Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu.

PERSPECTIVES AND SUMMARY . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 ORIGINS OF CONSERVED GENE REGULATORS . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 211 Steroids . . . . .. . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . .... . . . . . . .. . . . . . . . .. . . . . . . .. . 211 Steroid Receptors .... " . . . . . . . . . . . . . . . . . . . . . " . . . . . . . . . . . . . . . . . . . . . . . . "" . . . . . . . . . . . . . . . . . . . . . . " 211 FUNCTIONAL ASPECTS OF RECEPTOR STRUCTURE" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Activation and Intracellular Localization ... . ". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Steroid Binding .. ...... , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " . . . . . . . . . . . . . . . . . . , 214 Nonspecific DNA Binding . .. . ..... ....... . ....... " . . . . . . . . .. . . . . . . . . . . . . . . . . " 215 Functional Heterogeneity 216 SELECTIVE RECEPTOR:DNA INTERACTIONS ... . . .... .. .... . .... . 21 7 Receptor Binding at Genomic Sites of Action . " . . . . . . . " 218 Detection of Specific DNA Binding Sites """""",,, , , """""'" . . . . . . . . . . . . . . . . . . . . . . . 21 8 DNA Sequences Bound by Steroid Receptors .. . ......... . ..... . ..... . ..... . ... , 221 LOCALIZATION OF STEROID RESPONSE ELEMENTS (REs) . .... " . . . . . . . . . 222 Deletion Mapping .. . ..... ......... . ....... . " . . . . . . . . . . . . . . . . . . . . . ""." . . .... . . . 223 RE-Promoter Fusions .... ......... . " . . . . . . . . . . . . . . . . . ". . . . . . . . . . . . . . . . . . 223 In Vitro Mutagenesis" ...... . ........ ......... .. " . . . . . . . . . . . . . . . . . "" . . . . . . " . . . . . . . . . . . . . . . . " 224 TRANSCRIPTIONAL ENHANCEMENT BY RECEPTOR:RE COMPLEXES " " "'" ' ' 224 Glucocorticoid-Dependent Enhancement 225 Detection and Characterization of Enhancers. . .. . . . . . . .... . . .... . . . . . . .. . . . . . . .... . . ... . . . . 225 EFFECTS OF RECEPTOR BINDING ON GENOME STRUCTURE ... " . . ". . . . . . . . . . . . . . 227 Cytological and Nuclease Probes .. . . """""" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " . . . . . . . . . 227 Chromosomal Protein Alterations 229 DNA-Structure Alterations 230 New Experimental Approaches . . . . . ... """ . . ,, .. " . . . . ,,". . . . . . . . . . . . . . . . . . . . . . . . . 232 HIGHER-ORDER CONTROL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 233 Gene Networks ..... ...... ........ ....... . " . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ". . . . . . . . . 234 Multifactor Regulation ...... ... ,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

Steroid Receptor Regulated Transcription of Specific Genes and Gene Networks

A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective lambda prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42 degrees C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective lambda prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.

/pdf/an-efficient-recombination-system-for-chromosome-engineering-4acmootzcr.pdf

An efficient recombination system for chromosome engineering in Escherichia coli

https://mmbr.asm.org/content/mmbr/47/2/180.full.pdf

Linkage map of Escherichia coli K-12, edition 7.

http://zhiyuan.sjtu.edu.cn/file/course/20131031102853_Zhuang%20Yuan-Lecture%201-optional%20reading.pdf

The V(D)J Recombination Activating Gene, RAG-1

Take your vitamins with a pinch of RNA.

