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At least 1,207 records · Page 67Linked to original sources
PubMed1996

Overexpression of APOBEC-1 results in mooring sequence-dependent promiscuous RNA editing.

Apolipoprotein B (apoB) RNA editing involves site-specific deamination of a cytidine to a uridine. A mooring sequence, a spacer region, and a regulator region are components of the apoB RNA editing motif of which only the mooring sequence is both necessary and sufficient for editosome assembly and editing. The catalytic component of the editosome is APOBEC-1. In rat hepatoma, stable cell lines, overexpression of APOBEC-1 resulted in 3 6-fold stimulation of the editing efficiency on either rat endogenous apoB RNA or transiently expressed human apoB RNA. In these cell lines, cytidines in addition to the one at the wild type site were edited. The occurrence and efficiency of this "promiscuous" editing increased with increasing expression of APOBEC-1. Promiscuous editing was restricted to cytidines 5' of the mooring sequence and only occurred on RNAs that had been edited at the wild type site. Moreover, RNAs with mutant editing motifs supported high efficiency but low fidelity editing in the presence of high levels of APOBEC-1. This study demonstrates that overexpression of APOBEC-1 can increase the efficiency of site-specific editing but can also result in promiscuous editing.

APOBEC-1 Deaminase↗
PubMed1991

A practical approach to acid-base disorders.

This discussion was selected from the weekly staff conferences in the Department of Medicine, University of California, San Francisco. Taken from a transcription, it has been edited by Homer A. Boushey, MD, Professor of Medicine, and Nathan M. Bass, MD, PhD, Associate Professor of Medicine, under the direction of Lloyd H. Smith, Jr, MD, Professor of Medicine and Associate Dean in the School of Medicine.

Acid-Base Imbalance↗
PubMed1999

An ounce of prevention ... what are the returns? Second edition, 1999.

CONTEXT: Because human and financial resources are limited, health efforts must focus on prevention strategies that yield the most benefit for the investment. Many current strategies identified in the literature offer opportunities to promote health at a reasonable cost. OBJECTIVE: To present a literature-based review of evidence demonstrating that prevention can be an effective and wise use of resources through CDC's An Ounce of Prevention ... What Are the Returns? Second Edition. DESIGN: Systematic review of cost-effectiveness literature for a selected group of prevention strategies. SETTING: Prevention strategies relevant to the U.S. population. RESULTS: Data indicate that the health conditions considered can be addressed through prevention strategies that are either cost effective or cost saving. CONCLUSIONS: An Ounce of Prevention ... What Are the Returns? Second Edition can be used to conveniently access information on prevention strategies, the diseases and injuries they address, and their cost effectiveness. It also complements other comprehensive prevention guides. However, limitations of the available cost-effectiveness studies indicate that standardized procedures should be followed for studies of all recommended prevention strategies. Researchers must standardize review procedures to improve both the quality and comparability of studies.

Adolescent↗
PubMedDate not supplied

Prototype decision support system for a differential diagnosis of psychotic, mood, and organic mental disorders: Part II.

This paper presents a prototype parallel-processing decision support system, based on the ALICE graph reduction machine, for the differential diagnosis of psychotic, mood, and organic mental disorders in accordance with the American Psychiatric Association's revised third edition of the Diagnostic and Statistical Manual of Mental Disorders. The paper extends the authors' earlier work where the same domain was implemented on an expert-system shell using a rule-based representation.

Computer Graphics↗
PubMed1992

High frequency of single-base transitions and extreme frequency of precise multiple-base reversion mutations in poliovirus.

We employed independent clones of a temperature-sensitive mutant of type 1 poliovirus, 3AB-310/4, to quantitate the frequency of specific U----C transitions at nucleotide 5310, within the genomic region encoding polypeptide 3AB, which is involved in the initiation of RNA replication. Only this U----C base substitution restores the wild-type phenotypic ability to form plaques at 39 degrees C; the other two base substitutions at this site are lethal. The observed frequency of this specific transition averaged 2 x 10(-5), and all revertant viruses forming plaques at 39 degrees C contained the expected cytidine at nucleotide 5310. Incredibly, only 3 of 10 revertants exhibited this one specific U----C transition whereas 7 of 10 exhibited this same transition plus four additional base substitutions that precisely reverted temperature-sensitive 3AB-310/4 to wild-type poliovirus sequence (these latter four mutations had been introduced into 3AB-310/4 as silent third base mutations to provide new restriction sites in infectious cDNAs). No other mutations were detected in this polypeptide 3AB domain in either the single-base or the precise 5-base revertants. No intermediates were seen; all revertants exhibited either the single U----C transition at nucleotide 5310 or the same transition plus four precise reversions to the wild-type sequence at sites 8, 11, 43, and 46 bases distant from nucleotide 5310. Similar results were obtained after transfection of cDNA-derived transcripts. We discuss possible mechanisms for our data. These include (but may not be limited to) error-prone polymerase activity, sequential RNA recombination events joining independent mutations, or some unusual RNA editing process.

