A temperature-dependent pBR322 copy number mutant resulting from a Tn5 position effect.

In the process of randomly mutagenizing a recombinant pBR322 clone with transposon Tn5, a high copy number plasmid mutant, pLO88, has been isolated. The copy number phenotype of pLO88 is observed only at elevated temperatures, greater than or equal to 37 degrees C, and is due to the precise position of a Tn5 insertion. Nucleotide sequence of the Tn5-pBR322 junction reveals that Tn5-88 has inserted into an open reading frame that codes for a 63 amino acid protein previously shown to negatively regulate pBR322 plasmid copy number. By deleting portions of the Tn5 it is shown that the copy number phenotype is due not only to the insertion of Tn5 in pBR322 but also to the requirement that some Tn5 sequences remain intact. It appears that an outwardly directed Tn5 promoter initiates the synthesis of a transcript (RNA X) that interferes with the normal repressor RNA (RNA I)-primer RNA (RNA II) interaction at elevated temperatures.