Enzymatic techniques for the isolation of random single-base substitutions in vitro at high frequency.

Abstract:

:A general and efficient method has been developed to generate large numbers of single-base substitution mutations simply and rapidly. A unique f1 phage recombinant DNA cloning vector is described, which contains the phi X174 origin of viral strand DNA synthesis and allows one to direct mutagenesis to any specific segment of DNA. Gapped circular DNA is constructed by annealing viral single-stranded circular DNA [ss(c) DNA] with a mixture of linear duplex DNAs that have had their 3'-OH termini processively digested with Escherichia coli exonuclease III under conditions in which the resulting, newly generated 3'-OH termini present in the various hybrid molecules span the region of interest. Base changes are induced by misincorporation of an alpha-thiodeoxynucleoside triphosphate analog onto this primer-template, followed by DNA repair synthesis. The asymmetric segregation of mutants from wild-type sequences is accomplished by double-stranded replicative form DNA----ss(c) DNA synthesis in vitro, initiated from the phi X174 viral strand origin sequence present on the vector DNA. Mutated ss(c) DNA is screened by the dideoxy chain termination method. In one mutagenesis experiment, 21 independent single-base substitutions were isolated in a 72-nucleotide-long target region. DNA sequence analysis showed that all possible base transversions and transitions were represented.

authors

Abarzúa P,Marians KJ

doi

10.1073/pnas.81.7.2030

subject

Has Abstract

pub_date

1984-04-01 00:00:00

pages

2030-4

issue

7

eissn

0027-8424

issn

1091-6490

journal_volume

81

pub_type

杂志文章
  • NMR structures of three single-residue variants of the human prion protein.

    abstract::The NMR structures of three single-amino acid variants of the C-terminal domain of the human prion protein, hPrP(121-230), are presented. In hPrP(M166V) and hPrP(R220K) the substitution is with the corresponding residue in murine PrP, and in hPrP(S170N) it is with the corresponding Syrian hamster residue. All three su...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.97.15.8340

    authors: Calzolai L,Lysek DA,Guntert P,von Schroetter C,Riek R,Zahn R,Wüthrich K

    更新日期:2000-07-18 00:00:00

  • The selection of acceptable protein mutations.

    abstract::We have determined the general constraints that govern sequence divergence in proteins that retain entirely, or very largely, the same structure and function. To do this we collected data from three different groups of orthologous sequences: those found in humans and mice, in humans and chickens, and in Escherichia co...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.0703737104

    authors: Sasidharan R,Chothia C

    更新日期:2007-06-12 00:00:00

  • The human ERG1 channel polymorphism, K897T, creates a phosphorylation site that inhibits channel activity.

    abstract::Single-nucleotide polymorphisms (SNPs) in the human ether-a-go-go-related gene 1, hERG1, are associated with cardiac arrhythmias. The Kv11.1 channels encoded by hERG1 are also essential for rhythmic excitability of the pituitary, where they are regulated by thyroid hormone through a signal transduction cascade involvi...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.0802250105

    authors: Gentile S,Martin N,Scappini E,Williams J,Erxleben C,Armstrong DL

    更新日期:2008-09-23 00:00:00

  • Role of social wasps in Saccharomyces cerevisiae ecology and evolution.

    abstract::Saccharomyces cerevisiae is one of the most important model organisms and has been a valuable asset to human civilization. However, despite its extensive use in the last 9,000 y, the existence of a seasonal cycle outside human-made environments has not yet been described. We demonstrate the role of social wasps as vec...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1208362109

    authors: Stefanini I,Dapporto L,Legras JL,Calabretta A,Di Paola M,De Filippo C,Viola R,Capretti P,Polsinelli M,Turillazzi S,Cavalieri D

    更新日期:2012-08-14 00:00:00

  • Geological sulfur isotopes indicate elevated OCS in the Archean atmosphere, solving faint young sun paradox.

    abstract::Distributions of sulfur isotopes in geological samples would provide a record of atmospheric composition if the mechanism producing the isotope effects could be described quantitatively. We determined the UV absorption spectra of 32SO2, 33SO2, and 34SO2 and use them to interpret the geological record. The calculated i...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.0903518106

    authors: Ueno Y,Johnson MS,Danielache SO,Eskebjerg C,Pandey A,Yoshida N

    更新日期:2009-09-01 00:00:00

  • Monoclonal antibodies distinguish subtypes of retinal horizontal cells.

