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At least 325 records · Page 18Linked to original sources
PubMed2004

Complete genome sequence of Fer-de-Lance virus reveals a novel gene in reptilian paramyxoviruses.

The complete RNA genome sequence of the archetype reptilian paramyxovirus, Fer-de-Lance virus (FDLV), has been determined. The genome is 15,378 nucleotides in length and consists of seven nonoverlapping genes in the order 3' N-U-P-M-F-HN-L 5', coding for the nucleocapsid, unknown, phospho-, matrix, fusion, hemagglutinin-neuraminidase, and large polymerase proteins, respectively. The gene junctions contain highly conserved transcription start and stop signal sequences and tri-nucleotide intergenic regions similar to those of other Paramyxoviridae. The FDLV P gene expression strategy is like that of rubulaviruses, which express the accessory V protein from the primary transcript and edit a portion of the mRNA to encode P and I proteins. There is also an overlapping open reading frame potentially encoding a small basic protein in the P gene. The gene designated U (unknown), encodes a deduced protein of 19.4 kDa that has no counterpart in other paramyxoviruses and has no similarity with sequences in the National Center for Biotechnology Information database. Active transcription of the U gene in infected cells was demonstrated by Northern blot analysis, and bicistronic N-U mRNA was also evident. The genomes of two other snake paramyxovirus genotypes were also found to have U genes, with 11 to 16% nucleotide divergence from the FDLV U gene. Pairwise comparisons of amino acid identities and phylogenetic analyses of all deduced FDLV protein sequences with homologous sequences from other Paramyxoviridae indicate that FDLV represents a new genus within the subfamily Paramyxoviridae. We suggest the name Ferlavirus for the new genus, with FDLV as the type species.

Amino Acid Sequence↗
PubMed1990

Characterization of the apolipoprotein B mRNA editing activity in enterocyte extracts.

Apolipoprotein B (apoB) circulates in human plasma as two isoforms, apoB-100 (512 kDa) and apoB-48 (242 kDa). ApoB-48 is generated by a novel RNA editing mechanism which post-transcriptionally modifies apoB mRNA in the intestine by converting cytidine at nucleotide 6666 to uridine. This converts codon 2153 from glutamine (CAA) to a premature stop codon (UAA). To characterize the activity which edits apoB mRNA, extracts were prepared from enterocytes isolated from baboon small intestine. These extracts efficiently edit synthetic apoB RNA in vitro. Editing was detected by primer extension, and the specificity of the reaction was confirmed by DNA sequencing. Extracts prepared from other baboon tissues did not edit apoB RNA in vitro. The editing activity was partially purified by chromatography of the enterocyte extracts on DEAE-cellulose. The activity is sensitive to proteinase K but resistant to micrococcal nuclease and has an average molecular mass of 125 kDa when analyzed by gel filtration chromatography.

Animals↗
PubMed1995

Rapid corepressor exchange from the trp-repressor/operator complex: an NMR study of [ul-13C/15N]-L-tryptophan.

[ul-13C/15N]-L-tryptophan was prepared biosynthetically and its dynamic properties and intermolecular interaction with a complex of Escherichia coli trp-repressor and a 20 base-pair operator DNA were studied by heteronuclear isotope-edited NMR experiments. The resonances of the free and bound corepressor (L-Trp) were unambiguously identified from gradient-enhanced 15N-1H HSQC, 13C-1H HSQC, 13C- and 15N-edited 2D NOESY spectra. The exchange off-rate of the corepressor between the bound and free states was determined to be 3.4 +/- 0.52 s-1 at 45 degrees C, almost three orders of magnitude faster than the dissociation of the protein-DNA complex. Examination of the experimental NOE buildup curves indicates that it may be desirable to use longer mixing times than would normally be used for a large molecule, in order to detect weak intermolecular NOEs in the presence of exchange. Intermolecular NOEs from bound corepressor to trp-repressor and DNA were analyzed with respect to the mechanism of ligand exchange. This analysis suggests that, in order for the ligand to diffuse out of the complex, there must be significant movement or 'breathing' of the protein and/or DNA.

Bacterial Proteins↗
PubMed1991

Expression of the wheat mitochondrial nad3-rps12 transcription unit: correlation between editing and mRNA maturation.

