scholarly journals ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes

2020 ◽  
Vol 4 (2) ◽  
pp. e202000836
Author(s):  
Arnaud Rondelet ◽  
Andrei Pozniakovsky ◽  
Devika Namboodiri ◽  
Richard Cardoso da Silva ◽  
Divya Singh ◽  
...  
Keyword(s):  
Protein Function ◽  
High Efficiency ◽  
Marker Gene ◽  
Point Mutations ◽  
Artificial Chromosome ◽  
Single Step ◽  
Step Method ◽  
Mutation Site ◽  
Endogenous Gene ◽  
Artificial Chromosomes

Bacterial artificial chromosome (BAC)–based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. Although homologous recombination-based recombineering methods have streamlined the efficient integration of protein tags onto BAC transgenes, generating precise point mutations has remained less efficient and time-consuming. Here, we present a simplified method for inserting point mutations into BAC transgenes requiring a single recombineering step followed by antibiotic selection. This technique, which we call exogenous/synthetic intronization (ESI) mutagenesis, relies on co-integration of a mutation of interest along with a selectable marker gene, the latter of which is harboured in an artificial intron adjacent to the mutation site. Cell lines generated from ESI-mutated BACs express the transgenes equivalently to the endogenous gene, and all cells efficiently splice out the synthetic intron. Thus, ESI mutagenesis provides a robust and effective single-step method with high precision and high efficiency for mutating BAC transgenes.

scholarly journals ESI mutagenesis: A one-step method for introducing point mutations into bacterial artificial chromosome transgenes

2019 ◽  
Author(s):  
Arnaud Rondelet ◽  
Andrei Pozniakovsky ◽  
Marit Leuschner ◽  
Ina Poser ◽  
Andrea Ssykor ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Protein Function ◽  
High Efficiency ◽  
Marker Gene ◽  
Point Mutations ◽  
Artificial Chromosome ◽  
Single Step ◽  
Step Method ◽  
Mutation Site ◽  
Endogenous Gene

AbstractBacterial artificial chromosome (BAC)-based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. While homologous recombination-based recombineering methods have streamlined the efficient integration of protein tags onto BAC transgenes, generating precise point mutations has remained less efficient and time-consuming. Here we present a simplified method for inserting point mutations into BAC transgenes requiring a single recombineering step followed by antibiotic selection. This technique, which we call ESI (Exogenous/Synthetic Intronization) mutagenesis, relies on co-integration of a mutation of interest along with a selectable marker gene, the latter of which is harboured in an artificial intron adjacent to the mutation site. Cell lines generated from ESI-mutated BACs express the transgenes equivalently to the endogenous gene, and all cells efficiently splice out the synthetic intron. Thus, ESI-mutagenesis provides a robust and effective single-step method with high precision and high efficiency for mutating BAC transgenes.

Single-Step Conversion of P1 and P1 Artificial Chromosome Clones into Yeast Artificial Chromosomes

Genomics ◽  
2000 ◽  
Vol 68 (1) ◽  
pp. 106-110 ◽  
Author(s):  
Parvoneh Poorkaj ◽  
Kenneth R. Peterson ◽  
Gerard D. Schellenberg
Keyword(s):  
Artificial Chromosome ◽  
Single Step ◽  
Artificial Chromosomes ◽  
Yeast Artificial Chromosomes

scholarly journals Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR

1997 ◽  
Vol 43 (12) ◽  
pp. 2262-2267 ◽  
Author(s):  
Marla J Lay ◽  
Carl T Wittwer
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Factor V Leiden ◽  
Resonance Energy ◽  
Fluorescence Analysis ◽  
Single Step ◽  
Factor V ◽  
Step Method ◽  
Mutation Site ◽  
Thermal Cycler

Abstract A single-step method for factor V Leiden genotyping is presented that uses rapid-cycle PCR and simultaneous fluorescence analysis with resonance energy transfer probes. A fragment of the factor V gene containing the mutation is amplified asymmetrically through use of a primer labeled with Cy5TM in the presence of a 3′-fluorescein-labeled probe that covers the mutation site. When the fluorescein probe is annealed to the extension product of the Cy5-labeled primer, the fluorophores are brought into close enough contact for resonance energy transfer to occur. As the temperature increases, the probe melts from its target, decreasing the resonance energy transfer. When the probe is complementary to the product strand, it melts at 65 °C; if the single-base mutation is present, the probe melts at 57 °C. Concurrent amplification and analysis from genomic DNA takes 20–45 min and requires no sample manipulation after the fluorescence thermal cycler is loaded.

Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain

1983 ◽  
Vol 49 (01) ◽  
pp. 024-027 ◽  
Author(s):  
David Vetterlein ◽  
Gary J Calton
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Culture Media ◽  
Biological Fluids ◽  
Single Step ◽  
High Yield ◽  
Step Method ◽  
Commercial Preparation ◽  
E Coli ◽  
Tissue Culture Media

SummaryThe preparation of a monoclonal antibody (MAB) against high molecular weight (HMW) urokinase light chain (20,000 Mr) is described. This MAB was immobilized and the resulting immunosorbent was used to isolate urokinase starting with an impure commercial preparation, fresh urine, spent tissue culture media, or E. coli broth without preliminary dialysis or concentration steps. Monospecific antibodies appear to provide a rapid single step method of purifying urokinase, in high yield, from a variety of biological fluids.

