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At least 271 records · Page 15Linked to original sources
PubMed1996

The gene for a subunit of an ABC-type heme transporter is transcribed together with the gene for subunit 6 of NADH dehydrogenase in rice mitochondria.

We previously identified a chloroplast-derived (ct-derived) sequence of 32 base pairs (bp) in rice mitochondrial DNA that includes a part (30 bp; psitrnI) of a gene for isoleucine tRNA (CAU) of the chloroplast. Analyzing the ct-derived psitrnI, we found that an open reading frame (orf240), which was homologous to the gene for a subunit of an ATP-binding cassette-type (ABC-type) heme transporter, namely helC, of Rhodobacter capsulatus, and a gene for subunit 6 of NADH dehydrogenase (nad6) were located upstream of and downstream from the ct-derived psitrnI, respectively. Northern-blot hybridization and analysis by reverse transcription-polymerase chain reaction (RT-PCR) revealed that both orf240 and nad6 were co-transcribed in rice mitochondria. An analysis of PCR-amplified fragments of the region of orf240/nad6 from the DNA of some Gramineae suggests that the arrangement of orf240/nad6 was generated in the mitochondrial genome of the genus Oryza during evolution after its divergence from the other Gramineae. Most of the transcripts of orf240 are edited, with a change from cytidine to uridine, at 35 positions. Editing of the RNA changes 33 amino-acid residues among the 240 encoded amino-acid residues, suggesting that the orf240 gene is functional in rice mitochondria.

ATP-Binding Cassette Transporters↗
PubMed2006

Gene Designer: a synthetic biology tool for constructing artificial DNA segments.

BACKGROUND: Direct synthesis of genes is rapidly becoming the most efficient way to make functional genetic constructs and enables applications such as codon optimization, RNAi resistant genes and protein engineering. Here we introduce a software tool that drastically facilitates the design of synthetic genes. RESULTS: Gene Designer is a stand-alone software for fast and easy design of synthetic DNA segments. Users can easily add, edit and combine genetic elements such as promoters, open reading frames and tags through an intuitive drag-and-drop graphic interface and a hierarchical DNA/Protein object map. Using advanced optimization algorithms, open reading frames within the DNA construct can readily be codon optimized for protein expression in any host organism. Gene Designer also includes features such as a real-time sliding calculator of oligonucleotide annealing temperatures, sequencing primer generator, tools for avoidance or inclusion of restriction sites, and options to maximize or minimize sequence identity to a reference. CONCLUSION: Gene Designer is an expandable Synthetic Biology workbench suitable for molecular biologists interested in the de novo creation of genetic constructs.

Base Sequence↗
PubMed2025

A quick guide to evaluating prime editing efficiency in mammalian cells.

According to the Clinvar database, modeling the diseases associated with pathogenic mutations requires the installation of base substitutions, small insertions or deletions. Prime editor (PE) was recently developed to precisely install any base substitutions and/or small insertions/deletions (indels) in mammalian cells and animals without requiring DSBs or donor DNA templates. PE also offers greater editing and targeting flexibility compared to other precision CRISPR editing methods because the versatile editing information is encoded in the reverse-transcription template of its prime editing guide RNA. However, optimal PE system selection and experimental design can be complex, and there are various factors that can affect PE efficiency. This chapter serves as a rapid entry-level guideline for the application of PE, providing an experimental framework for using PE at a specific genomic locus. RUNX1 was selected as a representative target site to illustrate the detailed methodology for constructing PE plasmids and the process of transfecting these plasmids into 293FT cells. We further examined the efficiency of PE-mediated genome editing in mammalian cells by using next-generation sequencing.

Gene Editing↗
PubMed2003

Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts.

Viral replication usually requires that innate intracellular lines of defence be overcome, a task usually accomplished by specialized viral gene products. The virion infectivity factor (Vif) protein of human immunodeficiency virus (HIV) is required during the late stages of viral production to counter the antiviral activity of APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; also known as CEM15), a protein expressed notably in human T lymphocytes. When produced in the presence of APOBEC3G, vif-defective virus is non-infectious. APOBEC3G is closely related to APOBEC1, the central component of an RNA-editing complex that deaminates a cytosine residue in apoB messenger RNA. APOBEC family members also have potent DNA mutator activity through dC deamination; however, whether the editing potential of APOBEC3G has any relevance to HIV inhibition is unknown. Here, we demonstrate that it does, as APOBEC3G exerts its antiviral effect during reverse transcription to trigger G-to-A hypermutation in the nascent retroviral DNA. We also find that APOBEC3G can act on a broad range of retroviruses in addition to HIV, suggesting that hypermutation by editing is a general innate defence mechanism against this important group of pathogens.