In plant mitochondria, RNA editing involves the conversion of cytidines in the genomic DNA into uridines in the corresponding RNA. Analysis of cDNAs prepared by reverse transcription of mitochondrial RNAs has shown that partially edited RNAs are present in wheat mitochondria. The extent of this partial editing as well as its potential influence on the corresponding protein sequence were studied along with the expression of a wheat mitochondrial locus. The sequence, expression, and RNA editing of the wheat mitochondrial transcription unit containing four open reading frames (nad3, rps12, orf299, orf156), all cotranscribed into a same predominant precursor RNA, have been studied. The product of orf156 is an 18-kD mitochondrial membrane protein of unknown function, whereas the product of orf299 could not be detected and this sequence seems to be a pseudogene. Sequences of cDNA clones derived by the polymerase chain reaction technique show that nad3, rps12, and orf156 transcripts are edited, whereas orf299 is not edited, except for a sequence identical to part of the coxII gene. Analysis of cDNA clones obtained from the precursor RNA shows the presence of a large number of partially edited nad3-rps12 transcripts with no evident polarity for the editing process. This shows that RNA editing is a post-transcriptional event. In addition, study of partial editing at the level of precursor, mature, and polysomal transcripts shows that mainly mature, completely edited sequences are used for translation. Deletions of a nucleotide at editing sites were observed in a number of cDNA clones, suggesting that C----U RNA editing in plant mitochondria would be achieved by nucleotide replacement.

Amino Acid Sequence↗
PubMed2023

Polγ coordinates DNA synthesis and proofreading to ensure mitochondrial genome integrity.

Accurate replication of mitochondrial DNA (mtDNA) by DNA polymerase γ (Polγ) is essential for maintaining cellular energy supplies, metabolism, and cell cycle control. To illustrate the structural mechanism for Polγ coordinating polymerase (pol) and exonuclease (exo) activities to ensure rapid and accurate DNA synthesis, we determined four cryo-EM structures of Polγ captured after accurate or erroneous incorporation to a resolution of 2.4-3.0 Å. The structures show that Polγ employs a dual-checkpoint mechanism to sense nucleotide misincorporation and initiate proofreading. The transition from replication to error editing is accompanied by increased dynamics in both DNA and enzyme, in which the polymerase relaxes its processivity and the primer-template DNA unwinds, rotates, and backtracks to shuttle the mismatch-containing primer terminus 32 Å to the exo site for editing. Our structural and functional studies also provide a foundation for analyses of Polγ mutation-induced human diseases and aging.

Humans↗
PubMed1985

Translesion DNA synthesis: polymerase response to altered nucleotides.

A system for the determination of the specificity of incorporation opposite lesions during DNA synthesis past damaged bases has been used as a model of mutation. The system, based on the dideoxynucleotide sequence method, uses lesions in the template strand as chain terminators. As a first approximation, such lesions constitute non-instructive sites in the DNA. DNA synthesis catalysed by bacteriophage T4 DNA polymerase terminates one nucleotide 3' to the lesion on the template strand. With other polymerases, synthesis may terminate opposite the lesion. The details of termination site depend on the enzyme, the metal ion (Mg2+ or Mn2+), the lesion and the particular nucleotide sequence. The sequence effect for termination on normal templates has two components, one ascribable to secondary structure, the other intrinsic to the sequence itself. DNA molecules terminated before lesions may be used as substrates in 'second stage' reactions in which elongation can be detected on the addition of particular dNTPs (deoxynucleoside triphosphates) to a reaction mixture. The specificity of elongation depends on the polymerase, on the activity of the 3'--greater than 5' editing nuclease and on the particular lesion. DNA polymerases have a preference for the addition of purines, particularly adenine, opposite non-instructional sites. This preference suggests an explanation for the specificity of base substitution mutations: treatments that produce non-instructional sites from purines will lead to transversions, treatments that affect pyrimidines lead to transitions. Even though a base is added opposite a lesion, further elongation may be rate limiting. Whether or not elongation occurs is dependent on the sequence 5' to the lesion on the template strand. The interactions of the factors affecting bypass: polymerase, lesion and sequence, may well result in an idiosyncratic behaviour for each mutable site.

Animals↗
PubMed2005

Spectroscopic characterization of interstrand carbinolamine cross-links formed in the 5'-CpG-3' sequence by the acrolein-derived gamma-OH-1,N2-propano-2'-deoxyguanosine DNA adduct.

