Insertional activation markers

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.

In this case, intron insertions are selected on the basis of acquisition of resistance to erythromycin due to the incorporation of an inactivated ermB gene, or RAM (Retrotransposition-Activated Marker), that became functional as a consequence of the successful insertion of the group II intron-encoding DNA into the genomic target [133,134]. 

Methods of DNA manipulation now make it possible to insert DNA into prokaryotic, eukaryotic, or viral hosts, creating versatile marker systems that allow as- sessment of the survival and spread of strains, studies on gene transfer, and determinations of cell activity. A potential marker gene must be absent from the strain used in the study, and either absent or in sufficiently low abundance in the microbial population under study to allow detection of marked cells at an appropriate level. 

Fig. 6. Kinetics of immobilization of glutaryl-7-ACA-acylase on epoxy-activated polymethacrylate. The Gl-7-ACA-acylase was incubated with the epoxy-activated carrier. At definite times aliquots were taken from the reaction suspension. Supernatant and carrier-fixed enzyme were separated by centrifugation. The carrier-fixed enzyme was washed with water to remove non-covalently linked enzyme. The activities of the immobilized enzyme and supernatant were determined (5 mM potassium phosphate buffer pH 8,37°C, 2% glutaryl-7-amino cepha-losporanic acid, pH-stat 8.0). Simultaneously, an aliquot of carrier-fixed enzyme was boiled in sodium dodecylsulfate (SDS)/glycine buffer and the supernatant was subjected to SDS-polyacrylamide electrophoresis (see insert from left to right lane 1 Carrier-fixed enzyme, 2 h lane 2 Carrier-fixed enzyme, 4 h lane 3 Carrier-fixed enzyme, 6 h lane 4 Carrier-fixed enzyme, 21 h lane 5 Carrier-fixed enzyme, 69 h lane 6 Dialyzed enzyme lane 7 Supernatant, 2 h lane 8 Supernatant, 21 h lane 9 Supernatant, 69 h lane 10 Molecular weight calibration markers)...

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