New vectors for expression of theE.coli lacZgene inDictyostelium

The E. coli lacZ gene encoding /3-galactosidase has proven to be a useful reporter gene in Dictyostelium (1, 2). We describe a set of three vectors for the rapid construction of promoter ::lacZ fusions. These vectors improve on the previously described vector pDdlac-1 by: (1) the removal of the additional lacZ fragment responsible for instabiity of the polylinker when grown in E. coli; (ii) the presence of a neo gene in a suitable expression cassette to confer G418 resistance when transfected into Dictyostelium cells; (iii) the presence of an initiator codon in pDdGal-15 eliminating the need to fuse promoters through their coding sequence. These vectors consist of the lacZ gene and the Dictyostelium actin 8 terminator sequence from pDdlac-1 inserted into a derivative of the plasmid pB10TP2 (3, Fig. A). pB10TP2 is based on the plasmid pEMBL18+ and so contains a fragment of the lacZ gene. This fragment has been removed to avoid the stability problems experienced with pDdlac-1. As a result, the fl origin sequence has also been removed. The three vectors pDdGal-15, — 16 and —17 contain different polylinkers, shown in Fig. B. Each polylinker contains a Bgin site in a different reading-frame relative to the lacZ gene. To confirm the utility of the vectors the promoter of the Dictyostelium actin 15 gene was fused to lacZ. A comparable construct was made using the deleted promoter Actin 15ABam. This promoter contains an internal deletion known to completely abolish expression from the promoter. Both plasmids gave G418-resistant colonies when used to transfect Dictyostelium cells. Extracts from cells transfected with the wildtype promoter, but not the deleted form, had high levels of /3-galactosidase activity. The unmodified vectors pDdGal-15, —16 and —17 were also transfected into Dictyostelium cells and gave no /3galactosidase activity (Table 1).