Investiqation of an E. coli rRNA Promoter Inserted in Streptomyces lividans pKT565
Peterson, Gail E.
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An E. coli rrnA promoter, which was inserted into a Streptomyces lividans plasmid, was investigated to determine whether it could function. Transcriptional activity was detected by expression of a promoterless gene for. chloramphenicol (Cm) acetyltransferase (CAT) which had been inserted into the plasmid to derive Streptomyces plasmid pKT565. This plasmid pKT565 exhibited Cm resistance, so it was thought that the promoter was probably functioning. However, digestion of the plasmid with EcoRI, and EcoRI and HindIII showed that the CAT gene was transcribing towards the promoter, indicating that some other source initiated the transcription. Reversing the BgIII cassette containing the rrnA promoter and CAT gene resulted in transformed colonies that were Cm sensitive, indicating that transcription was coming from an upstream source. Digestion with SphI and EcoRI ensured that the transcriptional terminator from E. coli phage fd, which had been inserted upstream from the CAT gene to prevent significant transcriptional read-through from vector promoters, was still present. To determine whether the E. coli promoter could function in pKT565, an attempt was made to reverse the orientation of the CAT gene. However, increased resistance to Cm, which might have been expected, was not observed. Instead, the transformed colonies displayed resistance. It was therefore determined that the E. coli rrnA promoter could not function in plasmid pKT565.