E. coli mutants produced by the insertion of bacteriophage lambda DNA.

CONTENTS INTRODUCTION 452 DELETIONS .. , .. ,,, .. , ...... ," "'"'''''''''''''''''' "' .. , .. , .. ,'" "'''''' """''', .. , .. , .. ,'' '" ","""'" "'",''''''' '" 452 The Influence o[ Base Sequence .. " ... " .. "."."" ... "".""".".".".".""" .. "."" ... "."."""" 453 Deletion analysis of the gal operon of E. coli """"""""""""""" .. "" ........ ",, .... """"" 453 Deletions in the ara operon of E. coli """ " ,," 454 Deletion analysis of the lacI gene of E. coli 455 Genetic Control o[ Deletion Formation 457 The role of bacterial genes " 457 The role of phage A genes ...... ... . . ...... . . ... . 457 Site-Specific Deletions ..... ,""""""',.,"',',." .. , ' .. , .. ,., ,. 458 Int dependent deletions ........ " ..... " " ........ " 458 IS-associated deletions ..... .... ,.,.,., .... ,." ........ , ,., .. , ..... , 458

In bacteria and bacteriophage

Integration hostfactor (IHF)isa small, basic protein thatisneeded forefficient recombination of bacteriophage A,aswell asforother hostandviral functions. We haveconstructed strains inwhichthetwo subunits ofIHF,encoded bythehimAandhipgenes ofEscherichia coli, areexpressed underthecontrol ofthe XPLpromoter. Separate overexpression ofhimAandhipledtotheproduction ofunstable andinsoluble peptides, respectively. Incontrast, theoverexpression ofbothgenes conjointly ledtotheaccumulation oflarge amounts ofactive IHF.Extracts ofsuchcells provided thestarting material forarapid purification procedure thatresults inmilligram quantities ofapparently homogenous IHF. TheDNA ofbacteriophage Aintegrates into thechromosomeofits Escherichia coli hostduring theestablishment of thelysogenic state (2). Early genetic studies (for areview, seereference 8)showedthat aphagegene, int, isessential forthissite-specific recombination, andsubsequent biochemical studies uncovered anessential contribution from thehost(15, 17). Oneoftherequired hostproteins, integration hostfactor (IHF), isadirect participant insite-specific recombination reactions (10, 14). IHFappears tobeadimer containing twosmall, nonidentical polypeptides, IHF-aand IHF-,(18). Thegenesforthesepolypeptides, himAfor IHF-aandhip(orhimD)forIHF-P, arelocated farapart fromeachother ontheE.coli chromosome (14). Recently, bothgenes werecloned andsequenced (5,12). Thebinding ofIHFtoattP, thesegment ofthelambda chromosome that isessential forintegration, isneeded forefficient recombination (3,7),butthewayinwhichIHFbinding activates attPisunknown. IHFalso appears tobeimportant formany processes otherthanlambdaintegration (forreviews see references 4and6).Insomeofthese cases, anIHF-binding site isfound ataposition that suggests adirect involvement ofIHFintheprocess; inother cases, theroleofIHFis unclear. IHFisnotanabundant protein, andithasbeendifficult to obtain substantial quantities ofhighly purified material (18). Inthispaper, we describe theconstruction ofstrains in whichbothsubunits ofIHFareplaced underthecontrol of aregulatable promotor. We showthat whenthese strains are induced, large amounts ofactive IHFaccumulate. A simple purification schemestarting withsuchinduced cells leads to large amounts ofhomogeneous material. Interestingly, the overexpression ofeachgeneseparately wasnotausable methodforoverproducing theactive protein; thepresence of stoichiometric amounts ofIHF-ac appears tosolubilize intracellular IHF-P, andthepresence ofstoichiometric amounts ofIHF-,appears tostabilize intracellular IHF-oa. This finding provides further evidence thatthetwosubunits interact directly invivoandoffers a cautionary noteto workers attempting tooverproduce other proteins thatare composed ofmorethanonekindofsubunit.

Robert A. Weisberg

Papers

Take your vitamins with a pinch of RNA.

E. coli mutants produced by the insertion of bacteriophage lambda DNA.

In bacteria and bacteriophage

Overproduction ofEscherichia coli Integration HostFactor, a Protein withNonidentical Subunits

Transducing Viruses and Viral Integration: Techniques for Genetic Modification