APOBEC-3G Deaminase↗
PubMed1999

Age-dependent changes in DNA polymerase fidelity and proofreading activity during cellular aging.

DNA polymerase alpha and the 3'-->5' exonuclease involved in the proofreading of DNA synthesis were isolated from human diploid fetal lung fibroblast (TIG-1) cells at various population doubling levels (PDL). The final PDL of the TIG-1 cells used in these experiments was 70. The fidelity of DNA polymerase alpha remained high until late passage and fell suddenly just before the end of the life span between 65 and 69 PDL. The activities of the 3'-->5' exonuclease related to proofreading remained unchanged from 21 to 61 PDL, but the activity decreased rapidly in more aged cells. The 3'-->5' exonuclease activity at 69 PDL was about 50% of that in TIG cells at 21 PDL. In vitro DNA synthesis by DNA polymerase alpha from TIG-1 cells harvested at 69 PDL showed the amount of non-complementary nucleotides incorporated to be decreased by the addition of the 3'-->5' exonuclease from the same cells. However, not all errors were edited out since the ratio of DNA polymerase activity to 3'-->5' exonuclease activity was adjusted to reflect that in vivo and the infidelity of DNA synthesis by error-prone DNA polymerase alpha from aged cells was improved by the addition of the highly active 3'-->5' exonuclease from cells at 41 PDL. These results suggested that the mutation frequency rises just before the end of the life span of TIG-1 cells.

Cell Line↗
PubMed2000

APOBEC-1 dependent cytidine to uridine editing of apolipoprotein B RNA in yeast.

Cytidine to uridine editing of apolipoprotein B (apoB) mRNA requires the cytidine deaminase APOBEC-1 as well as a tripartite sequence motif flanking a target cytidine in apoB mRNA and an undefined number of auxiliary proteins that mediate RNA recognition and determine site-specific editing. Yeast engineered to express APOBEC-1 and apoB mRNA supported editing under conditions of late log phase growth and stationary phase. The cis -acting sequence requirements and the intracellular distribution of APOBEC-1 in yeast were similar to those described in mammalian cells. These findings suggest that auxiliary protein functions necessary for the assembly of editing complexes, or 'editosomes', are expressed in yeast and that the distribution of editing activity is to the cell nucleus.

APOBEC-1 Deaminase↗
PubMed1996

Extensive RNA editing of U to C in addition to C to U substitution in the rbcL transcripts of hornwort chloroplasts and the origin of RNA editing in green plants.

We cloned and sequenced a portion of chloroplast DNA from the hornwort Anthoceros formosae. A nucleotide sequence of 7556 bp contained structures similar to those of ndhK, ndhC, trnV, trnM, atpE, atpB, rbcL, trnR and accD. The arrangement of these was the same as that of other chloroplast DNA. However, two nonsense codons were located within the putative coding region of rbcL, although they were used as putative termination codons of the genes. RNA was extensively edited in the transcripts of rbcL when cDNA sequences were analyzed. The unusual nonsense codons of TGA and TAA became CGA and CAA respectively. These are examples of U to C type RNA editing, which was never been found before in chloroplast mRNA. In general, 13 Cs of genomic DNA were found as Ts in the cDNA sequence and seven Ts were found as Cs. This is the first finding of RNA editing on the transcripts of rbcL and also in bryophytes. This event had been thought to arise in land plants after the split of bryophytes. The origin of RNA editing is discussed in relation to the landing of green plants.

Amino Acid Sequence↗
PubMed2004

The PCNA-RFC families of DNA clamps and clamp loaders.

