Summary: In two separate studies a /I-generated DNA fragment coding for the enzyme tyrosinase, responsible for melanin synthesis, was cloned from DNA into two SLP1.2-based plasmid vectors (pIJ37 and pIJ41) to generate the hybrid plasmids, designated pIJ700 and pIJ701, using 66 as the host. The fragment (1.55 kb) was subcloned into the multicopy plasmid pIJ350 (which carries thiostrepton resistance and has two non-essential /I sites) to generate four new plasmids (pIJ702-pIJ705) with the tyrosinase insert located in either orientation at each site. All six plasmids conferred melanin production (the Mel phenotype) on their host. As in the parent, strains of carrying the gene specifying tyrosinase synthesis possessed an enzyme activity which was inducible. Most of the tyrosinase activity was secreted during growth of ; in contrast, the majority remained intracellular in the clones. The specific activity of the induced tyrosinase activity (intracellular) was higher (up to 36-fold) when the gene was present on the multicopy vector in comparison with its location on the low copy plasmids, pIJ700 or pIJ701, or in .

Restriction mapping of the tyrosinase fragment in pIJ702 revealed endonuclease cleavage sites for several enzymes, including single sites for /II, I and I that are absent from the parent vector (pIJ350). Insertion of DNA fragments at any one of these sites abolished the Mel phenotype. The results indicate that pIJ702 is a useful cloning vector with insertional inactivation of the Mel character as the basis of clone recognition.


Article metrics loading...

Loading full text...

Full text loading...


Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error