Insertional inactivation

Insertional inactivation is the inactivation of a gene by the insertion of another DNA sequence. 

Deretic, V., O. Francetic, and V. Glisin, "Instability of the plasmid carrying active penicillin acylase gene from Escherichia coil conditions inducing insertional inactivation," FEMS Microbiology Letters 24 (1984) 173 - 177. 

Plasmid MW(x 10- ) Pertinent antibiotic markers Sites at which insertion inactivates a marker... 

Figure V-18 Insertional inactivation of the tetracycline resistance gene following cloning in pBR322.

Insertional inactivation markers. These allow selection of recombinant plasmids from non-recombi-nants. The most commonly used is the lacZ coding for P-galactosidase activity. This enzyme can cleave a substrate that mimics galactose known as X-Gal, which results in the generation... 

Fig. 24.1 Simplified diagram of the plasmid pUCl 8. lacZ represents the insertional inactivation marker coding for fl-galactosidase activity. A multiple cloning site (MCS) is present within the LacZ gene to enable the cloning of DNA fragments. Ori represents the origin of replication which, in this case, works in Escherichia coli. Finally, Ampr represents an ampicillin resistance marker.

The pUC vectors contain an ampicillin-resistance gene and a multiple cloning site at the 5 -end of the lacZ gene. Insertional inactivation of lacZ allows... 

Insertional inactivation of the mglA gene on the So. cellulosum So ce26 chromosome by homologous recombination led to a mutant that displayed a non-... 

Lombo F, K. Siems, A.F. Brana, C. Mendez, K. Bindscil, J. A. Salas (1997). Cloning and insertional inactivation of Slreptomyces argillaceus genes involved in the earliest steps of biosynthesis of the sugar moieties of the antitumor polyketide mithramycin. J. Bacteriol. 179 3354-3357. 

Vrilbloed JW, Zerbe-Brnkhardt K, Ratnatilleke A, Grubelnik-Leiser A, Robinson JA (1999) Insertional inactivation of methylmalonyl coenzyme A (CoA) mutase and isobutyryl-CoA mutase genes in Streptomyces cinnamonensis-. influence on polyketide antibiotic biosynthesis. J Bacteriol 181 5600 - 5605... 

Distinguish between screening and selection. Explain insertional inactivation. Understand the role of drug resistance genes in cloning. 

An instance of pathway manipulation with potentially high commercial significance was the production of epirubidn in S.peucetius [13]. The (4S)-TDP-4-keto-6-deoxyhexose reductase gene, dnmV, was disrupted by insertional inactivation, and the gene for a homologous reductase from erythromycin (EryBIV) or avermectin (AvrE) biosynthesis, with different product stereochemistry (4R), was introduced to the production strain (Scheme 9). 

Boles, B.R. and McCarter, L.L. (2000). Insertional inactivation of genes encoding components of the sodium-type flagellar motor and switch of Vibrio parahaemolyticus. J. Bacteriol. 182, 1035-1045. 

McDaniel LS, Yother J, Vijayakumar M et aL Use of insertional inactivation to facilitate studies of biological properties of pneumococcal surface protein A (PspA). J Exp Med 1987 165(2) 381-394. 

---