Mapping of regions on cloned Saccharomyces cerevisiae 2-mum DNA coding for polypeptides synthesized in Escherichia coli minicells.

Saccharomyces cerevisiae 2-mum DNA and some of its restriction fragments were integrated in vector pCR1 ,pBR313 or pBR322 and their expression in Escherichia coli P678-54 minicells was analyzed. 2mum DNA inserted at the EcoRI site of pCR1 or pBR313 and at the PstI site of pBR322 promoted the synthesis of polypeptides of 48,000, 37,000, 35,000 and 19,000 daltons. The DNA regions coding for these polypeptides were mapped on the 2-mum DNA molecule by insertion of single EcoRI or HindIII restriction fragments and comparison of the polypeptides produced. For the synthesis of the 37,000 dalton polypeptide, intact sites RIB and H3 were required. The disappearance of the 37,000 dalton polypeptide on interruption of one of these sites by insertion of the vector, was correlated with the appearance of a polypeptide of 22,000 or 23,500 daltons respectively. The DNA sequence coding for the 37,000 daltons polypeptide, therefore, has to be located in the S-loop region close to or overlapping with the site RIB and H3. Assuming that the 22,000 and the 23,500 dalton polypeptides are truncated forms of the 37,000 dalton polypeptide, the last polypeptide can be exactly mapped. The polypeptide of 48,000 daltons was mapped to that half of the L-loop segment containing the sites H1 and H2. If, however, HindIII fragment H1-H2 was expressed, the 48,000 dalton polypeptide was lost and concomitantly a 43,000 dalton polypeptide appeared. We assume that this polypeptide results from early termination of the polypeptide of 48,000 daltons. The 35,000 and 9,000 dalton polypeptides were mapped to the S-loop region. The integrated inverted repeat sequence of yeast 2-mum Dna did not induce any detectable insert-specific polypeptide synthesis.