Showing papers by "Robert A. Weisberg published in 1987"
TL;DR: Two strains are constructed in which the two subunits of IHF, encoded by the himA and hip genes of Escherichia coli, are expressed under the control of the lambda rho L promoter, resulting in milligram quantities of apparently homogeneous IHF.
Abstract: Integration host factor (IHF) is a small, basic protein that is needed for efficient recombination of bacteriophage lambda, as well as for other host and viral functions. We have constructed strains in which the two subunits of IHF, encoded by the himA and hip genes of Escherichia coli, are expressed under the control of the lambda rho L promoter. Separate overexpression of himA and hip led to the production of unstable and insoluble peptides, respectively. In contrast, the overexpression of both genes conjointly led to the accumulation of large amounts of active IHF. Extracts of such cells provided the starting material for a rapid purification procedure that results in milligram quantities of apparently homogeneous IHF.
196 citations
TL;DR: The various activities of gene 3 endonuclease are consistent with the known role of this enzyme in genetic recombination, in maturation and packaging of T7DNA, and in degradation of host DNA, and suggest that the enzyme recognizes a specific structural feature in DNA.
107 citations
TL;DR: N Nun appears to be a transcription termination factor with a remarkable specificity; it converts the lambda nut sequences, which normally interact with lambda N protein to suppress transcription termination, into terminators as discussed by the authors.
67 citations
01 Jan 1987
TL;DR: Constructed strains in which the two subunits of integration hostfactor,encoded by thehimA andhipgenes ofEscherichia coli, are expressed under the control of the XPLpromoter, providing further evidence that thetwosubunits interact directly invivo and a cautionary noteto workers attempting tooverproduce other proteins that are composed of morethan onekind ofsubunit.
Abstract: 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.
2 citations