The interstrand N2,N2-dG DNA cross-linking chemistry of the acrolein-derived gamma-OH-1,N2-propanodeoxyguanosine (gamma-OH-PdG) adduct in the 5'-CpG-3' sequence was monitored within a dodecamer duplex by NMR spectroscopy, in situ, using a series of site-specific 13C- and 15N-edited experiments. At equilibrium 40% of the DNA was cross-linked, with the carbinolamine form of the cross-link predominating. The cross-link existed in equilibrium with the non-crosslinked N2-(3-oxo-propyl)-dG aldehyde and its geminal diol hydrate. The ratio of aldehyde/diol increased at higher temperatures. The 1,N2-dG cyclic adduct was not detected. Molecular modeling suggested that the carbinolamine linkage should be capable of maintaining Watson-Crick hydrogen bonding at both of the tandem C x G base pairs. In contrast, dehydration of the carbinolamine cross-link to an imine (Schiff base) cross-link, or cyclization of the latter to form a pyrimidopurinone cross-link, was predicted to require disruption of Watson-Crick hydrogen bonding at one or both of the tandem cross-linked C x G base pairs. When the gamma-OH-PdG adduct contained within the 5'-CpG-3' sequence was instead annealed into duplex DNA opposite T, a mixture of the 1,N2-dG cyclic adduct, the aldehyde, and the diol, but no cross-link, was observed. With this mismatched duplex, reaction with the tetrapeptide KWKK formed DNA-peptide cross-links efficiently. When annealed opposite dA, gamma-OH-PdG remained as the 1,N2-dG cyclic adduct although transient epimerization was detected by trapping with the peptide KWKK. The results provide a rationale for the stability of interstrand cross-links formed by acrolein and perhaps other alpha,beta-unsaturated aldehydes. These sequence-specific carbinolamine cross-links are anticipated to interfere with DNA replication and contribute to acrolein-mediated genotoxicity.

Acrolein↗
PubMed1997

Efficient enzymatic synthesis of 13C,15N-labeled DNA for NMR studies.

The power of heteronuclear NMR spectroscopy to study macromolecules and their complexes has been amply demonstrated over the last decade. The obstacle to routinely applying these techniques to the study of DNA has been the synthesis of 13C,15N-labeled DNA. Here we present a simple and efficient method to generate isotope-labeled DNA for NMR studies that is as easy as that for isotope labeling of RNA. The method was used to synthesize a uniformly 13C,15N-labeled 32-nucleotide DNA that binds to human basic fibroblast growth factor with high affinity and specificity. Isotope-edited experiments were applied to the 13C,15N-labeled DNA bound to unlabeled protein, and the 13C,15N-labeled DNA was also examined in complex with 15N-labeled protein. The NMR experiments show that the DNA adopts a well-defined stable structure when bound to the protein, and illustrate the potential of 13C,15N-labeled DNA for structural studies of DNA-protein complexes.

Carbon Isotopes↗
PubMed1992

Characterization of apolipoprotein B mRNA editing from rabbit intestine.

Apolipoprotein (apo) B-48 is generated by a unique physiological process. Cytidine 6,666 of the apo B primary transcript is posttranscriptionally converted to a uridine by an RNA editing mechanism that transforms the codon for glutamine 2,153 to a termination codon. The editing reaction can be duplicated in a cell-free extract. In this study, the apo B-48 mRNA editing activity derived from partially purified extracts of rabbit enterocytes was characterized. The optimum conditions for the editing reaction were determined to be a salt concentration of 0.125-0.150 M NaCl or KCl, a pH of 8-8.5, and a temperature of 30 degrees C. The reaction rate was linear up to 45 minutes and was proportional to the editing extract concentration. No metal ion cofactors, DNA or RNA cofactors, or energy requirements were identified. At optimum conditions, the reaction followed Michaelis-Menten kinetics, with a Km of 0.4 nM for the rabbit RNA substrate. In addition, the reaction rate was enhanced by the addition of 25 micrograms/ml heparin or 40% glycerol. The characteristics of the editing reaction suggest that it is catalyzed by a nucleotide sequence-specific cytidine deaminase that is either a single enzyme or a multimeric protein.

Animals↗
PubMed2003

Extra-pair sires as identified by means of standardized across-gel comparisons of multilocus DNA fingerprints.

Molecular analyses of genetic parentage in wild bird populations demonstrate that extra-pair paternity (EPP) is common, even in socially monogamous species. After having identified extra-pair offspring (EPO), an important step is to uncover the identity of extra-pair sires (EPS). Studies relying on multilocus DNA fingerprinting face the problem that simple between-gel comparisons of fingerprint banding patterns are imprecise in assigning parentage across gels. To tackle this problem, we developed a method to identify EPS by means of standardized across-gel comparisons of multilocus DNA fingerprints and applied it in a socially monogamous bird species with high levels of EPP, the coal tit (Parus ater). Scanned DNA fingerprint gels and standard image-editing software allowed to screen and reliably identify EPS. Overall, EPP was successfully assigned to 32 different EPS for 143 out of 210 (68.1%) EPO detected. This corresponds well to extra-pair parentage assignment rates revealed by microsatellite-based studies in congeneric species. Our approach proves especially useful whenever multilocus DNA fingerprints for parentage exclusion exist while microsatellite markers are not available.

