Abstract:
:About 40% of the genes in the nematode Caenorhabditis elegans have homologs in humans. Based on the history of this model system, it is clear that the application of genetic methods to the study of this set of genes would provide important clues to their function in humans. To facilitate such genetic studies, we are engaged in a project to derive deletion alleles in every gene in this set. Our standard methods make use of nested PCR to hunt for animals in mutagenized populations that carry deletions at a given locus. The deletion bearing animals exist initially in mixed populations where the majority of the animals are wild type at the target. Therefore, the production of the PCR fragment representing the deletion allele competes with the production of the wild type fragment. The size of the deletion fragment relative to wild type determines whether it can compete to a level where it can be detected above the background. Using our standard conditions, we have found that when the deletion is <600 bp, the deletion fragment does not compete effectively with the production of the wild type fragment in PCR. Therefore, although our standard methods work well to detect mutants with deletions >600 bp, they do not work well to detect mutants with smaller deletions. Here we report a new strategy to detect small deletion alleles in complex DNA pools. Our new strategy is a modification of our standard PCR based screens. In the first round of the nested PCR, we include a third PCR primer between the two external primers. The presence of this third primer leads to the production of three fragments from wild type DNA. We configure the system so that two of these three fragments cannot serve as a template in the second round of the nested PCR. The addition of this third primer, therefore, handicaps the amplification from wild type template. On the other hand, the amplification of mutant fragments where the binding site for the third primer is deleted is unabated. Overall, we see at least a 500-fold increase in the sensitivity for small deletion fragments using our new method. Using this new method, we report the recovery of new deletion alleles within 12 C.elegans genes.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Edgley M,D'Souza A,Moulder G,McKay S,Shen B,Gilchrist E,Moerman D,Barstead Rdoi
10.1093/nar/gnf051keywords:
subject
Has Abstractpub_date
2002-06-15 00:00:00pages
e52issue
12eissn
0305-1048issn
1362-4962journal_volume
30pub_type
杂志文章abstract::mRNA expression involves transcription initiation, elongation and degradation. In cells, these dynamic processes are highly regulated. However, experimental characterization of the dynamic processes in vivo is difficult due to the paucity of methods capable of direct measurements. We present a highly sensitive and ver...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkw596
更新日期:2016-10-14 00:00:00
abstract::Light-inducible systems allow spatiotemporal control of a variety of biological activities. Here, we report newly optimized optogenetic tools to induce transcription with light in mammalian cells, using the Arabidopsis photoreceptor Flavin Kelch-repeat F-box 1 (FKF1) and its binding partner GIGANTEA (GI) as well as CR...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkx804
更新日期:2017-11-16 00:00:00
abstract::A new system, ZCURVE 1.0, for finding protein- coding genes in bacterial and archaeal genomes has been proposed. The current algorithm, which is based on the Z curve representation of the DNA sequences, lays stress on the global statistical features of protein-coding genes by taking the frequencies of bases at three c...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkg254
更新日期:2003-03-15 00:00:00
abstract::Alfalfa mosaic virus RNA 4, the subgenomic messenger for viral coat protein, was partially digested with RNase T1 or RNase A and the sequence of a number of fragments was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. From overlapping fragments, the complete prima...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/8.10.2213
更新日期:1980-05-24 00:00:00
abstract::Post-transcriptional methylation of ribose at position O-2' is one of the most common and conserved types of RNA modification. Details of the functional roles of these methylations are far from clear, although in tRNA they are involved at position 34 in regulation of codon recognition and in eukaryotic rRNAs they are ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/27.18.e20
更新日期:1999-09-15 00:00:00
abstract::We define phosphovariants as genetic variations that change phosphorylation sites or their interacting kinases. Considering the essential role of phosphorylation in protein functions, it is highly likely that phosphovariants change protein functions. Therefore, a comparison of phosphovariants between individuals or be...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkn1008