Coaction of Electrostatic and Hydrophobic Interactions: Dynamic Constraints on Disordered TrkA Juxtamembrane Domain

2019 ◽  
Author(s):  
Zichen Wang ◽  
Huaxun Fan ◽  
Xiao Hu ◽  
John Khamo ◽  
Jiajie Diao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Hydrophobic Interactions ◽  
Transmembrane Helix ◽  
Point Mutations ◽  
Salt Bridge ◽  
Receptor Activation ◽  
Membrane Dynamics ◽  
Juxtamembrane Domain ◽  
Joint Work

<p>The receptor tyrosine kinase family transmits signals into cell via a single transmembrane helix and a flexible juxtamembrane domain (JMD). Membrane dynamics makes it challenging to study the structural mechanism of receptor activation experimentally. In this study, we employ all-atom molecular dynamics with Highly Mobile Membrane-Mimetic to capture membrane interactions with the JMD of tropomyosin receptor kinase A (TrkA). We find that PIP<sub>2 </sub>lipids engage in lasting binding to multiple basic residues and compete with salt bridge within the peptide. We discover three residues insertion into the membrane, and perturb it through computationally designed point mutations. Single-molecule experiments indicate the contribution from hydrophobic insertion is comparable to electrostatic binding, and in-cell experiments show that enhanced TrkA-JMD insertion promotes receptor ubiquitination. Our joint work points to a scenario where basic and hydrophobic residues on disordered domains interact with lipid headgroups and tails, respectively, to restrain flexibility and potentially modulate protein function.</p>

scholarly journals CHROMOSOMAL BASIS OF THE MEROZYGOSITY IN A PARTIALLY DIPLOID MUTANT OF PNEUMOCOCCUS

Genetics ◽  
1975 ◽  
Vol 80 (4) ◽  
pp. 667-678
Author(s):  
Mary Lee S Ledbetter ◽  
Rollin D Hotchkiss
Keyword(s):  
High Efficiency ◽  
Point Mutations ◽  
Resistant Mutant ◽  
Chromosomal Marker ◽  
Structural Abnormality ◽  
C Cells ◽  
Wild Type ◽  
Chromosomal Locus ◽  
Low Efficiency ◽  
Derivatives Of

ABSTRACT A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.

scholarly journals Glutamine synthetase as a central element in hepatic glutamine and ammonia metabolism: novel aspects

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Benedikt Frieg ◽  
Boris Görg ◽  
Holger Gohlke ◽  
Dieter Häussinger
Keyword(s):  
Glutamine Synthetase ◽  
Protein Function ◽  
Nitrosative Stress ◽  
Point Mutations ◽  
Central Element ◽  
Ph Sensitive ◽  
Tyrosine Nitration ◽  
Ammonia Metabolism ◽  
Functional Consequences

Abstract Glutamine synthetase (GS) in the liver is expressed in a small perivenous, highly specialized hepatocyte population and is essential for the maintenance of low, non-toxic ammonia levels in the organism. However, GS activity can be impaired by tyrosine nitration of the enzyme in response to oxidative/nitrosative stress in a pH-sensitive way. The underlying molecular mechanism as investigated by combined molecular simulations and in vitro experiments indicates that tyrosine nitration can lead to a fully reversible and pH-sensitive regulation of protein function. This approach was also used to understand the functional consequences of several recently described point mutations of human GS with clinical relevance and to suggest an approach to restore impaired GS activity.

scholarly journals High-resolution limited-angle phase tomography of dense layered objects using deep neural networks

2019 ◽  
Vol 116 (40) ◽  
pp. 19848-19856 ◽  
Author(s):  
Alexandre Goy ◽  
Girish Rughoobur ◽  
Shuai Li ◽  
Kwabena Arthur ◽  
Akintunde I. Akinwande ◽  
...  
Keyword(s):  
Integrated Circuit ◽  
Deep Neural Networks ◽  
Tomographic Reconstruction ◽  
Synthetic Data ◽  
Single Step ◽  
Training Set ◽  
Step Method ◽  
3 Dimensional ◽  
Dimensional Reconstruction ◽  
Optical Domain

We present a machine learning-based method for tomographic reconstruction of dense layered objects, with range of projection angles limited to ±10○. Whereas previous approaches to phase tomography generally require 2 steps, first to retrieve phase projections from intensity projections and then to perform tomographic reconstruction on the retrieved phase projections, in our work a physics-informed preprocessor followed by a deep neural network (DNN) conduct the 3-dimensional reconstruction directly from the intensity projections. We demonstrate this single-step method experimentally in the visible optical domain on a scaled-up integrated circuit phantom. We show that even under conditions of highly attenuated photon fluxes a DNN trained only on synthetic data can be used to successfully reconstruct physical samples disjoint from the synthetic training set. Thus, the need for producing a large number of physical examples for training is ameliorated. The method is generally applicable to tomography with electromagnetic or other types of radiation at all bands.

A Single-Step Method for the Isolation of Antithrombin IIP

Vox Sanguinis ◽  
1984 ◽  
Vol 47 (6) ◽  
pp. 397-405
Author(s):  
Milan Wickerhauser ◽  
Craigenne Williams
Keyword(s):  
Single Step ◽  
Step Method
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