Base Sequence↗
PubMed1997

Disruption of a gene encoding a novel mitochondrial DEAD-box protein in Trypanosoma brucei affects edited mRNAs.

The majority of mitochondrial pre-mRNAs in kinetoplastid protozoa such as Trypanosoma, Leishmania, and Crithidia are substrates of a posttranscriptional processing reaction referred to as RNA editing. The process results in the insertion and, to a lesser extent, deletion of uridylates, thereby completing the informational content of the mRNAs. The specificity of the RNA editing reaction is provided by guide RNAs (gRNAs), which serve as templates for the editing apparatus. In addition, the process relies on mitochondrial proteins, presumably acting within a high-molecular-mass ribonucleoprotein complex. Although several enzymatic activities have been implicated in the editing process, no protein has been identified to date. Here we report the identification of a novel mitochondrial DEAD-box protein, which we termed mHel61p. Disruption of the mHEL61 alleles in insect-stage Trypanosoma brucei cells resulted in a reduced growth rate phenotype. On a molecular level, the null mutant showed significantly reduced amounts of edited mRNAs, whereas never-edited and nuclear mRNAs were unaffected. Reexpression of mHel61p in the knockout cell line restored the ability to efficiently synthesize edited mRNAs. The results suggest an involvement of mHel61p in the control of the abundance of edited mRNAs and thus reveal a novel function for DEAD-box proteins.

Alleles↗
PubMed1991

In vitro mutagenesis in the lacI gene of Escherichia coli: fate of 3'-terminal mispairs versus internal base mispairs in a transfection assay.

The fate of G.T mismatches and frameshifts, present at the 3'-terminus of primer-template or internally, has been studied with a combined transfection and electrophoretic assay following in vitro polymerization by DNA polymerase I (Klenow enzyme) of Escherichia coli. Several synthetic oligodeoxynucleotide primers were synthesized and annealed to uracil-containing single-stranded DNA of M13 phage bearing the lacI gene, to produce 1-3 consecutive G.T mismatches in the middle of the duplex region or at the 3'-OH end of the primer. Additional mismatched primer-templates were prepared, in which the primer had a deleted nucleotide, an extra nucleotide or both G.T mismatch and an extra nucleotide. The extension or degradation of these primers during in vitro DNA synthesis in the presence of all 4 dNTPs ('complete' reaction) or in the absence of dATP ('-A' reaction) was monitored by gel electrophoresis. Duplex DNA products were used in a transfection assay and the nucleotide changes in i-mutant progeny were determined by sequence analysis. The results suggest that whereas a single 3'-terminal G.T mismatch is relatively stable in chain elongation by Klenow enzyme, multiple terminal G.T mismatches are degraded by the 3'-exonuclease activity of this polymerase prior to primer extension. This editing activity is increased with the number of 3'-terminal mispairs. Single, double and triple T----C base substitutions were efficiently recovered when the mismatches occurred internally. Also, single-base eliminations or additions were readily recovered when the mutagenic primers contained an internal base deletion or addition, respectively. When products of the '-A' misincorporation reaction (catalyzed by Klenow enzyme) were assayed by transfection, base substitutions (exclusively T----C), but no frameshifts, were recovered. The results indicate that the absence of multiple tandem base substitutions among i- mutants recovered following primer elongation under mutagenic 'minus' conditions was due to the efficient action of the 3'-exonuclease activity of the Klenow enzyme on multiple terminal mismatches during in vitro polymerization, rather than to in vivo events (lack of expression or occurrence of mismatch repair) in the M13-lacI transfection assay.

