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 proteins for improved or modified characteristics has successfully been applied in the areas of protein-ligand binding, improving protein stability and modifying enzyme selectivity. A wide range of techniques are now available for generating gene libraries with different characteristics. This review will discuss these different methodologies, their accessibility and applicability to non-expert laboratories and the characteristics of the libraries they produce. The aim is to provide an up to date resource to allow groups interested in using directed evolution to identify the most appropriate methods for their purposes and to guide those moving on from initial experiments to more ambitious targets in the selection of library construction techniques. References are provided to original methodology papers and other recent examples from the primary literature that provide details of experimental methods.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Neylon Cdoi
10.1093/nar/gkh315keywords:
subject
Has Abstractpub_date
2004-02-27 00:00:00pages
1448-59issue
4eissn
0305-1048issn
1362-4962pii
32/4/1448journal_volume
32pub_type
杂志文章,评审abstract::DrugCentral (http://drugcentral.org) is an open-access online drug compendium. DrugCentral integrates structure, bioactivity, regulatory, pharmacologic actions and indications for active pharmaceutical ingredients approved by FDA and other regulatory agencies. Monitoring of regulatory agencies for new drugs approvals ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkw993
更新日期:2017-01-04 00:00:00
abstract::We devised an indicator gene for retrotransposition based on an autocatalytic ribozyme element--the Tetrahymena thermophila 23S rRNA group I intron--which can self-splice in vitro and does not require--at variance with nuclear mRNA introns--any specific pathway and cellular component for the completion of the splicing...
journal_title:Nucleic acids research
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doi:10.1093/nar/30.11.e49
更新日期:2002-06-01 00:00:00
abstract::Nucleotide sequences of large T1 ribonuclease fragments of 18S ribosomal RNA of Novikoff rat ascites hepatoma cells and chicken lymphoblastoid cells were determined and compared. Among the 19 large T1 ribonuclease fragments examined of rat 18S ribosomal RNA, 12 fragments were found to be the same in chicken 18S riboso...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/7.5.1131
更新日期:1979-11-10 00:00:00
abstract::The sarcomeric myosin gene, Myh7b, encodes an intronic microRNA, miR-499, which regulates cardiac and skeletal muscle biology, yet little is known about its transcriptional regulation. To identify the transcription factors involved in regulating Myh7b/miR-499 gene expression, we have mapped the transcriptional start s...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gks466
更新日期:2012-08-01 00:00:00
abstract::We have measured the amount of 5S-ribosomal DNA in the genomes of Xenopus laevis, Triturus cristatus carnifex and Ambystoma mexicanum, three species of Amphibians which have widely different C-values. Our best estimate is that these organisms have about 24,000, 32,000 and 61,000 5S-genes per haploid genome respectivel...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/11.8.2381
更新日期:1983-04-25 00:00:00
abstract::Sox proteins belong to the HMG box superfamily of DNA-binding proteins and are found throughout the animal kingdom. They are involved in the regulation of such diverse developmental processes as germ layer formation, organ development and cell type specifi-cation. Hence, deletion or mutation of Sox proteins often resu...
journal_title:Nucleic acids research
pub_type: 杂志文章,评审
doi:10.1093/nar/27.6.1409
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abstract::Structures of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 have been determined by NMR spectroscopy under low salt conditions. All protons and phosphorus nuclei resonances have been assigned. Signals of H5'/5" have been assigned stereospecifically. All 3JH,H and 3JP,H coupling constants have been measured. The structures were dete...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/25.22.4589
更新日期:1997-11-15 00:00:00
abstract::In diverse bacterial species, the turnover and processing of many RNAs is mediated by the ribonuclease RNase J, a member of the widely occurring metallo-β-lactamase enzyme family. We present crystal structures of Streptomyces coelicolor RNase J with bound RNA in pre- and post-cleavage states, at 2.27 Å and 2.80 Å reso...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkv732
更新日期:2015-09-18 00:00:00
abstract::We have created a database of comparatively derived group I intron secondary structure diagrams. This collection currently contains a broad sampling of phylogenetically and structurally similar and diverse structures from over 200 publicly available intron sequences. As more group I introns are sequenced and added to ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/22.17.3508
更新日期:1994-09-01 00:00:00
abstract::The human prothrombin G20210A polymorphism located at the 3' cleavage site of the mRNA results in elevated plasma prothrombin levels and increased risk of venous thrombosis. This polymorphism has been shown to directly influence a variety of processes related to prothrombin mRNA metabolism. We have constructed plasmid...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gki245
更新日期:2005-02-17 00:00:00
abstract::Hexokinase 2 is an essential factor for signalling repression through the Saccharomyces cerevisiae high-glucose sensing pathway. The main regulatory mechanism that controls the HXK2 gene expression in yeast is mediated by the Rgt1 and Med8 transcription factors, which repress HXK2 expression in low-glucose containing ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkl028
更新日期:2006-03-09 00:00:00
abstract::The HhaI methyltransferase recognizes the sequence GCGC and transfers a methyl group to C5 of the first cytosine residue. All m5C-methyltransferases contain a highly conserved sequence motif called the P-C motif. The cysteine residue of this motif is involved in catalysis by forming a covalent bond with the 6-position...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/21.10.2459
更新日期:1993-05-25 00:00:00