Animals↗
PubMed1993

The 5'-terminal region of the apocytochrome b transcript in Crithidia fasciculata is successively edited by two guide RNAs in the 3' to 5' direction.

We analyzed the chimeric guide RNA (gRNA)-mRNA molecules in Crithidia fasciculata that are predicted to transiently exist in editing of the 5'-terminal domain of apocytochrome b (CYb) mRNA, by polymerase chain reaction amplification and DNA sequencing, and obtained evidence suggesting that among the 14 editing sites numbered from 3' to 5', the sequence in the 3'-half of the sites (3' block) was specified by one guide RNA species (gRNA-I) and that in the remaining half of the sites (5' block) by the other guide RNA species (gRNA-II) and that the direction of editing in each block was 3' to 5'. The predicted transition site of editing by two gRNAs was between the first and second U residues from the 3' end within editing site 7. We found that a stretch of the edited sequence in the 3' block of mRNA could form a stable duplex with a stretch immediately upstream of the guide sequence in gRNA-II. The result leads to a successive editing model that the 3' block of pre-edited mRNA is first edited by gRNA-I, and after completion of editing, the 5' portion of gRNA-II pairs with the edited mRNA for editing of the 5' block.

Animals↗
PubMed1986

Analysis of a sea urchin gene cluster coding for the small nuclear U7 RNA, a rare RNA species implicated in the 3' editing of histone precursor mRNAs.

A genomic 9.3-kilobase DNA fragment of the sea urchin Psammechinus miliaris, containing a cluster of five U7-RNA genes (or pseudogenes), has been isolated and analyzed by partial DNA sequencing. The U7-RNA coding sequences differ from one another by one or two nucleotides, one of the five gene sequences being identical to those of the cDNA U73 clone prepared earlier [Strub, K., Galli, G., Busslinger, M. & Birnstiel, M. L. (1984) EMBO J. 3, 2801-2807]. The spacer sequences separating the genes have, on the whole, a low degree of homology; hence, the five genes must have arisen by an ancient duplication event. The sequences preceding the coding portion contain three highly conserved sequence motifs but no "TATA box." The 3' flanking sequences include a highly conserved AAAGNNAGA sequence that is held in common with other U-RNA genes from both sea urchins and vertebrates. Our findings confirm our classification of the U7 RNA as a genuine, if sparsely represented, member of the U-RNA family.

Animals↗
PubMed1988

Overproduction of the epsilon subunit of DNA polymerase III counteracts the SOS mutagenic response of Escherichia coli.

It has been found that the mutator phenotype of the recA441 and recA730 strains that express the SOS response constitutively is suppressed by pIP1, a high-copy plasmid carrying the dnaQ gene encoding the 3'----5' exonuclease subunit (epsilon) of DNA polymerase III. We have constructed plasmid pIP11, in which the dnaQ gene is fused to the strong tac (trp-lac) promoter. Enhanced synthesis of the epsilon subunit stimulated by isopropyl beta-D-thiogalactopyranoside, the inducer of tac, prevents expression of the mutator phenotype of recA441 and markedly decreases the frequency of UV-induced mutations. These results strongly suggest that a loss of editing capacity by the epsilon subunit of DNA polymerase III holoenzyme plays a crucial role in generation of mutations during the SOS response.

DNA Polymerase III↗
PubMed2000

Sequence bias in edited kinetoplastid RNAs.

Uridylate residues (Us) are inserted and deleted at precise positions in mitochondrial transcripts of Trypanosoma brucei. These sequence changes are determined by interactions with small guide RNAs (gRNAs) that are complementary to edited sequence. Adenylate (A) and guanylate (G) residues in gRNAs across from editing sites pair with inserted Us. We evaluate whether sequence bias exists in the bases surrounding insertion sites. Upon analyzing all reported insertion sites in T. brucei, we find that the predicted base pairs flanking insertion sites show a strong bias. Specifically, guiding As and Gs tend to be flanked by cytosine residues and Us. This bias is expected if precise base-pair interactions at the editing site determine the number of inserted Us.

Adenine↗
PubMed2004

Identification of substrates for adenosine deaminases that act on RNA.