The proliferating cell nuclear antigen PCNA functions at multiple levels in directing DNA metabolic pathways. Unbound to DNA, PCNA promotes localization of replication factors with a consensus PCNA-binding domain to replication factories. When bound to DNA, PCNA organizes various proteins involved in DNA replication, DNA repair, DNA modification, and chromatin modeling. Its modification by ubiquitin directs the cellular response to DNA damage. The ring-like PCNA homotrimer encircles double-stranded DNA and slides spontaneously across it. Loading of PCNA onto DNA at template-primer junctions is performed in an ATP-dependent process by replication factor C (RFC), a heteropentameric AAA+ protein complex consisting of the Rfc1, Rfc2, Rfc3, Rfc4, and Rfc5 subunits. Loading of yeast PCNA (POL30) is mechanistically distinct from analogous processes in E. coli (beta subunit by the gamma complex) and bacteriophage T4 (gp45 by gp44/62). Multiple stepwise ATP-binding events to RFC are required to load PCNA onto primed DNA. This stepwise mechanism should permit editing of this process at individual steps and allow for divergence of the default process into more specialized modes. Indeed, alternative RFC complexes consisting of the small RFC subunits together with an alternative Rfc1-like subunit have been identified. A complex required for the DNA damage checkpoint contains the Rad24 subunit, a complex required for sister chromatid cohesion contains the Ctf18 subunit, and a complex that aids in genome stability contains the Elg1 subunit. Only the RFC-Rad24 complex has a known associated clamp, a heterotrimeric complex consisting of Rad17, Mec3, and Ddc1. The other putative clamp loaders could either act on clamps yet to be identified or act on the two known clamps.

DNA↗
PubMed1998

V(H) gene replacement occurs in the spleen and bone marrow of non-autoimmune quasi-monoclonal mice.

Genes encoding the heavy chain portion of immunoglobulin molecules arise from the combinatorial association of V, D and J gene segments, which occurs during discrete stages of B lineage development in the bone marrow. Recently, V(H) replacement, a form of receptor editing, has been described, in which the variable region of an existing VDJ(H) rearrangement is replaced by another V(H) gene segment in a recombination event believed to involve an embedded heptamer within the coding region of the V(H). Studies of transgenic mice with "knocked-in" VDJ(H) genes encoding anti-DNA specificity have demonstrated that receptor editing of the heavy chain is one mechanism by which autoreactive B cell receptors can be modified. Another mouse, the "quasi-monoclonal", which encodes a "knocked-in" VDJ(H) for the hapten NP also contains B lineage cells that undergo V(H) replacement. This suggests that V(H) replacement may play a role in the normal diversification of the antibody repertoire. Using a ligation-mediated PCR assay, we have identified V(QM) double-stranded DNA breaks indicative of V(H) replacement intermediates from bone marrow and splenic B lineage cells of quasi-monoclonal mice in the absence of immunization. V(QM) to J558 recombination deletion products consistent with V(H) replacement were also detected in both the bone marrow and spleen of non-immunized quasi-monoclonal mice. Moreover, RAG-1 transcripts were detected in the spleen. These data suggest that V(H) replacement can be part of the mechanism(s) used by B lineage cells to generate diversity throughout B lineage development, including later stages occurring in secondary lymphoid tissues.

Animals↗
PubMed1999

RNA editing sites in tobacco chloroplast transcripts: editing as a possible regulator of chloroplast RNA polymerase activity.

Genetic information in chloroplast DNA is sometimes altered at the transcript level by a process known as RNA editing. Sequence analysis of amplified cDNAs for 69 potential editing sites revealed 13 real editing sites in transcripts of 11 tobacco chloroplast genes. Together with those reported previously, these bring the total of edited sites observed in tobacco chloroplast transcripts to 31 (all involve C to U conversion). Alignment of sequences around the 31 editing sites revealed no obvious consensus, apart from an apparent bias for U or C at position -1 and A at position +2. Editing in tobacco rpoA mRNA restores the conserved leucine residue which is known to be important for transcriptional activation of the alpha subunit of E. coli RNA polymerase. Editing of this site is partial and the extent of editing depends on developmental conditions, suggesting that editing is, at least in part, involved in the regulation of chloroplast-encoded RNA polymerase activity.

Amino Acid Sequence↗
PubMed2025

Biochemical reconstitution of a type I-B CRISPR-associated transposon.

