Fusion-PCR generates attL recombination site adaptors and allows Rapid One-Step Gateway (ROG) cloning

Abstract The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

Download Full-text

Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing

Molecular Biotechnology ◽

10.1007/s12033-017-0050-7 ◽

2017 ◽

Vol 60 (2) ◽

pp. 92-99 ◽

Cited By ~ 2

Author(s):

Yilan Liu ◽

Jinjin Chen ◽

Anders Thygesen

Keyword(s):

Fusion Pcr ◽

One Step

Download Full-text

Marker-fusion PCR for one-step mutagenesis of essential genes in yeast

Yeast ◽

10.1002/yea.806 ◽

2002 ◽

Vol 19 (2) ◽

pp. 141-149 ◽

Cited By ~ 18

Author(s):

Ana A. Kitazono ◽

Brian T. D. Tobe ◽

Helen Kalton ◽

Noam Diamant ◽

Stephen J. Kron

Keyword(s):

Essential Genes ◽

Fusion Pcr ◽

One Step

Download Full-text

Fusion PCR, a One-Step Variant of the “Megaprimer” Method of Mutagenesis

BioTechniques ◽

10.2144/98245bm08 ◽

1998 ◽

Vol 24 (5) ◽

pp. 736-742 ◽

Cited By ~ 23

Author(s):

Christiaan Karreman

Keyword(s):

Fusion Pcr ◽

One Step

Download Full-text

An Interactive Minicomputer Program for the Indexing and Simulation of Electron Diffraction Patterns

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100092670 ◽

1976 ◽

Vol 34 ◽

pp. 542-543

Author(s):

R.P. Goehner ◽

W.T. Hatfield ◽

Prakash Rao

Keyword(s):

Electron Diffraction ◽

Real Time ◽

Pattern Analysis ◽

Flow Diagram ◽

Hard Copy ◽

Time Analysis ◽

Real Time Analysis ◽

Transmission Electron ◽

One Step ◽

Diffraction Patterns

Computer programs are now available in various laboratories for the indexing and simulation of transmission electron diffraction patterns. Although these programs address themselves to the solution of various aspects of the indexing and simulation process, the ultimate goal is to perform real time diffraction pattern analysis directly off of the imaging screen of the transmission electron microscope. The program to be described in this paper represents one step prior to real time analysis. It involves the combination of two programs, described in an earlier paper(l), into a single program for use on an interactive basis with a minicomputer. In our case, the minicomputer is an INTERDATA 70 equipped with a Tektronix 4010-1 graphical display terminal and hard copy unit.A simplified flow diagram of the combined program, written in Fortran IV, is shown in Figure 1. It consists of two programs INDEX and TEDP which index and simulate electron diffraction patterns respectively. The user has the option of choosing either the indexing or simulating aspects of the combined program.

Download Full-text

Nutrient-regulated gene expression in eukaryotes

Biochemical Society Symposium ◽

10.1042/bss0730085 ◽

2006 ◽

Vol 73 ◽

pp. 85-96 ◽

Cited By ~ 8

Author(s):

Richard J. Reece ◽

Laila Beynon ◽

Stacey Holden ◽

Amanda D. Hughes ◽

Karine Rébora ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Control ◽

Direct Interaction ◽

Signalling Pathways ◽

A Cell ◽

One Step ◽

Higher Eukaryotes ◽

Regulated Gene Expression

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.

Download Full-text