1887

Abstract

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.

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/content/journal/micro/10.1099/00221287-129-9-2703
1983-09-01
2020-01-22
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http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-129-9-2703
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