutaneously into the coronary arteries of canines. The existing algorithms compared were the ArTrek and the Coronary Measurement System (CMS). The latter was applied in two modes: a mode based on a minimal cost analysis algorithm and a mode based on a gradient field transform. The new algorithm (CorTrek) was also applied in two modes: a mode called the ArTrek compatible mode and a mode known as the 'regression' mode. The latter mode uses a look-up table, based on a phantom calibration step, to readjust the measured MLD to overcome system nonlinearities (overestimation of small diameters and underestimation of large diameters). RESULTS: In the absence of editing, the optimal accuracy (no significant bias) was achieved with the ArTrek compatible algorithm (-0.03 mm for an MLD between 0.83 and 1.83 mm). All other algorithms showed significant under- or overestimation of the MLD within this range. The precision ranged from 0.18 to 0.40 mm without editing, and was best with the ArTrek algorithm. CONCLUSIONS: The present study provides in vivo validation and comparative performance characteristics of a new, accurate coronary quantitative angiography system.

Algorithms↗
PubMed2002

Chemical modification of nucleotide bases and mRNA editing depend on hexamer or nucleoprotein phase in Sendai virus nucleocapsids.

The minus-strand genome of Sendai virus is an assembly of the nucleocapsid protein (N) and RNA, in which each N subunit is associated with precisely 6 nt. Only genomes that are a multiple of 6 nt long replicate efficiently or are found naturally, and their replication promoters contain sequence elements with hexamer repeats. Paramyxoviruses that are governed by this hexamer rule also edit their P gene mRNA during its synthesis, by G insertions, via a controlled form of viral RNA polymerase "stuttering" (pseudo-templated transcription). This stuttering is directed by a cis-acting sequence (3' UNN UUUUUU CCC), whose hexamer phase is conserved within each virus group. To determine whether the hexamer phase of a given nucleotide sequence within nucleocapsids affected its sensitivity to chemical modification, and whether hexamer phase of the mRNA editing site was important for the editing process, we prepared a matched set of viruses in which a model editing site was displaced 1 nt at a time relative to the genome ends. The relative abilities of these Sendai viruses to edit their mRNAs in cell culture infections were examined, and the ability of DMS to chemically modify the nucleotides of this cis-acting signal within resting viral nucleocapsids was also studied. Cytidines at hexamer phases 1 and 6 were the most accessible to chemical modification, whereas mRNA editing was most extensive when the stutter-site C was in positions 2 to 5. Apparently, the N subunit imprints the nucleotide sequence it is associated with, and affects both the initiation of viral RNA synthesis and mRNA editing. The N-subunit assembly thus appears to superimpose another code upon the genetic code.

Base Sequence↗
PubMed1970

[Not Available].

Determination is made of the chronological limits and authorship of the widely known and popular during the Middle Ages treatise "De viribus herbarum", containing extensive medicamentous references. The author of the paper proceeds from the critical analyses of opinions and commentaries made by his predecessors, compares various editions of the translations of the poem, resorts to the linguistical method and determines with greater precision a number of additional indirect evidence and documental data. A discussion is made of the influence of the poem exerted upon the school of Salerno and the "Salernian Health Code".

Europe↗
PubMed1993

[How to edit a scientific article and where to publish it?].

The editorial aspect of scientific writing cannot be dissociated from the scientific message it carries. Precision, clearness and conciseness are qualities which express the level of rigour reached in the scientific work presented. By gaining knowledge of the universally accepted rules for medical writing, and abiding by them, authors can expect optimal diffusion of the scientific content and facilitate reader comprehension. This paper outlines the precise rules common to all scientific articles and gives details on the elements on which a biomedical journal bases its decision on whether or not to publish the results of the author's work.

Humans↗
PubMedDate not supplied

[Contribution of 3'---5'-exonuclease from rat liver nuclei in precision of DNA synthesis, catalyzed by mammalian DNA polymerase alpha].

Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of editing 3'-->5'exonucleolytic activity. Presumably this activity could be effected by the exonucleases non-associated covalently with DNA polymerases. Two 3'-->5'exonucleases with molecular masses of 40 and 50 kDa have been isolated from rat liver nuclei and purified to near homogeneity. They are shown to excise mismatched nucleotides from a poly [d(A-T)] template respectively 10- and 2-fold faster than the matched ones. Upon addition of any of these exonucleases to DNA polymerase alpha from rat liver or calf thymus, the fidelity of in vitro reproduction of primed DNA from bacteriophage phi X174 amber 3 is increased 5-10-fold, the levels of exonuclease and polymerase activities being approximately the same. The extrapolation of replication fidelity to cellular activities of the exonucleases and alpha-polymerase suggests that exonuclease proofreading augments the accuracy of DNA synthesis at least by three orders of magnitude.