abstract::In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through NCBI's Web site. NCBI resources include Entrez, the Entrez Programming Utili...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkj158
更新日期:2006-01-01 00:00:00
abstract::Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkaa195
更新日期:2020-06-04 00:00:00
abstract::The Eco57I restriction endonuclease and methylase were purified to homogeneity from the E.coli RR1 strain carrying the eco57IRM genes on a recombinant plasmid. The molecular weight of the denaturated methylase is 63 kDa. The restriction endonuclease exists in a monomeric form with an apparent molecular weight of 104-1...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/20.22.6043
更新日期:1992-11-25 00:00:00
abstract::Tight cell-cycle regulation of the histone H4-K20 methyltransferase PR-Set7 is essential for the maintenance of genome integrity. In mammals, this mainly involves the interaction of PR-Set7 with the replication factor PCNA, which triggers the degradation of the enzyme by the CRL4CDT2 E3 ubiquitin ligase. PR-Set7 is al...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gky034
更新日期:2018-04-06 00:00:00
abstract::Linkage analysis is a successful procedure to associate diseases with specific genomic regions. These regions are often large, containing hundreds of genes, which make experimental methods employed to identify the disease gene arduous and expensive. We present two methods to prioritize candidates for further experimen...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkl707
更新日期:2006-01-01 00:00:00
abstract::Reversible methylation of the N6 or N1 position of adenine in RNA has recently been shown to play significant roles in regulating the functions of RNA. RNA can also be alkylated upon exposure to endogenous and exogenous alkylating agents. Here we examined how regio-specific methylation at the hydrogen bonding edge of ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkx515
更新日期:2017-09-06 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::In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DS...
journal_title:Nucleic acids research
pub_type: 杂志文章
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更新日期:2013-03-01 00:00:00
abstract::Non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSBs) is mediated by two protein complexes comprising Ku80/Ku70/DNA-PKcs/Artemis and XRCC4/LigaseIV/XLF. Loss of Ku or XRCC4/LigaseIV function compromises the rejoining of radiation-induced DSBs and leads to defective V(D)J recombination. In this study, we...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkn094
更新日期:2008-05-01 00:00:00
abstract::A reporter assay was developed to detect and quantify nonsense codon suppression by chemically aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gnf128
更新日期:2002-12-01 00:00:00
abstract::Alpha-1-microglobulin and bikunin are two plasma glycoproteins encoded by an alpha-1-microglobulin/bikunin precursor (AMBP) gene. The strict liver-specific expression of the AMBP gene is controlled by a potent enhancer made of six clustered boxes numbered 1-6 that have been reported to be proven or potential binding s...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/23.3.395
更新日期:1995-02-11 00:00:00
abstract::Scalable and cost-effective production of error-free DNA is critical to meet the increased demand for such DNA in the field of biological science. Methods based on 'Dial-out PCR' have enabled the high-throughput error-free DNA synthesis from a microarray-synthesized DNA pool by labeling with retrieval PCR tags, and re...
journal_title:Nucleic acids research
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doi:10.1093/nar/gky016
更新日期:2018-04-20 00:00:00
abstract::Human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, contains conserved motifs common to retroviral reverse transcriptases and telomerases. Within the C motif of hTERT is the Leu866-Val867-Asp868-Asp869 tetrapeptide that includes a catalytically essential aspartate dyad. Site-dire...
journal_title:Nucleic acids research
pub_type: 杂志文章
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更新日期:2007-01-01 00:00:00
abstract::The ability to store and interconnect all available information on proteins is crucial to modern biological research. Accordingly, the Universal Protein Resource (UniProt) plays an increasingly important role by providing a stable, comprehensive, freely accessible central resource on protein sequences and functional a...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkl929
更新日期:2007-01-01 00:00:00
abstract::Ovarian poly (A) + RNA from Xenopus laevis and Xenopus borealis was used to construct two cDNA libraries which were screened for histone sequences. cDNA clones to H4 mRNA were obtained from both species and an H3 cDNA clone from Xenopus laevis. The complete DNA sequences of these clones have been determined and are pr...
journal_title:Nucleic acids research
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doi:10.1093/nar/10.12.3769
更新日期:1982-06-25 00:00:00
abstract::RNA editing by adensosine deaminases is a widespread mechanism to alter genetic information in metazoa. In addition to modifications in non-coding regions, editing contributes to diversification of protein function, in analogy to alternative splicing. However, although splicing programs respond to external signals, fa...
journal_title:Nucleic acids research
pub_type: 杂志文章
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更新日期:2013-01-01 00:00:00
abstract::Microsatellites, such as (TG)n found at random throughout the genome, or as 3' extensions of Alu sequences are being increasingly used as genetic markers because of their pluriallelic character. The search for polymorphic microsatellites is time consuming, however, as it is necessary to sequence clones containing the ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/20.6.1333
更新日期:1992-03-25 00:00:00
abstract::Gene trap mutagenesis of mouse embryonic stem cells generates random loss-of-function mutations, which can be identified by a sequence tag and can often report the endogenous expression of the mutated gene. The Centre for Modeling Human Disease is performing expression- and sequence-based screens of gene trap insertio...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkh106
更新日期:2004-01-01 00:00:00