Bacterial Proteins↗
PubMed1999

Goldfish brain GluR2: multiple forms, RNA editing, and alternative splicing.

cDNA coding for a full-length goldfish alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit, GluR2, was cloned by screening unidirectional and bidirectional goldfish brain cDNA libraries. The clone has an open reading frame of 2679 bp, encoding a protein of 893 amino acids. Partial cDNA clones for three other GluR2 subunits were identified. GluR2 from goldfish brain exhibits RNA editing and alternative splicing. RNA editing occurred at the two sites demonstrated for mammalian GluR2 (Q/R and R/G). Unlike rat GluR2, GFGluR2a has a long (68 amino acids) C-terminal tail. Analysis of genomic DNA suggests that an alternatively spliced shorter C-terminal tail can be produced, similar to the rat protein. Thus, in goldfish brain, GluR2 exhibits diversity arising from multiple subtypes, RNA editing, and alternative splicing.

Alternative Splicing↗
PubMed2001

Molecular Biocomputing Suite: a word processor add-in for the analysis and manipulation of nucleic acid and protein sequence data.

In all fields of molecular biology, researchers are increasingly challenged by experiments planned and evaluated on the basis of nucleic acid and protein sequence data generally retrieved from public databases. Despite the wide spectrum of available Web-based software tools for sequence analysis, the routine use of these tools has disadvantages, particularly because of the elaborate and heterogeneous ways of data input, output, and storage. Here we present a Visual Basic-encoded Microsoft Word Add-In, the Molecular BioComputing Suite (MBCS), available at the BioTechniques Software Library (www.BioTechniques.com). The MBCS software aims to manage and expedite a wide range of sequence analyses and manipulations using an integrated text editor environment including menu-guided commands. Its independence of sequence formats enables MBCS to be used as a pivotal application between other software tools for sequence analysis, manipulation, annotation, and editing.

Amino Acid Sequence↗
PubMedDate not supplied

RNA editing site recognition in higher plant mitochondria.

RNA editing is a process by which genomically encoded cytidines are converted to uridines in plant mitochondrial transcripts. This conversion usually changes the amino acid specified by a codon and converts an "aberrant" residue to the evolutionarily conserved amino acid. The selection of the edited cytidine is highly specific. The cis-acting sequences for editing site recognition have been examined in ribosomal protein S12 (rps12) transcripts and in transcripts for a second copy of an internal portion of the ribosomal protein S12 (rps12b). rps12b was created by recombination at 7 and 9 nucleotide sequences that included editing sites I and IV of rps12, thus affording an opportunity to study the editing of chimeric transcripts with rearrangement very near C to U editing sites. Rearrangements downstream of editing site IV did not affect the editing of that sequence, while rearrangement upstream of editing site I ablated editing at that cytidine residue. Secondary structure predictions indicated that RNA structure did not correlate with the editing of these substrates. These results taken together with other studies in the literature suggest that RNA editing site recognition is primarily dependent on the 5' flanking RNA sequence.

Amino Acid Sequence↗
PubMed2006

Comparison of stage-specific outcome of breast cancer based on 5th and 6th AJCC staging system.

BACKGROUND AND OBJECTIVES: To investigate the appropriateness of the new staging system (AJCC 6th edition) for breast carcinoma. METHODS: We reviewed the clinicopathologic data of 1,768 breast cancer patients, and their disease stages were re-categorized by the new system. The overall survival (OS) and distant relapse free survival (DRFS) rates were compared between those patients whose stages by the old system (AJCC 5th edition) remained the same (the remainders) and those patients whose stages moved up (the upstaged cases) as well as between the subgroups in the new system. RESULTS: The 10-year DRFS rates of the upstaged cases in each stage were poorer than those of the remainders, and statistical significance was demonstrated for stage IIB and stage IIIA. The 10-year OS rates were also poorer in the upstaged cases, and statistical significance was demonstrated for stage IIIA. Subgroup analysis within the new system between the node-negative versus node-positive subgroups in stages IIA and IIB showed a significant OS difference. The DRFS difference was also shown between the subgroups in stage IIA. CONCLUSIONS: The new staging system seems to more accurately reflect disease outcome, however, a re-evaluation might be required to reflect the impact of nodal involvement upon the new staging system.

Breast Neoplasms↗
PubMed2025

Validation of caprine H11 and the Rosa26 platform for transgene integration via CRISPR-based system: investigations on stable transgene expression and genetic biosafety.