    abstract::Sixteen hybridomas have been identified that secrete antibodies specific to horizontal cells in the carp retina. The hybridomas have been classified into three groups based on their antibody staining patterns: group I, staining associated with all horizontal cells; group II, staining associated with the most abundant ...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.81.19.6255

    authors: Young LH,Dowling JE

    更新日期:1984-10-01 00:00:00

  • Molecular random tilings as glasses.

    abstract::We have recently shown that p-terphenyl-3,5,3',5'-tetracarboxylic acid adsorbed on graphite self-assembles into a two-dimensional rhombus random tiling. This tiling is close to ideal, displaying long-range correlations punctuated by sparse localized tiling defects. In this article we explore the analogy between dynami...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.0902443106

    authors: Garrahan JP,Stannard A,Blunt MO,Beton PH

    更新日期:2009-09-08 00:00:00

  • Depletion layer dynamics of polyelectrolyte solutions under Poiseuille flow.

    abstract::Complex liquids flow through channels faster than expected, an effect attributed to the formation of low-viscosity depletion layers at the boundaries. Characterization of depletion layer length scale, concentration, and dynamics has remained elusive due in large part to the lack of suitable real-space experimental tec...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1900623116

    authors: Park SJ,Shakya A,King JT

    更新日期:2019-08-13 00:00:00

  • Magnetic resonance studies of spin-labeled creatine kinase system and interaction of two paramagnetic probes.

    abstract::Creatine kinase from rabbit muscle has been specifically labeled with a derivative of a nitroxide free radical on the two essential sulfhydryl groups, one per active site. The conformation in the environment of the enzyme-bound free radical and the change in conformation induced upon binding of the substrate, metal ad...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.64.1.219

    authors: Taylor JS,Leigh JS Jr,Cohn M

    更新日期:1969-09-01 00:00:00

  • A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis.

    abstract::Seed dormancy is a trait of considerable adaptive significance because it maximizes seedling survival by preventing premature germination under unfavorable conditions. Understanding how seeds break dormancy and initiate growth is also of great agricultural and biotechnological interest. Abscisic acid (ABA) plays prima...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.081594298

    authors: Lopez-Molina L,Mongrand S,Chua NH

    更新日期:2001-04-10 00:00:00

  • Visualizing the formation of the Kondo lattice and the hidden order in URu(2)Si(2).

    abstract::Heavy electronic states originating from the f atomic orbitals underlie a rich variety of quantum phases of matter. We use atomic scale imaging and spectroscopy with the scanning tunneling microscope to examine the novel electronic states that emerge from the uranium f states in URu(2)Si(2). We find that, as the tempe...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1005892107

    authors: Aynajian P,da Silva Neto EH,Parker CV,Huang Y,Pasupathy A,Mydosh J,Yazdani A

    更新日期:2010-06-08 00:00:00

  • The RNA element encoded by the trans-activation-responsive region of human immunodeficiency virus type 1 is functional when displaced downstream of the start of transcription.

    abstract::The human immunodeficiency virus type 1 (HIV-1) trans-activator protein, Tat, specifically stimulates transcription from the viral long terminal repeat. Tat binds to an RNA stem-loop structure encoded by the trans-activation response region (TAR). To test whether TAR is functional when displaced downstream of the star...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.92.6.2408

    authors: Churcher MJ,Lowe AD,Gait MJ,Karn J

    更新日期:1995-03-14 00:00:00

  • Competition between noggin and bone morphogenetic protein 4 activities may regulate dorsalization during Xenopus development.

    abstract::Bone morphogenetic protein 4 (BMP-4) induces ventral mesoderm but represses dorsal mesoderm formation in Xenopus embryos. We show that BMP-4 inhibits two signaling pathways regulating dorsal mesoderm formation, the induction of dorsal mesoderm (Spemann organizer) and the dorsalization of ventral mesoderm. Ectopic expr...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.92.26.12141

    authors: Re'em-Kalma Y,Lamb T,Frank D

    更新日期:1995-12-19 00:00:00

  • Pausing and premature termination of human RNA polymerase II during transcription of adenovirus in vivo and in vitro.

    abstract::The major late transcriptional unit of adenovirus type 2 has served as a model for studying transcription in eukaryotes. We report that pausing and premature termination are intrinsic to the transcription of this transcriptional unit by RNA polymerase II. In vivo and in isolated nuclei, transcription pauses at discret...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.81.19.5931

    authors: Maderious A,Chen-Kiang S

    更新日期:1984-10-01 00:00:00

  • The Great Recession, genetic sensitivity, and maternal harsh parenting.