更新日期:2009-03-01 00:00:00
abstract::Here, with the aim of obtaining insight into the intriguing selectivity of G-quadruplex (G4) ligands toward cancer compared to normal cells, a genetically controlled system of progressive transformation in human BJ fibroblasts was analyzed. Among the different comparative evaluations, we found a progressive increase o...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkv006
更新日期:2015-02-18 00:00:00
abstract::Hfq, a bacterial RNA-binding protein, was recently shown to contain the Sm1 motif, a characteristic of Sm and LSm proteins that function in RNA processing events in archaea and eukaryotes. In this report, comparative structural modeling was used to predict a three-dimensional structure of the Hfq core sequence. The pr...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkf508
更新日期:2002-09-01 00:00:00
abstract::Ribotoxins are potent inhibitors of protein biosynthesis and inactivate ribosomes from a variety of organisms. The ribotoxin alpha-sarcin cleaves the large 23S ribosomal RNA (rRNA) at the universally conserved sarcin-ricin loop (SRL) leading to complete inactivation of the ribosome and cellular death. The SRL interact...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkq151
更新日期:2010-07-01 00:00:00
abstract::RegulonDB is a DataBase that integrates biological knowledge of the mechanisms that regulate the transcription initiation in Escherichia coli , as well as knowledge on the organization of the genes and regulatory signals into operons in the chromosome. The operon is the basic structure used in RegulonDB to describe th...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/26.1.55
更新日期:1998-01-01 00:00:00
abstract::We used the high resolution and accuracy of the Cambridge Structural Database (CSD) to provide detailed information regarding base pairing interactions of selected nucleobases. We searched for base pairs in which nucleobases interact with each other through two or more hydrogen bonds and form more or less planar struc...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkaa629
更新日期:2020-09-04 00:00:00
abstract::pS194 is a naturally occurring Staphylococcus aureus plasmid encoding streptomycin resistance. The plasmid has a copy number of about 25 per cell, and belongs to the inc5 incompatibility group. The nucleotide sequence of pS194 has been determined and consists of 4397 base pairs including four open reading frames poten...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/16.5.2179
更新日期:1988-03-25 00:00:00
abstract::In this paper we report the characterization of a newly sequenced 5' upstream region of the human HLA-DRA gene. We performed (i) search for transcription factor motifs, (ii) analysis of CpG display and observed/expected frequency ratios, (iii) search for regions homologous to the 5' upstream sequences of the murine EA...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/23.10.1671
更新日期:1995-05-25 00:00:00
abstract::Prokaryotes use primed CRISPR adaptation to update their memory bank of spacers against invading genetic elements that have escaped CRISPR interference through mutations in their protospacer target site. We previously observed a trend that nucleotide-dependent mismatches between crRNA and the protospacer strongly infl...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gky687
更新日期:2018-11-02 00:00:00
abstract::The Genomes On Line Database (GOLD) is a web resource for comprehensive access to information regarding complete and ongoing genome sequencing projects worldwide. The database currently incorporates information on over 1500 sequencing projects, of which 294 have been completed and the data deposited in the public data...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkj145
更新日期:2006-01-01 00:00:00
abstract::TIGRFAMs is a collection of protein family definitions built to aid in high-throughput annotation of specific protein functions. Each family is based on a hidden Markov model (HMM), where both cutoff scores and membership in the seed alignment are chosen so that the HMMs can classify numerous proteins according to the...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkl1043
更新日期:2007-01-01 00:00:00
abstract::Here we employ a set of RNA Polymerase II (Pol II) activity mutants to determine the consequences of increased or decreased Pol II catalysis on gene expression in Saccharomyces cerevisiae. We find that alteration of Pol II catalytic rate, either fast or slow, leads to decreased Pol II occupancy and apparent reduction ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkx037
更新日期:2017-05-05 00:00:00
abstract::We have examined the absorption and circular dichroism spectra of intact Penicillium chrysogenum virus, empty capsid particles, and isolated double-stranded RNA. The absorbance at 260 nm of intact virus was less than 4% hypochromic relative to the absorbances of the free double-stranded RNA and free viral protein, ind...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/11.1.175
