A transcriptional barrier to expression of cloned toxin genes of the linear plasmid k1 of<i>Kluyveromyces lactis</i>: evidence that native k1 has novel promoters

Abstract

The killer toxin of Kluyveromyces lactis consists of three polypeptides encoded by the linear plasmid k1. We re-introduced the entire k1 sequence, cloned on a circular replicating plasmid, into K. lactis strains lacking k1, and found that the resulting transformants did not produce toxin. The barrier to expression was found to be transcriptional: the four transcripts of native k1 were absent, and instead shorter, aberrant k1 transcripts were made. We determined the precise initiation sites of the four transcripts of native k1: these had very short untranslated leaders and mapped about 14bp downstream of an "upstream conserved sequence" (UCS). It appears that k1 has novel promoters which are inactive on circular plasmids which replicate in the nucleus. This is consistent with the suggestion that native k1 resides in the cytoplasm.