Animals↗
PubMed1999

The versatility of paramyxovirus RNA polymerase stuttering.

Paramyxoviruses cotranscriptionally edit their P gene mRNAs by expanding the number of Gs of a conserved AnGn run. Different viruses insert different distributions of guanylates, e.g., Sendai virus inserts a single G, whereas parainfluenza virus type 3 inserts one to six Gs. The sequences conserved at the editing site, as well as the experimental evidence, suggest that the insertions occur by a stuttering process, i.e., by pseudotemplated transcription. The number of times the polymerase "stutters" at the editing site before continuing strictly templated elongation is directed by a cis-acting sequence found upstream of the insertions. We have examined the stuttering process during natural virus infections by constructing recombinant Sendai viruses with mutations in their cis-acting sequences. We found that the template stutter site is precisely determined (C1052) and that a relatively short region (approximately 6 nucleotides) just upstream of the AnGn run can modulate the overall frequency of mRNA editing as well as the distribution of the nucleotide insertions. The positions more proximal to the 5' AnGn run are the most important in this respect. We also provide evidence that the stability of the mRNA/template hybrid plays a determining role in the overall frequency and range of mRNA editing. When the template U run is extended all the way to the stutter site, adenylates rather than guanylates are added at the editing site and their distribution begins to resemble the polyadenylation associated with mRNA 3' end formation by the viral polymerase. Our data suggest how paramyxovirus mRNA editing and polyadenylation are related mechanistically and how editing sites may have evolved from poly(A)-termination sites or vice versa.

5' Untranslated Regions↗
PubMed2026

Ultrastructural Insights Into the Reproductive Anatomy and Eggs of Cotton Pink Bollworm, Pectinophora gossypiella Saunders (Lepidoptera: Gelechiidae).

The pink bollworm, Pectinophora gossypiella Saunders is a major pest of cotton, notorious for its high reproductive potential and rapid evolution of resistance to Bacillus thuringiensis (Bt) toxins. Despite its economic significance, detailed knowledge of its reproductive anatomy and egg ultrastructure has remained limited, constraining the development of advanced molecular control strategies such as CRISPR/Cas9-based genome editing. The present study provides the first comprehensive characterization of the reproductive system and egg surface morphology of P. gossypiella using stereomicroscopy and scanning electron microscopy (SEM) techniques. The male reproductive system consists of fused, bean-shaped testes, seminal vesicles, duplex and simplex ejaculatory ducts, and paired accessory glands. The female reproductive system comprises paired ovaries with four polytrophic ovarioles per ovary, lateral and common oviducts, accessory glands, corpus bursae, and spermathecal glands. Eggs are oval, dorsoventrally flattened, exhibit a reticulated chorion with distinct micropylar and aeropylar regions. SEM images revealed 6-9 rosette cells encircling a circular micropylar plate, 14-19 first order and 17-23 s order ribs, and 250-291 polygonal surface cells. The structural features of P. gossypiella eggs reveal key sites for sperm entry, aeropylar respiration, and candidate zones for microinjection in gene editing applications. These findings establish a morphological baseline critical for optimizing embryo manipulation and ribonucleoprotein (RNP) delivery in lepidopteran genome editing. This study represents a pioneering effort to integrate classical egg morphology with molecular entomology, thereby advancing precision genetic interventions aimed at resistance management and population suppression in P. gossypiella.

Animals↗
PubMed1998

Construction of a 2.5-Mb integrated physical and gene map of distal 21q22.3.