    abstract::Using data from the Fragile Families and Child Wellbeing Study, this study examined the effects of the Great Recession on maternal harsh parenting. We found that changes in macroeconomic conditions, rather than current conditions, affected harsh parenting, that declines in macroeconomic conditions had a stronger impac...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 历史文章,杂志文章

    doi:10.1073/pnas.1312398110

    authors: Lee D,Brooks-Gunn J,McLanahan SS,Notterman D,Garfinkel I

    更新日期:2013-08-20 00:00:00

  • Germ-cell deficient (gcd), an insertional mutation manifested as infertility in transgenic mice.

    abstract::A genetic analysis is necessary to gain a greater understanding of the complex developmental processes in mammals. Toward this end, an insertional transgenic mouse mutant has been isolated that results in abnormal germ-cell development. This recessive mutation manifests as infertility in both males and females and is ...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.88.19.8787

    authors: Pellas TC,Ramachandran B,Duncan M,Pan SS,Marone M,Chada K

    更新日期:1991-10-01 00:00:00

  • Human ribosomal RNA gene: nucleotide sequence of the transcription initiation region and comparison of three mammalian genes.

    abstract::The transcription initiation site of the human ribosomal RNA gene (rDNA) was located by using the single-strand specific nuclease protection method and by determining the first nucleotide of the in vitro capped 45S preribosomal RNA. The sequence of 1,211 nucleotides surrounding the initiation site was determined. The ...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.79.10.3092

    authors: Financsek I,Mizumoto K,Mishima Y,Muramatsu M

    更新日期:1982-05-01 00:00:00

  • Genetic dating indicates that the Asian-Papuan admixture through Eastern Indonesia corresponds to the Austronesian expansion.

    abstract::Although the Austronesian expansion had a major impact on the languages of Island Southeast Asia, controversy still exists over the genetic impact of this expansion. The coexistence of both Asian and Papuan genetic ancestry in Eastern Indonesia provides a unique opportunity to address this issue. Here, we estimate rec...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1118892109

    authors: Xu S,Pugach I,Stoneking M,Kayser M,Jin L,HUGO Pan-Asian SNP Consortium.

    更新日期:2012-03-20 00:00:00

  • Radiometric dating of the type-site for Homo heidelbergensis at Mauer, Germany.

    abstract::The Mauer mandible, holotype of Homo heidelbergensis, was found in 1907 in fluvial sands deposited by the Neckar River 10 km southeast of Heidelberg, Germany. The fossil is an important key to understanding early human occupation of Europe north of the Alps. Given the associated mammal fauna and the geological context...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 历史文章,杂志文章

    doi:10.1073/pnas.1012722107

    authors: Wagner GA,Krbetschek M,Degering D,Bahain JJ,Shao Q,Falguères C,Voinchet P,Dolo JM,Garcia T,Rightmire GP

    更新日期:2010-11-16 00:00:00

  • Functional heterogeneity of the four voltage sensors of a human L-type calcium channel.

    abstract::Excitation-evoked Ca(2+) influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca(2+) entry are thought to be functions of voltage-gated calcium (CaV) channels composed of a ce...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1411127112

    authors: Pantazis A,Savalli N,Sigg D,Neely A,Olcese R

    更新日期:2014-12-23 00:00:00

  • Body map proto-organization in newborn macaques.

    abstract::Topographic sensory maps are a prominent feature of the adult primate brain. Here, we asked whether topographic representations of the body are present at birth. Using functional MRI (fMRI), we find that the newborn somatomotor system, spanning frontoparietal cortex and subcortex, comprises multiple topographic repres...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1912636116

    authors: Arcaro MJ,Schade PF,Livingstone MS

    更新日期:2019-12-03 00:00:00

  • Regulation of human EGF receptor by lipids.

    abstract::The human epidermal growth factor receptor (EGFR) is a key representative of tyrosine kinase receptors, ubiquitous actors in cell signaling, proliferation, differentiation, and migration. Although the receptor is well-studied, a central issue remains: How does the compositional diversity and functional diversity of th...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1105666108

    authors: Coskun Ü,Grzybek M,Drechsel D,Simons K

    更新日期:2011-05-31 00:00:00

  • The A-kinase anchoring protein (AKAP)-Lbc-signaling complex mediates alpha1 adrenergic receptor-induced cardiomyocyte hypertrophy.