Adenosine deaminases that acts on RNA (ADARs) are RNA-editing enzymes that convert adenosine to inosine in double-stranded RNA. This chapter provides a detailed protocol for identifying inosine-containing RNAs. Candidate ADAR substrates are identified by cleaving poly (A)+ RNA specifically after inosine and using differential display to detect cleaved molecules. To confirm the presence of inosine, each individual candidate substrate is amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and the PCR product is directly sequenced. Sites that contain inosine at the RNA level appear as a mixture of adenosine and guanosine in the cDNA. The relative peak areas provide an estimate of the extent of editing at each site.

Adenosine Deaminase↗
PubMedDate not supplied

Mutation rate: some biological and biochemical considerations.

This article discusses ideas about the ways in which the high fidelity of DNA replication is achieved: base selection, exonucleolytic editing, and postreplicative proofreading. I also review possible mechanisms for the enhanced mutation rate associated with SOS induction. The concept of environmental control of mutation rate and other modes of genetic variation is also considered from the point of view that SOS induction is an example of "genetic revolution".

Base Composition↗
PubMed1996

Structure of Taq polymerase with DNA at the polymerase active site.

The DNA polymerase from Thermus aquaticus (Taq polymerase) is homologous to Escherichia coli DNA polymerase I (Pol I) and likewise has domains responsible for DNA polymerase and 5' nuclease activities. The structures to the polymerase domains of Taq polymerase and of the Klenow fragment (KF) of Pol I are almost identical, whereas the structure of a vestigial editing 3'-5' exonuclease domain of Taq polymerase that lies between the other two domains is dramatically altered, resulting in the absence of this activity in the thermostable enzyme. The structures have been solved for editing complexes between KF and single-stranded DNA and for duplex DNA with a 3' overhanging single strand, but not for a complex containing duplex DNA at the polymerase active-site. Here we present the co-crystal structure of Taq polymerase with a blunt-ended duplex DNA bound to the polymerase active-site cleft; the DNA neither bends nor goes through the large polymerase cleft, and the structural form of the bound DNA is between the B and A forms. A wide minor groove allows access to protein side chains that hydrogen-bond to the N3 of purines and the O2 of pyrimidines at the blunt-end terminus. Part of the DNA bound to the polymerase site shares a common binding site with DNA bound to the exonuclease site, but they are translated relative to each other by several angstroms along their helix axes.

Binding Sites↗
PubMed2025

Mitochondrial DNA homeostasis: A novel therapeutic target for neurodegenerative diseases.

The mitochondrial genomic homeostasis is essential for the function of the oxidative phosphorylation system and cellular homeostasis. Mitochondrial DNA is particularly susceptible to aging-related oxidative stress due to the lack of a histone coat. Disturbances in mitochondrial DNA may contribute to functional decline during the aging process and in neurodegenerative diseases, leading to further impairment of mitochondrial DNA and initiating a vicious cycle. To date, it remains unclear how disturbed mitochondrial DNA is involved in the etiology of pathological aging and neurodegenerative diseases. The purpose of this review is to clarify the crucial roles of mitochondrial DNA homeostasis in the pathogenesis of neurodegenerative diseases. Mitochondrial DNA is distributed within nucleoids and is then transcribed into polycistronic mitochondrial DNA molecules within the mitochondrial granule region. Within the ultrastructure of the mitochondrial nucleoid and granule, a group of essential mitochondrial proteins involved in DNA replication, DNA transcription, RNA translation, RNA surveillance, and RNA degradation plays a crucial role in maintaining mitochondrial structure, genome integrity, and mitochondrial DNA processing. The uniparentally inherited mitochondrial DNA undergoes heritable polyploid variations, which include homoplasmy and heteroplasmy. Accumulating mitochondrial DNA alterations, such as deletions, point mutations, and methylations, occur during the pathogenic processes of neurodegenerative diseases. The increased mitochondrial DNA alterations can be propagated by the rise of deleterious heteroplasmy in neurodegenerative diseases, ultimately resulting in impairment to the oxidative phosphorylation system, biogenesis defects, and cellular metabolic dysfunction. Therefore, developing appropriate gene editing tools to rectify aberrant alterations in mitochondrial DNA and targeting the key proteins involved in maintaining mitochondrial DNA homeostasis can be considered promising therapeutic strategies for neurodegenerative diseases. Although therapeutic strategies targeting mitochondrial DNA in diseases show great potential, challenges related to efficacy and safety require a better understanding of the mechanisms underlying mitochondrial DNA alterations in aging and neurodegenerative diseases.

Alzheimer’s disease↗