CRISPR/Cas9 technology is an efficient tool for site-specific livestock gene editing. However, to minimize potential disruption of host genome function, exogenous genes should be integrated into well-characterized genomic loci, such as H11 or Rosa26, which have been empirically validated for stable transgene expression. This study established a multi-dimensional assessment system to evaluate the biological applicability of the H11 locus and the widely used Rosa26 targeting platform as sites for targeted integration of exogenous genes in goats. Donor cells carrying the enhanced green fluorescent protein (EGFP) reporter gene at the H11 and Rosa26 loci were generated via CRISPR/Cas9-mediated homology-directed repair; this was followed by somatic cell nuclear transfer to produce transgenic cloned embryos and healthy offspring. Multi-dimensional analyses revealed the following. At the cellular level, there was stable and efficient EGFP expression at integration sites, with donor cells maintaining normal cell cycle progression, proliferation capacity, and apoptosis levels, and with no alterations in the transcriptional integrity of adjacent genes. At the embryonic level, there was sustained EGFP expression across pre-implantation embryonic stages, with developmental metrics statistically indistinguishable from wild-type embryos. Finally, at the individual level, cloned offspring exhibited growth phenotypes consistent with wild-type counterparts, and EGFP showed broad-spectrum expression in eight tissues. This study establishes the first CRISPR/Cas9-based crossscale (cellular-embryonic-individual) validation in goats, demonstrating that the H11 and Rosa26 loci support efficient and stable transgene integration in goats. These results provide a precise and predictable technical framework for livestock genetic improvement.

Animals↗
PubMed2000

Editing data: what difference do consistency checks make?

In 1998, the Florida Department of Health undertook a self-administered school-based survey of tobacco use, attitudes, and behaviors among nearly 23,000 public school students in grades 6-12. The survey design did not use skip patterns; therefore, students had multiple opportunities to contradict themselves. By using examples from the high school portion (grades 9-12) of the survey, the authors examined five possible approaches to handling data inconsistencies and the effect that each has on point estimates. Use of these approaches resulted in point estimates of current cigarette use ranging from 25.6% to 29.7%. The number of missing respondents varied from 33 (less than 1%) to 1,374 (13%), depending on which approach was used. After stratification by gender and race, the prevalence estimates changed marginally for girls but strikingly for boys. Non-Hispanic White students were substantially more likely than non-Hispanic Black students to report current cigarette use, but the magnitude of this difference varied significantly according to the analytical approach used. The approach used to check data consistency may influence point estimates and comparability with other studies. Therefore, this issue should be addressed when findings are reported.

Adolescent↗
PubMed2026

Efficient homologous replacement and deletion of large genomic fragments through template-jumping prime editing in rice.

Homologous replacement of genomic sequences with large DNA fragments (> 100 bp) holds great potential for crop breeding, yet an efficient method to achieve such edits is lacking in plants. Here, in rice, we developed template-jumping prime editing (TJ-PE), a recently reported PE strategy for large targeted insertion, as an efficient tool for homologous replacement with DNA fragments ranging from dozens to hundreds of base pairs, and using TJ-PE, we replaced genomic fragments of up to 340 bp with homologous fragments of the same length. In addition, our TJ-PE tool also enabled precise deletion of 944- to 2024-bp fragments in rice, with efficiencies of up to 34.6% for c. 2000-bp precise deletions. Collectively, this study expands the editing scope of PE in rice and establishes TJ-PE as a generalist tool for precise deletion and replacement of large DNA fragments.

Oryza↗
PubMed1993

Implications of novel guide RNA features for the mechanism of RNA editing in Crithidia fasciculata.

We have determined the relative steady state concentration of the two Crithidia fasciculata guide (g)RNAs involved in editing the two domains of mRNAs for NADH dehydrogenase (ND) subunit 7. We found that, although there was an 8-fold difference between the molar ratio of these two gRNAs relative to the (pre)-mRNA, the two domains are edited with a very similar frequency (around 50%). Also, for the editing of a given domain, many gRNA species exist with the same 5' end but with a different 3' uridylation site. Approximately 20% of these short gRNAs do not contain the information required for editing a complete domain, which may explain the high incidence of partially edited RNAs. Remarkably, genomically encoded Us are missing from two sites of a few of the gRNAs involved in editing apocytochrome b RNA. We speculate that these species are created by editing-like events. Both the short and complete forms of the ND7 gRNAs are found in chimeric molecules, in which the gRNA is covalently linked via its 3'-terminus to an editing site of pre-edited ND7 RNA. Some features of the chimeric molecules are at odds with current models of RNA editing: (i) U residues are completely absent from the connecting sequence of a number of these molecules, (ii) the ND7 gRNAs are frequently hooked up to the wrong editing domain of ND7 RNA, although other gRNAs are not found at these positions and (iii) in some chimeric molecules the gRNA appears to be linked to the 5' end of pre-edited RNA.