The gene-rich telomeric region of 21q harbors several loci relevant to human diseases including autoimmune polyglandular disease type I, nonsyndromic deafness, Knobloch syndrome, holoprosencephaly, and bipolar affective disorder. A contig of genomic clones in this region would facilitate the isolation of these genes. However, distal 21q22.3 has yet been poorly mapped, presumably due to the presence of sequences that are underrepresented in yeast artificial chromosome (YAC) libraries. We generated a framework of YACs and used these clones as starting points for the isolation of a combination of bacterial artificial chromosome clones, P1-derived artificial chromosome clones, and cosmid clones by chromosome walking procedures. These studies resulted in the construction of a high-resolution contig map spanning the 2.5-Mb region from PFKL to the telomere, approximately 2 Mb of which are covered by ready-to-sequence contigs. Within this map we determined the location and relative distance of 21 markers. These include 9 established genetic markers, the order of which is cen-PFKL-D21S154-D21S170-D21S171-D21S1903- D21S1897- D21S112-D21S1446-D21S1575-tel. Moreover, we established the precise map position of 13 genes and 4 ESTs including the recently isolated genes C21ORF2, SMT3H1, RNA editing deaminase 1 (ADARB1), folate transporter (SLC19A1), COL18A1, lanosterol synthase (LSS-PEN), pericentrin (PCNT), and arginine methyltransferase (HRMT1L1). This integrated map provides a useful resource for the mapping and isolation of disease genes and for the construction of a complete transcription map of distal 21q as well as for large-scale sequencing efforts.

Chromosome Mapping↗
PubMed2025

General and robust sample preparation strategies for cryo-EM studies of CRISPR-Cas9 and Cas12 enzymes.

Cas9 and Cas12 are RNA-guided DNA endonucleases derived from prokaryotic CRISPR-Cas adaptive immune systems that have been repurposed as versatile genome-engineering tools. Computational mining of genomes and metagenomes has expanded the diversity of Cas9 and Cas12 enzymes that can be used to develop versatile, orthogonal molecular toolboxes. Structural information is pivotal to uncovering the precise molecular mechanisms of newly discovered Cas enzymes and providing a foundation for their application in genome editing. In this chapter, we describe detailed protocols for the preparation of Cas9 and Cas12 enzymes for cryo-electron microscopy. These methods will enable fast and robust structural determination of newly discovered Cas9 and Cas12 enzymes, which will enhance the understanding of diverse CRISPR-Cas effectors and provide a molecular framework for expanding CRISPR-based genome-editing technologies.

Cryoelectron Microscopy↗
PubMed1994

Definitions of panic attacks and panic disorder in the DSM-IV: implications for research.

The authors review various definitions of panic and recent conceptual and empirical evidence that led to the revised definitions of panic in the Diagnostic and Statistical Manual of Mental Disorders. Fourth Edition (DSM-IV; American Psychiatric Association, 1994). The DSM-IV definitions of panic are more precise and more empirically based than those contained in the DSM-III-R, but are designed primarily for clinicians rather than researchers. Thus, it is possible that definitions of panic used in research under the DSM-IV system will encompass a heterogeneous mix of emotional phenomena. Recommendations on essential components of a conservative definition of panic are proposed for research purposes. Implications of this definition for limited symptom attacks and "non-fearful" panic are elaborated and related to final definitions of panic attacks and panic disorder in the DSM-IV.

Electromyography↗
PubMedDate not supplied

Single-laboratory validation of EPA Method 8150 for determination of chlorinated herbicides in hazardous waste.

Method 8150, published in the second edition of Test Methods for Evaluating Solid Waste, Manual SW-846, required optimization, ruggedness testing, linearity determinations, precision tests, bias testing, gas chromatographic/mass spectrometric confirmation, and quality control guidelines for validation of the protocol. This single-laboratory validation, which is applicable to the determination of the herbicides dicamba, silvex, 2,4-D, 2,4-DB, 2,4,5-T, dinoseb, MCPP, MCPA, and dichlorprop in hazardous waste extracts, was completed and is described in this report. Final ruggedness testing of the optimized procedure gave a mean recovery of 89.3% with a standard deviation of 4.3%. Percent relative standard deviations are less than 10 (n = 20, each analyte) over a 10(2) linear range of concentration for MCPP and MCPA and over a 10(3) linear range of concentration for the other target herbicide esters. Instrumental detection limits for electron capture detection and mass spectrometric identity confirmation were determined and found to be matrix-dependent.

Chromatography, Gas↗
PubMed1982

A generalized plotting program, written in BASIC for scientific data.

A software package for the hardcopy graphic display of two-dimensional data had been developed. It is written in BASIC and is compatible with a microcomputer interfaced to an intelligent digital plotter. The core of the package is a plotting program. It features a command menu that displays 15 user-controllable parameters. Most of these parameters are automatically tabulated and can be changed in an interactive manner. The data which is plotted is either entered by hand or read from pre-existing data files. The package has several other important features. One subroutine within the plotting program determines and draws the best fit of a curve through a set of points. Using another program, the user can edit and manipulate the information contained in data files. All the program commands are written in simple and precise language, therefore the user needs no previous experience with computers. Furthermore the programs can be easily modified to suit personalized needs.

Computers↗