Abstract：
:A simple and efficient gene replacement method, based on the recombination and repair activities of the cell, was developed. The method permits the targeted construction of markerless deletions, insertions and point mutations in the Escherichia coli chromosome. A suicide plasmid, carrying the mutant allele and the recognition site of meganuclease I- Sce I, is inserted into the genome by homologous recombination between the mutant and the wild-type (wt) alleles. Resolution of this cointegrate by intramolecular recombination of the allele pair results in either a mutant or a wt chromosome which can be distinguished by allele-specific PCR screening. The resolution process is stimulated by introducing a unique double-strand break (DSB) into the chromosome at the I- Sce I site. Cleavage by the nuclease not only enhances the frequency of resolution by two to three orders of magnitude, but also selects for the resolved products. The DSB-stimulated gene replacement method can be used in recombination-proficient E.coli cells, does not require specific growth conditions, and is potentially applicable in other microorganisms. Use of the method was demonstrated by constructing a 17-bp and a 62-kb deletion in the MG1655 chromosome. Cleavage of the chromosome induces the SOS response but does not lead to an increased mutation rate.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Pósfai G,Kolisnychenko V,Bereczki Z,Blattner FRdoi
10.1093/nar/27.22.4409keywords:
subject
Has Abstractpub_date
1999-11-15 00:00:00pages
4409-15issue
22eissn
0305-1048issn
1362-4962pii
gkc658journal_volume
27pub_type
杂志文章abstract：:Our database on large ribosomal subunit RNA contained 334 sequences in July, 1995. All sequences in the database are aligned, taking into account secondary structure. The aligned sequences are provided, together with incorporated secondary structure information, in several computer-readable formats. These data can eas...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/24.1.92
更新日期：1996-01-01 00:00:00
abstract：:Molecular models of six anthracycline antibiotics and their complexes with 32 distinct DNA octamer sequences were created and analyzed using HINT (Hydropathic INTeractions) to describe binding. The averaged binding scores were then used to calculate the free energies of binding for comparison with experimentally deter...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkg645
更新日期：2003-08-01 00:00:00
abstract：:Chromatin modulation at various cis-acting elements is critical for V(D)J recombination during T and B cell development. MARbeta, a matrix-associated region (MAR) located upstream of the T cell receptor beta (TCRbeta) enhancer (Ebeta), serves a crucial role in silencing Ebeta-mediated TCR activation. By DNaseI hyperse...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkh807
更新日期：2004-09-15 00:00:00
abstract：:The ribosomal frameshifting signal of the mouse embryonal carcinoma differentiation regulated (Edr) gene represents the sole documented example of programmed -1 frameshifting in mammalian cellular genes [Shigemoto,K., Brennan,J., Walls,E,. Watson,C.J., Stott,D., Rigby,P.W. and Reith,A.D. (2001), Nucleic Acids Res., 29...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gki299
更新日期：2005-03-14 00:00:00
abstract：:Dimerization of HIV-1 genomic RNA is an essential step of the viral cycle, initiated at a conserved stem-loop structure which forms a 'kissing complex' involving loop-loop interactions (dimerization initiation site, DIS). A 19mer RNA oligonucleotide (DIS-19) has been synthesized which forms a stable symmetrical dimer ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/26.15.3567
更新日期：1998-08-01 00:00:00
abstract：:Tetramers of the arginine-rich histones H3 and H4 associate with supercoiled SV40 DNA either singly, giving tetrameric nucleoprotein complexes or in pairs giving octameric complexes, both of which are visualized as beads in the electron microscope. The relative amounts of the two complexes may be revealed by complete ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/7.3.611
更新日期：1979-10-10 00:00:00
abstract：:Sequencing of RNAs (RNA-Seq) has revolutionized the field of transcriptomics, but the reads obtained often contain errors. Read error correction can have a large impact on our ability to accurately assemble transcripts. This is especially true for de novo transcriptome analysis, where a reference genome is not availab...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkt215
更新日期：2013-05-01 00:00:00
abstract：:We have recently developed a novel method for the affinity purification of the complete suite of translating mRNA from genetically labeled cell populations. This method permits comprehensive quantitative comparisons of the genes employed by each specific cell type. We provide a detailed description of tools for analys...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkq130
更新日期：2010-07-01 00:00:00
abstract：:Short interfering RNAs (siRNAs) are valuable reagents for sequence-specific inhibition of gene expression via the RNA interference (RNAi) pathway. Although it has been proposed that the relative thermodynamic stability at the 5'-ends of siRNAs plays a crucial role in siRNA strand selection, we demonstrate here that a ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkn584
更新日期：2008-10-01 00:00:00
abstract：:The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3'-5' exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkv260
更新日期：2015-04-30 00:00:00
abstract：:Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA's 5'-terminal 'cap'. The minimal 'cap0' consists of N7-methylguanosine linked to the first nucleotide via a 5'-5' triphosphate (ppp) bridge. Cap0 is further modified by 2'-O-methylation of the next two riboses, yielding 'cap1' (m7GpppNmN) ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkt1321
更新日期：2014-03-01 00:00:00
abstract：:The replacement of thymidine with 5-bromo-2'-deoxyuridine (BrdU) is well-known to sensitize cells to ionizing radiation and photoirradiation. We reported here the sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex oligodeoxynucleotides harboring a BrdU or its closely rela...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkl892
更新日期：2006-01-01 00:00:00
abstract：:Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of thei...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/18.19.5667
更新日期：1990-10-11 00:00:00