CRISPR-associated transposons (CASTs) are potential gene editing tools because of their RNA-guided DNA insertion activity. It is essential to understand the mechanisms underlying the transposition for the application of CASTs. Here, we provide protocols for the biochemical reconstitution of a type I-B CAST for RNA-guided transposition. The procedures may be applicable to other types of CASTs and facilitate the mechanism studies of various CASTs.

DNA Transposable Elements↗
PubMed2000

Conservation of RNA editing between rice and maize plastids: are most editing events dispensable?

The extent of conservation of RNA editing sites in the plastid genome of rice was determined by comparing the genomic sequence with that of the cDNA. The presence of a T in the cDNA predicted to be a C by the DNA sequence of the plastid genome, indicated C to U editing. In the 11 plastid transcripts of rice a total of 21 editing sites were found. In maize, a closely related grass species, 26 editing sites have been reported in 13 plastid transcripts. Most editing sites are conserved between the two species, although differences in RNA editing were found at eight sites. In seven cases the T was already encoded at the DNA level, eliminating the requirement for RNA editing. In one case (rpoB, codon 206) the RNA sequence was conserved between the two species, but the mRNA is still not edited in rice. It appears that, although evolutionarily conserved, RNA editing is essential only for a few plastid editing sites. Information about RNA editing in rice plastids will facilitate the design of plastid vectors with broad applicability in grass species.

DNA-Directed RNA Polymerases↗
PubMed1993

RNA editing in maize chloroplasts is a processing step independent of splicing and cleavage to monocistronic mRNAs.

The psbB operon contained in the plastomes of higher plants consists of the genes psbB, psbH, petB and petD. The primary transcript of this operon is subject to a series of processing steps which include cleavages resulting in four monocistronic mRNAs and splicing of the petB and petD transcripts. A search for editing sites within the two latter transcripts from maize led us to the detection of one editing site within the petB coding region which is conserved at the DNA level in other graminean species and in tobacco. This shows that editing must be considered as an additional processing step of the psbB operon encoded primary transcript. As is evident from cDNA sequences derived from the dicistronic and/or unspliced petB/D transcripts which are completely edited, editing is an early step of mRNA processing which precedes both splicing and cleavage to the monocistronic mRNAs and which must, therefore, be independent of the latter two steps. This conclusion is confirmed by a similar observation with the editing site of the rpl2 transcript which is contained in the polycistronic transcript of the rpoA operon, although here only partial editing is observed for the unspliced dicistronic rpl23/rpl2 transcript.

Animals↗
PubMed2002

Differences between the European carp (Cyprinus carpio carpio) and the coloured carp (Cyprinus carpio haematopterus) in susceptibility to Thelohanellus nikolskii (Myxosporea) infection.

Thelohanellus nikolskii infection of the common carp (Cyprinus carpio L.) has been a common parasitosis in the Central European fish farms since the first detection of the parasite about 20 years ago. This parasite, introduced from the Far East, causes intensive infection on the fins of fingerlings of the carp subspecies cultured in Europe (European carp, Cyprinus carpio carpio). This infection of the common carp occurs in the Hungarian fish farms every year. Until the present study, this parasite had not been recorded from the fins of koi or coloured carp (Cyprinus carpio haematopterus), a carp of Far Eastern origin, which is cultured in Hungary as an ornamental fish. A recent survey conducted in common carp, koi and goldfish stocks demonstrated that T. nikolskii infection of low prevalence and intensity occurs also in koi populations, but its prevalence and intensity are markedly lower than in common carp kept in the same ponds. It is suggested that the observed differences are due to disparities in the susceptibility of the two carp subspecies to T. nikolskii, and that the koi is less susceptible to this infection. Other signs of susceptibility can also be observed in the European subspecies, since in 15% of the fish plasmodium development was arrested at an early stage. Thelohanellus nikolskii infection could not be demonstrated on goldfish (Carassius auratus).

Animals↗
PubMed1992

[Advances in molecular parasitology].

Recent studies on molecular biology of parasites revealed novel mechanisms for the control of expression of parasitic genes. In trypanosomatid protozoa, unusual characteristics of gene regulation, including polycistronic transcription, trans-RNA splicing and RNA editing have been discovered. This review describes the recent progress of RNA editing in trypanosomes, and trans-splicing in both trypanosomes and nematodes. Studies of the editing of mRNAs in trypanosomatid mitochondria indicate the involvement of small guide RNA molecules, which are encoded in the minicircles and/or maxicircles of kinetoplast DNA. A proposed transesterification model for RNA editing suggests an analogous reaction between RNA editing and RNA splicing. In addition, the development of stable transfection system and success of homologous gene targeting in Trypanosoma and Leishmania are reported.