更新日期:1983-01-11 00:00:00
abstract::The ability to integrate 'omics' (i.e. transcriptomics and proteomics) is becoming increasingly important to the understanding of regulatory mechanisms. There are currently no tools available to identify differentially expressed genes (DEGs) across different 'omics' data types or multi-dimensional data including time ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkw157
更新日期:2016-06-02 00:00:00
abstract::For the isolation of single stranded plasmid DNA, various E. coli and E. coli-Streptomyces shuttle plasmids were equipped with the phage f1 replication origin. The transformation of some representative Streptomyces species with plasmid vectors occurred irrespective of whether single or double stranded DNA was used. In...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/19.4.727
更新日期:1991-02-25 00:00:00
abstract::Gene expression circuitries, which enable cells to detect precise levels within a morphogen concentration gradient, have a pivotal impact on biological processes such as embryonic pattern formation, paracrine and autocrine signalling, and cellular migration. We present the rational synthesis of a synthetic genetic cir...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkq671
更新日期:2010-10-01 00:00:00
abstract::By successively assembling genetic parts such as BioBrick according to grammatical models, complex genetic constructs composed of dozens of functional blocks can be built. However, usually every category of genetic parts includes a few or many parts. With increasing quantity of genetic parts, the process of assembling...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkx859
更新日期:2017-12-15 00:00:00
abstract::A recently reported cDNA clone coding for human promyelocytic L apoferritin, shows some differences with a liver L apoferritin cDNA. We have investigated if these differences are due to the expression of different genes or to an alternative transcription of an unique gene. In this paper we report data suggesting that ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/14.7.2863
更新日期:1986-04-11 00:00:00
abstract::Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (posi...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkq341
更新日期:2010-09-01 00:00:00
abstract::Although subtelomeric regions in humans are heterochromatic, the epigenetic nature of human telomeres remains controversial. This controversy might have been influenced by the confounding effect of subtelomeric regions and interstitial telomeric sequences (ITSs) on telomeric chromatin structure analyses. In addition, ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gky006
更新日期:2018-03-16 00:00:00
abstract::The H19 lncRNA has been implicated in development and growth control and is associated with human genetic disorders and cancer. Acting as a molecular sponge, H19 inhibits microRNA (miRNA) let-7. Here we report that H19 is significantly decreased in muscle of human subjects with type-2 diabetes and insulin resistant ro...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gku1160
更新日期:2014-12-16 00:00:00
abstract::A downstream G-rich sequence (GRS), GGGGGAGGUGUGGG, has been previously shown to influence the efficiency of 3' end processing of the SV40 late polyadenylation signal. We have now defined several important parameters for GRS-mediated polyadenylation. The ability of the GRS to influence 3' end processing efficiency was...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/23.9.1625
更新日期:1995-05-11 00:00:00
abstract::Three clones containing both the human G gamma and A gamma globlin genes have been isolated and characterized from a library of DNA fragments generated by partial Eco RI digestion of cellular DNA using charon 4A phage as vector. Two of the clones (NY 2 and 3) are identical and have an insert of 14.0 kb. The third clon...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/7.5.1147
更新日期:1979-11-10 00:00:00
abstract::Homologous recombination is essential for the preservation of genome stability, thereby preventing cancer. The recombination protein RAD51 drives DNA strand exchange, which requires the assembly, rearrangement and disassembly of a RAD51 filament on DNA, coupled to ATP binding and hydrolysis. This process is facilitate...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkr295
更新日期:2011-08-01 00:00:00
abstract::Eukaryotic elongation factor 1 A (eEF1A, formerly elongation factor-1 alpha) is an important component of the protein synthesis apparatus. Here we report the isolation and characterization of the cDNA sequence encoding rabbit eEF1A-2, an isoform of eEF1A, as well as a structural and functional comparison of the two ra...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/26.8.1884
更新日期:1998-04-15 00:00:00