Abstract:
:In the last decade polycistronic vectors have become essential tools for both basic science and gene therapy applications. In order to co-express heterologous polypeptides, different systems have been developed from Internal Ribosome Entry Site (IRES) based vectors to the use of the 2A peptide. Unfortunately, these methods are not fully suitable for the efficient and reproducible modulation of the ratio between the proteins of interest. Here we describe a novel bicistronic vector type based on the use of alternative splicing. By modifying the consensus sequence that governs splicing, we demonstrate that the ratio between the synthesized proteins could easily vary from 1 : 10 to 10 : 1. We have established this system with luciferase genes and we extended its application to the production of recombinant monoclonal antibodies. We have shown that these vectors could be used in several typical cell lines with similar efficiencies. We also present an adaptation of these vectors to hybrid alternative splicing/IRES constructs that allow a ratio-controlled expression of proteins of interest in stably transfected cell lines.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Fallot S,Ben Naya R,Hieblot C,Mondon P,Lacazette E,Bouayadi K,Kharrat A,Touriol C,Prats Hdoi
10.1093/nar/gkp716subject
Has Abstractpub_date
2009-11-01 00:00:00pages
e134issue
20eissn
0305-1048issn
1362-4962pii
gkp716journal_volume
37pub_type
杂志文章abstract::We have studied the effect of temperature on transcription initiation in vitro at two related promoters ga/Pcon and ga/P1, which have the same nucleotide sequence around the -10 region and transcription start site, but differ in upstream sequences. One of the promoters, ga/Pcon, carries the consensus -35 hexamer, 5'TT...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/19.22.6113
更新日期:1991-11-25 00:00:00
abstract::The single nucleotide polymorphisms (SNPs) in conserved protein regions have been thought to be strong candidates that alter protein functions. Thus, we have developed SNP@Domain, a web resource, to identify SNPs within human protein domains. We annotated SNPs from dbSNP with protein structure-based as well as sequenc...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkl323
更新日期:2006-07-01 00:00:00
abstract::Activating sequence factor 1 (ASF-1) is a nuclear DNA-binding activity that is found in monocots and dicots. It interacts with several TGACG-containing elements that have been characterized from viral and T-DNA genes, the prototypes of which are the as-1 element of the CaMV 35S promoter and the ocs element from the oc...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/23.18.3778
更新日期:1995-09-25 00:00:00
abstract::A fundamental problem with applying Consensus, Weight-Matrix or hidden Markov models as search tools for biosequences is that there is no way to know, from the model, if the modeled sequences display any dependencies between positional identities. In some instances, these dependencies are crucial in correctly acceptin...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gki374
更新日期:2005-07-01 00:00:00
abstract::The organelle genome database GOBASE, now in its 21st release (June 2008), contains all published mitochondrion-encoded sequences (approximately 913,000) and chloroplast-encoded sequences (approximately 250,000) from a wide range of eukaryotic taxa. For all sequences, information on related genes, exons, introns, gene...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkn819
更新日期:2009-01-01 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::Methionyl-tRNA synthetase belongs to the class I aminoacyl-tRNA synthetase family characterized both by a catalytic center built around a Rossmann Fold and by the presence of the two peptidic marker sequences HIGH and KMSKS. In this study, the role of the 21HLGH24 motif of Escherichia coli methionyl-tRNA synthetase wa...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/23.23.4793
更新日期:1995-12-11 00:00:00
abstract::A cDNA that encodes a transcription factor able to recognize the thyroglobulin gene promoter in vitro was isolated from a dog thyroid cDNA expression library in lambda gt11. The library was screened with a multimerized 20 bp-oligonucleotide probe corresponding to the -126 to -107 bp region of the bovine thyroglobulin ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/19.5.1121
更新日期:1991-03-11 00:00:00
abstract::Despite the extensive use of Saccharomyces cerevisiae as a platform for synthetic biology, strain engineering remains slow and laborious. Here, we employ CRISPR/Cas9 technology to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locu...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkw1023
更新日期:2017-01-09 00:00:00
abstract::Targeted gene silencing by RNA interference allows the study of gene function in plants and animals. In cell culture and small animal models, genetic screens can be performed--even tissue-specifically in Drosophila--with genome-wide RNAi libraries. However, a major problem with the use of RNAi approaches is the unavoi...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkp450
更新日期:2009-07-01 00:00:00
abstract::2'-O-(2-methoxyethyl) (2'-MOE) RNA possesses favorable pharmocokinetic properties that make it a promising option for the design of oligonucleotide drugs. Telomerase is a ribonucleoprotein that is up-regulated in many types of cancer, but its potential as a target for chemotherapy awaits the development of potent and ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/29.8.1683
更新日期:2001-04-15 00:00:00
abstract::Mutations help us to understand the molecular origins of diseases. Researchers, therefore, both publish and seek disease-relevant mutations in public databases and in scientific literature, e.g. Medline. The retrieval tends to be time-consuming and incomplete. Automated screening of the literature is more efficient. W...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkh162
更新日期:2004-01-02 00:00:00
abstract::Vaccinia virus DNA topoisomerase catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5'-CCCTT/, that are opposed within a partially mobile four-way junction. Efficient resolution occurs on a junction with a 10 bp segment of branc...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/28.14.2658
更新日期:2000-07-15 00:00:00
abstract::An immunofluorescence assay was developed to identify proteins specifically binding to oligonucleoside phosphorodithioate (ODN) aptamers from a bead-bound ODN library. Accordingly, NF-kappaB p50 protein was incubated with either bead-bound NF-kappaB consensus sequence or a bead-bound ODN combinatorial library and adso...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gng054