abstract：:The pyrrole-imidazole (Py-Im) triamide-cyclopropa pyrroloindole (CPI) conjugates ImPyImLDu86 (7) and ImImPyLDu86 (14) were synthesized and their alkylating activities and inhibitory effects on DNA hydrolysis by restriction endonucleases were examined. Sequencing gel analysis demonstrated that conjugates 7 and 14 speci...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkf486
更新日期：2002-09-01 00:00:00
abstract：:Plants have large diverse families of small secreted proteins (SSPs) that play critical roles in the processes of development, differentiation, defense, flowering, stress response, symbiosis, etc. Oryza sativa is one of the major crops worldwide and an excellent model for monocotyledonous plants. However, there had no...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gks1090
更新日期：2013-01-01 00:00:00
abstract：: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 co...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/24.6.1008
更新日期：1996-03-15 00:00:00
abstract：:Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polkappa encoded by the DINB1 gene and examined its response to damaged DNA templates...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/28.21.4138
更新日期：2000-11-01 00:00:00
abstract：:Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging DNA template strand. The enzyme discriminates at the DNA binding step between substrates containing a 5'-phosphate versus a 5'-hydroxyl at the nick. Mu...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/26.2.525
更新日期：1998-01-15 00:00:00
abstract：:Programmed chromosome breakage occurs at 50-200 specific sites in the genome of Tetrahymena thermo-phila during somatic nuclear (macronuclear) differentiation. Previous studies have identified a 15 bp sequence, the Cbs (for chromosome breakage sequence), that is necessary and sufficient to specify these sites. In this...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/28.4.895
更新日期：2000-02-15 00:00:00
abstract：:Chicken erythrocyte inner histones (H2A, H2B, H3 and H4) were associated with the two complementary homopolymeric polydeoxyribonucleotides and the two alternating copolymeric polydeoxyribonucleotides. No evidence for formation of chromatin-like structures was obtained for the complexes with poly(dG) . poly(dC) or poly...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/6.4.1387
更新日期：1979-04-01 00:00:00
abstract：:SIRT6 is critical for activating transcription of Nuclear factor (erythroid-derived 2)-like 2 (NRF2) responsive genes during oxidative stress. However, while the mechanism of SIRT6-mediated silencing is well understood, the mechanism of SIRT6-mediated transcriptional activation is unknown. Here, we employed SIRT6 sepa...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkz528
更新日期：2019-09-05 00:00:00
abstract：:In squid (Loligo bleekeri) mitochondria, the two 3'-terminal nucleotides (G72-G73) of the tRNA(Tyr) gene overlap with the two 5'-terminal nucleotides (G1-G2) of the downstream tRNA(Cys) gene. To elucidate the processing mechanism(s) of the tRNA molecules derived from this region, tRNAs were analyzed by sequencing cDNA...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/24.24.4987
更新日期：1996-12-15 00:00:00
abstract：:We have isolated and characterized a S. pombe promoter using a functional heterologous gene product assay. Random S. pombe genomic fragments were cloned upstream from the promoterless 'lacZ gene and tested in vivo for their efficiency to promote expression of the beta-galactosidase protein in the fission yeast. An eff...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/16.17.8603
更新日期：1988-09-12 00:00:00
abstract：:Among a collection of 74 aprt mutations induced by treatment of plateau phase Chinese hamster ovary CHO cells with the radiomimetic DNA double-strand cleaving agent neocarzinostatin, nine were large-scale rearrangements. Molecular analysis indicated that all nine were highly conservative, non-homologous reciprocal exc...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkf369
更新日期：2002-06-15 00:00:00
abstract：:We show how to speed up sequence alignment algorithms of the type introduced by Needleman and Wunsch (and generalized by Sellers and others). Faster alignment algorithms have been introduced, but always at the cost of possibly getting sub-optimal alignments. Our modification results in the optimal alignment still bein...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/12.1part1.175
更新日期：1984-01-11 00:00:00
abstract：:Signals setting the level of transcription of the tRNA-tufB operon have been studied by deletion mapping. TufB transcription was measured in vivo with plasmid-borne tRNA-tufB:galk operon fusions. Removal of the sequences from -133 to -58 with respect to the transcription start point, results in a 90% decrease of tufB ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/15.22.9515
更新日期：1987-11-25 00:00:00
abstract：:A new series of DNA bis-intercalators is reported in which acridine moieties are connected by rigid and extended pyridine-based linkers of varied length. Cross-linking of DNA by bis-intercalation is inferred from the unwinding and folding of linear DNA induced by the compounds; after ligation and removal of the bis-in...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/20.5.983
更新日期：1992-03-11 00:00:00
abstract：:While biallelic mutations in the PALB2 tumor suppressor cause Fanconi anemia subtype FA-N, monoallelic mutations predispose to breast and familial pancreatic cancer. Although hundreds of missense variants in PALB2 have been identified in patients to date, only a few have clear functional and clinical relevance. Herein...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkz780
更新日期：2019-11-18 00:00:00
abstract：:Directed molecular evolution and combinatorial methodologies are playing an increasingly important role in the field of protein engineering. The general approach of generating a library of partially randomized genes, expressing the gene library to generate the proteins the library encodes and then screening the protei...
journal_title：Nucleic acids research
pub_type： 杂志文章,评审
doi：10.1093/nar/gkh315
更新日期：2004-02-27 00:00:00
abstract：:A new E. coli strain has been constructed that contains the dinD1::LacZ+ fusion and is deficient in methylation-dependent restriction systems (McrA-, McrBC-, Mrr-). This strain has been used to clone restriction endonuclease genes directly into E. coli. When E. coli cells are not fully protected by the cognate methyla...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/22.12.2399
更新日期：1994-06-25 00:00:00
