Two vectors which facilitate gene manipulation and a simplified transformation procedure for Dictyostelium discoideum

Abstract

We have constructed and characterized two Dictyostelium transformation vectors (pB10TP1 and pB10TP2) designed for the facile sequence determination, mutagenesis and functional analysis of Dictyostelium genes. The vectors incorporate the B10 neomycin-resistance (neo) gene [Nellen et al., Mol. Cell. Biol. 4 (1984) 2890-2898] and sequences derived pEMBL18+ [Dente et al., Nucl. Acids Res. 11 (1983) 1645-1655], enabling the production of single-stranded template and increasing the yield of double-stranded DNA. A new multiple cloning site (MCS) has been inserted adjacent to the M13 sequence primer binding site so that single-stranded template DNA isolated from recombinants prepared using these vectors is suitable for sequence analysis and site-directed mutagenesis. The linker incorporates restriction sites suitable for the preparation of a directed deletion series and useful in cloning, including some sites with recognition sequences frequent in the extremely A + T-rich Dictyostelium genome. A Dictyostelium genomic fragment has been included to provide transcription termination signals for the neo gene. One of the two vectors (pB10TP1) contains the 3'-proximal portion of a constitutively expressed mRNA of unknown function. It is located downstream from the MCS so that 5'-proximal fragments of genes, cloned into the MCS, generate fusion transcripts which are distinguishable from transcripts of the corresponding endogenous genes. The complete nucleotide sequence of the two vectors has been established and a comprehensive restriction map deduced. We also describe a modification of the published transformation system, which allows it to be applied to the commonly used strain Ax-2, and another generally applicable modification which greatly reduces the time required to obtain stable transformants.