更新日期:2003-05-15 00:00:00
abstract::RNA editing in protozoan parasites is a mitochondrial RNA processing reaction in which exclusively uridylate residues are inserted into, and less frequently deleted from, pre-mRNAs. Molecules central to the process are so-called guide RNAs (gRNAs) which function as templates in the reaction. For a detailed molecular u...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/25.12.2311
更新日期:1997-06-15 00:00:00
abstract::Soil salinity is a significant threat to sustainable agricultural production worldwide. Plants must adjust their developmental and physiological processes to cope with salt stress. Although the capacity for adaptation ultimately depends on the genome, the exceptional versatility in gene regulation provided by the spli...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkx1229
更新日期:2018-02-28 00:00:00
abstract::The somatic V(D)J recombination for the assembly of the Ig and TCR genes is mediated by the recombination signal sequences (Rss) and the V(D)J recombinase. A cDNA clone was isolated from a lambda gt11 expression library made from mouse thymocyte poly(A)+ RNA, using the Rss as a ligand. The deduced amino acid sequence ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/21.22.5067
更新日期:1993-11-11 00:00:00
abstract::We examined the 3'-flanking regions required for accurate 3'-end formation of wheat histone H3 mRNA using gene expression in transformed sunflower cells. The introduction of mutations into the conserved sequence AATGGAAATG in the 3'-flanking region of plant histone genes, located 22 bp upstream from the polyadenylatio...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/22.6.1052
更新日期:1994-03-25 00:00:00
abstract::Alternative splicing (AS) and alternative polyadenylation (APA) generate diverse transcripts in mammalian genomes during development and differentiation. Epigenetic marks such as trimethylation of histone H3 lysine 36 (H3K36me3) and DNA methylation play a role in generating transcriptome diversity. Intragenic CpG isla...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkaa556
更新日期:2020-09-04 00:00:00
abstract::dITP may be generated from dATP by a slow, nonenzymatic hydrolysis. While [3H]dITP was degraded rapidly to [3H]deoxyinosine by HeLa cell nuclear extracts, no net degradation of [3H]dITP was observed in the presence of physiological concentrations of ATP, apparently because the extract contained deoxynucleoside diphosp...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/10.12.3693
更新日期:1982-06-25 00:00:00
abstract::Members of several classes of proto-oncogenes replicate during the first third of S-phase in two human (K562 erythroleukemia and HeLa), one Chinese hamster (CHO) and eight mouse cell lines. These cell lines exhibit a variety of specialized functions characteristic of pre-B and B cells, T cells and erythroid cells. The...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/15.1.87
更新日期:1987-01-12 00:00:00
abstract::Tomato golden mosaic geminivirus has a genome of two single-stranded (ss) DNA components, A and B. An almost identical 'common' region in DNA A and DNA B is thought to contain sequence elements controlling replication and transcription. Hence investigation of sequences important for DNA replication by in vitro mutagen...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/17.24.10213
更新日期:1989-12-25 00:00:00
abstract::We have formed complexes of relaxed closed circular Col E1 DNA with various combinations of histones, and examined the effects of treating the complexes with nicking-closing enzyme. Germond et al (1) have shown that when a mixture of the four core histones of the nucleosome (HIA, H2B, H3 and H4) is used in such an exp...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/4.5.1159-a
更新日期:1977-01-01 00:00:00
abstract::The history and mechanism of molecular evolution in DNA have been greatly elucidated by contributions from genetics, probability theory and bioinformatics--indeed, mathematical developments such as Kimura's neutral theory, Kingman's coalescent theory and efficient software such as BLAST, ClustalW, Phylip, etc., provid...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkp477
更新日期:2009-07-01 00:00:00
abstract::The Vienna RNA secondary structure server provides a web interface to the most frequently used functions of the Vienna RNA software package for the analysis of RNA secondary structures. It currently offers prediction of secondary structure from a single sequence, prediction of the consensus secondary structure for a s...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkg599
更新日期:2003-07-01 00:00:00
abstract::DNA polymerase lambda (pol lambda) is a member of the X family DNA polymerases and is endowed with multiple enzymatic activities. In this work we investigated the in vitro miscoding properties of full-length, human pol lambda either in the absence or in the presence of the human auxiliary proteins proliferating cell n...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkl032
更新日期:2006-03-06 00:00:00
abstract::The abundance and cell cycle dependent expression of the mRNA for human nonhistone protein HMG-17 were studied in synchronized HeLa cells. Slot blot analysis indicates that the HMG-17 mRNA is a very abundant message, significantly more so than histone or actin mRNA. RNA prepared from tissue culture cells contains high...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/15.8.3549
更新日期:1987-04-24 00:00:00
abstract::The gene, rpb2, encoding the second largest subunit, subunit 2, of RNA polymerase II has been cloned from Schizosaccharomyces pombe using the corresponding gene, RPB2, of Saccharomyces cerevisiae as a probe for cross-hybridization. We have determined the complete nucleotide sequence of rpb2, and parts of the PCR-ampli...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/21.3.469
更新日期:1993-02-11 00:00:00
abstract::Modification of chromatin and related transcription factors by histone deacetylases (HDACs) is one of the major strategies for controlling gene expression in eukaryotes. The HDAC domains of class IIa HDACs repress the respective target genes by interacting with the C-terminal region of the silencing mediator for retin...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gky926
更新日期:2018-12-14 00:00:00
abstract::Human single-strand (ss) DNA binding proteins 1 (hSSB1) has been shown to participate in DNA damage response and maintenance of genome stability by regulating the initiation of ATM-dependent signaling. ATM phosphorylates hSSB1 and prevents hSSB1 from ubiquitin-proteasome-mediated degradation. However, the E3 ligase th...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gku876
更新日期:2014-10-01 00:00:00