Animals↗
PubMed1993

Sequence determination of the P gene of simian virus 41: presence of irregular deletions near the RNA-editing sites of paramyxoviruses.

The complete nucleotide sequence of the P gene of simian virus 41 (SV41) was determined. The gene was found to be 1406 nucleotides long and to contain a relatively small open reading frame encoding a cysteine-rich V protein with a calculated M(r) of 24076. We have demonstrated that RNA-editing events occur in SV41 P gene transcripts and that the ratio of edited mRNAs to faithfully copied mRNA (P-mRNA:V-mRNA) is about 1:5 at either 24 or 40 h post-infection. The mRNA with two G insertions was capable of encoding a P protein of 395 amino acids with a predicted M(r) of 41,992. A kinetic study of P and V proteins by Western blot analysis showed that in virus-infected cells the amounts of both proteins were almost equal although the V-mRNA was considerably more abundant than the P-mRNA. Alignment of the SV41 P and V proteins with those of nine other paramyxoviruses demonstrated that irregular gaps were present around the RNA-editing sites.

Amino Acid Sequence↗
PubMed2004

NMR spectroscopic filtration of polypeptides and proteins in complex mixtures.

Due to the inherent complexity of the natural biological environment, most studies on polypeptides, proteins and nucleic acids have so far been performed in vitro, away from physiologically relevant conditions. Nuclear magnetic resonance is an ideal technique to extend the in vitro analysis of simple model systems to the more complex biological context. This work shows how diffusion-based spectroscopic selection can be combined with isotopic labeling to tackle and optimize the NMR analysis of specific macromolecules in multicomponent mixtures. Typical media include cell-free systems containing overexpressed proteins, lysates and proteolytic mixtures. We present a few variants of diffusion-edited HSQC pulse sequences for the selective spectroscopic detection of protein and polypeptide resonances within complex mixtures containing undesired species of smaller molecular weight. Due to diffusion-based filtering, peak intensities of fast diffusing small molecules are attenuated more than peaks due to large molecules. The basic sequence, denoted as PFGSTE-HSQC, combines translational diffusion-ordering with two dimensional heteronuclear single quantum correlation spectroscopy. The GCSTE-HSQC and BPPSTE-HSQC sequences include bipolar gradients and are therefore suitable for both diffusion-based filtering and determination of diffusion coefficients of individual mixture components. Practical applications range from protein stability/folding investigations in physiologically relevant contexts to prescreening of tertiary fold and resonance assignments in structural genomics studies. A few applications of diffusion-edited HSQC to an E. coli cell lysate containing the (15)N-labeled B domain of streptococcal protein G (GB1), and to a (15)N-labeled N-acetylglycine/apomyoglobin mixture are presented. In addition, we provide specific guidelines for experimental setup and parameter optimization.

Data Interpretation, Statistical↗
PubMed2003

Computational geometry for patient-specific reconstruction and meshing of blood vessels from MR and CT angiography.

Investigation of three-dimensional (3-D) geometry and fluid-dynamics in human arteries is an important issue in vascular disease characterization and assessment. Thanks to recent advances in magnetic resonance (MR) and computed tomography (CT), it is now possible to address the problem of patient-specific modeling of blood vessels, in order to take into account interindividual anatomic variability of vasculature. Generation of models suitable for computational fluid dynamics is still commonly performed by semiautomatic procedures, in general based on operator-dependent tasks, which cannot be easily extended to a significant number of clinical cases. In this paper, we overcome these limitations making use of computational geometry techniques. In particular, 3-D modeling was carried out by means of 3-D level sets approach. Model editing was also implemented ensuring harmonic mean curvature vectors distribution on the surface, and model geometric analysis was performed with a novel approach, based on solving Eikonal equation on Voronoi diagram. This approach provides calculation of central paths, maximum inscribed sphere estimation and geometric characterization of the surface. Generation of adaptive-thickness boundary layer finite elements is finally presented. The use of the techniques presented here makes it possible to introduce patient-specific modeling of blood vessels at clinical level.

Algorithms↗