    abstract::In response to various pathological stresses, the heart undergoes a pathological remodeling process that is associated with cardiomyocyte hypertrophy. Because cardiac hypertrophy can progress to heart failure, a major cause of lethality worldwide, the intracellular signaling pathways that control cardiomyocyte growth ...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.0701099104

    authors: Appert-Collin A,Cotecchia S,Nenniger-Tosato M,Pedrazzini T,Diviani D

    更新日期:2007-06-12 00:00:00

  • Disassembly of the Staphylococcus aureus hibernating 100S ribosome by an evolutionarily conserved GTPase.

    abstract::The bacterial hibernating 100S ribosome is a poorly understood form of the dimeric 70S particle that has been linked to pathogenesis, translational repression, starvation responses, and ribosome turnover. In the opportunistic pathogen Staphylococcus aureus and most other bacteria, hibernation-promoting factor (HPF) ho...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1709588114

    authors: Basu A,Yap MN

    更新日期:2017-09-26 00:00:00

  • Maternal viral infection causes global alterations in porcine fetal microglia.

    abstract::Maternal infections during pregnancy are associated with increased risk of neurodevelopmental disorders, although the precise mechanisms remain to be elucidated. Previously, we established a maternal immune activation (MIA) model using swine, which results in altered social behaviors of piglet offspring. These behavio...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1817014116

    authors: Antonson AM,Lawson MA,Caputo MP,Matt SM,Leyshon BJ,Johnson RW

    更新日期:2019-10-01 00:00:00

  • Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide.

    abstract::Escherichia coli lipopolysaccharide (LPS)-induced nitrate biosynthesis was studied in LPS-sensitive C3H/He and LPS-resistant C3H/HeJ mice. Intraperitoneal injection of 15 micrograms of LPS led to a temporary 5- to 6-fold increase in blood nitrate concentration in the C3H/He strain. Levels of nitrate excreted in the ur...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.82.22.7738

    authors: Stuehr DJ,Marletta MA

    更新日期:1985-11-01 00:00:00

  • Anatomy of funded research in science.

    abstract::Seeking research funding is an essential part of academic life. Funded projects are primarily collaborative in nature through internal and external partnerships, but what role does funding play in the formulation of these partnerships? Here, by examining over 43,000 scientific projects funded over the past three decad...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1513651112

    authors: Ma A,Mondragón RJ,Latora V

    更新日期:2015-12-01 00:00:00

  • Observations on the pH dependence of the glutaminase activity of a glutamine amidotransferase, carbamylphosphate synthetase.

    abstract::Glutamine-dependent carbamylphosphate synthetase (Escherichia coli) is composed of a heavy subunit (molecular weight about 130,000) and a light subunit (molecular weight about 42,000), which can be separated with retention of catalytic activities. The separated heavy subunit can catalyze activation of CO(2) by ATP and...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.70.10.2717

    authors: Trotta PP,Wellner VP,Pinkus LM,Meister A

    更新日期:1973-10-01 00:00:00

  • Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium.

    abstract::Nitrogenase is an ATP-requiring enzyme capable of carrying out multielectron reductions of inert molecules. A purified remodeled nitrogenase containing two amino acid substitutions near the site of its FeMo cofactor was recently described as having the capacity to reduce carbon dioxide (CO2) to methane (CH4). Here, we...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.1611043113

    authors: Fixen KR,Zheng Y,Harris DF,Shaw S,Yang ZY,Dean DR,Seefeldt LC,Harwood CS

    更新日期:2016-09-06 00:00:00

  • Conformations of arsanilazotyrosine-248 carboxypeptidase A alpha, beta, gamma, comparison of crystals and solution.

    abstract::The spectra of the alpha, beta, and gamma forms of zinc monoarsanilazotyrosine-248 carboxypeptidase A are indistinguishable. At pH 8.2 their crystals are yellow, while their solutions are red, lambda(max) 510 nm. Absorption and circular dichroism-pH titrations of the modified zinc and apoenzymes demonstrate that the a...

    journal_title:Proceedings of the National Academy of Sciences of the United States of America

    pub_type: 杂志文章

    doi:10.1073/pnas.70.7.2006

    authors: Johansen JT,Vallee BL

    更新日期:1973-07-01 00:00:00