Random generation of β-galactosidase gene fusions using Tn 3

Random generation of β-galactosidase gene fusions using Tn 3

TIG - - technicaltip June 1985 Random generation of ~galactosidase gene fusions using Tn 3 A Tn3-/ac transposon, Tn3Hollo1, can serve as a transp...

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TIG

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-

technicaltip

June 1985

Random generation of ~galactosidase gene fusions using Tn 3 A Tn3-/ac transposon, Tn3Hollo1, can serve as a transposon mutagen and provide a new and useful system for the random generation of both transcriptional and translational /acZ gene fusions. This method supplements those using ,I and ~.-Mn derivatives. The production of 3-galactosidase, the/acZ gene product, is placed under the control of the gene into which Tn2-HoHol has inserted. The expression of the gene can thus be analysed by

Twin promoter transcription system*,

monitoring 3-galactosidase activity. Tn3-HoHol carries a

non-functional gene;

transpo~se

consequently,

it can

transpose only if transposase activity is supplied in trans, and is stable in the absence of this activity. 2¢~achel, S. E., An, G., Flores, C. and Nester, E. W. (1985) A Tr'.3 /acZ transposon for the random] generation of O-ffalactosidasegene fusions: application to the analysis of gene expression in AgrobacteriNnt EMBOJ 4,891-898

The system gives a choice of two highly promoter-specific RNA polymerases from phages The Riboprobe Gemini tran- SP6 and TT. These are used scription system allows any with either one of two plasmids. DNA fragment to be tran- The plasmids contain both SP6 scribed selectively from either and T7 promoters pointing in strand in either direction follow- opposite directions on either ing a single plasmid cor~struc- side of a multiple cloning site t-ion. (MCS). The plasmids are identi-

Immobilization of DNA

substrate for the assay of nuclease activities. Enzyme purification and isolation of

DNA immobilization on Sephadex G200 in the presence of water-soluble carbodiimide is significandy increased when the incubation temperature of the mixture is increased from 42 to 60°C. Native DNA is immobilized with higher efficiency than denatured DNA. The DNA-Sephadex complex can be stored as a fine powder. The DNA-Sephadex complex can be used directly as a

ribo- and deoxyribo-n ucleotides by hybridization using affinity chromatography techniques is also possible.

cal except for the orientation of the MCS. The system allows the choice of which strand of an inserted DNA fragment is to be transcribed and in which direction. A positive control template also allows RNA marker synthesis and monitoring of system performance.

Mykoniatis, M. G. (1985)Immobilizationofnative and denatured DNA on Sepbadex (;200. J Btochem. Biophys. Methods 10, 321-328 Distributorsin UK: P & S Biochemicals, 38 Queensland Street, Liverpool 1.7 3JG, UK, for PromegaBiotech, 2800 S. Fish Hatchery Road, Madison, W153711, USA.

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