Animals↗
PubMed1998

Mechanism and control of interspecies recombination in Escherichia coli. I. Mismatch repair, methylation, recombination and replication functions.

A genetic analysis of interspecies recombination in Escherichia coli between the linear Hfr DNA from Salmonella typhimurium and the circular recipient chromosome reveals some fundamental aspects of recombination between related DNA sequences. The MutS and MutL mismatch binding proteins edit (prevent) homeologous recombination between these 16% diverged genomes by at least two distinct mechanisms. One is MutH independent and presumably acts by aborting the initiated recombination through the UvrD helicase activity. The RecBCD nuclease might contribute to this editing step, presumably by preventing reiterated initiations of recombination at a given locus. The other editing mechanism is MutH dependent, requires unmethylated GATC sequences, and probably corresponds to an incomplete long-patch mismatch repair process that does not depend on UvrD helicase activity. Insignificant effects of the Dam methylation of parental DNAs suggest that unmethylated GATC sequences involved in the MutH-dependent editing are newly synthesized in the course of recombination. This hypothetical, recombination-associated DNA synthesis involves PriA and RecF functions, which, therefore, determine the extent of MutH effect on interspecies recombination. Sequence divergence of recombining DNAs appears to limit the frequency, length, and stability of early heteroduplex intermediates, which can be stabilized, and the recombinants mature via the initiation of DNA replication.

Adenosine Triphosphatases↗
PubMed2026

Minimizing Off-Target Effects of CRISPR-Cas9 With Optimized sgRNA: Evaluation of Efficiency and Specificity in the Tumor Protein 53 (TP53) Region.

CRISPR-Cas9 is a widely used genetic tool with therapeutic potential in molecular biology. CRISPR-Cas9 enables precise genome editing by its ability to target specific DNA sequence. After off-target and on-target regions are identified, CRISPR-Cas9 is applied to these regions based on the match between the guide RNA (gRNA) and target DNA sequence. This study points to the off-target impact of mismatches between the gRNA and target DNA on exon regions of the TP53 gene, which are involved in regulating multiple genes and cellular functions. Off-target positions are typically evaluated using scoring methods. In this study, we have used latent class analysis to reveal subclasses of off-target positions. Thus, we have created the levels of off-target positions and evaluated the effects of mismatching positions within these classes using machine learning classifiers. The results revealed that mismatching positions could be categorized into three levels: low, middle, and high off-target positions. We have improved a computational framework to minimize off-target effects and to identify the PAM sequences in the gRNA design. Thus, carefully designed gRNAs will ensure that desired genetic edits are performed and target variants are achieved. This work will avail the future research aimed at optimizing genome editing by customizing CRISPR-Cas9 to target specific protospacer DNA through gRNA.

CRISPR-Cas Systems↗
PubMed1995

Genetic map of Salmonella typhimurium, edition VIII.

We present edition VIII of the genetic map of Salmonella typhimurium LT2. We list a total of 1,159 genes, 1,080 of which have been located on the circular chromosome and 29 of which are on pSLT, the 90-kb plasmid usually found in LT2 lines. The remaining 50 genes are not yet mapped. The coordinate system used in this edition is neither minutes of transfer time in conjugation crosses nor units representing "phage lengths" of DNA of the transducing phage P22, as used in earlier editions, but centisomes and kilobases based on physical analysis of the lengths of DNA segments between genes. Some of these lengths have been determined by digestion of DNA by rare-cutting endonucleases and separation of fragments by pulsed-field gel electrophoresis. Other lengths have been determined by analysis of DNA sequences in GenBank. We have constructed StySeq1, which incorporates all Salmonella DNA sequence data known to us. StySeq1 comprises over 548 kb of nonredundant chromosomal genomic sequences, representing 11.4% of the chromosome, which is estimated to be just over 4,800 kb in length. Most of these sequences were assigned locations on the chromosome, in some cases by analogy with mapped Escherichia coli sequences.

Chromosome Mapping↗