Two distinct populations of prestalk cells within the tip of the migratory Dictyostelium slug with differing fates at culmination.

The ecmA gene of Dictyostelium encodes an extracellular matrix protein and is selectively expressed in prestalk cells. We show that its promoter contains discrete elements that direct expression in different subpopulations of prestalk cells. Prestalk(pst)A cells occupy the front half of the prestalk region. Expression in pstA cells requires DNA sequences close to the cap site of the gene and a separate, upstream region that acts in combination with the gene proximal sequences. PstO cells are situated in the rear half of the prestalk region and at least two separate and redundant promoter regions direct expression within them. All constructs that are expressed in pstO cells are also expressed in anterior-like cells (ALCs); cells that resemble prestalk cells but which are scattered throughout the prespore region. This observation suggests that pstO cells and ALCs may be very similar in their properties. If development occurs under conditions in which a migratory slug is not formed, there is an ordered movement of cells into the stalk tube. PstA cells enter the stalk tube first, followed by a proportion of the pstO cells. The remainder of the pstO cells contribute to the upper cup, an ALC-derived subpopulation of prestalk cells which is located at the apex of the spore head. After prolonged slug migration, a discrete pstO zone appears not to be maintained and, at culmination, pstO cells are found scattered throughout the stalk.

Unexpected localisation of cells expressing a prespore marker of Dictyostelium discoideum.

We show that the anterior, prestalk region of the Dictyostelium slug contains cells which express, or have expressed, a prespore-specific marker. We term these cells "prespore-like cells" (PLC). In newly formed slugs there is a sharp prespore/prestalk boundary, with very few PLC, but after several days of migration the clear demarcation between prespore and prestalk zones breaks down because the number of PLC increases dramatically. This is consistent with previous observations showing there to be rapid interchange of cells between the prestalk and prespore regions. This is not, however, their only source, as a scattering of PLC appear when separate prestalk and prespore regions first become apparent at the time of tip formation. Also, at culmination, there is respecification of "prespore" cells at the prestalk/prespore boundary to form part of the mature stalk. The existence of these cells, and of PLC, may explain why we find prespore-specific mRNAs in mature stalk cells.

Identification of sequences regulating the transcription of a Dictyostelium gene selectively expressed in prespore cells.

There has been considerable debate about the relative contributions of transcriptional and post-transcriptional mechanisms to the regulation of prespore gene expression in Dictyostelium. We have determined the DNA sequence upstream of D19, the Dictyostelium gene encoding PsA, a prespore-specific, cell surface protein of unknown function. Our analysis of gene fusions, in which D19 upstream sequences are placed adjacent to a heterologous reporter gene, indicates that transcriptional signals alone are sufficient for the correct temporal and cell-type specific expression of this gene. We also show that the 5' and 3' boundaries of the minimal sequences necessary for correct developmental regulation lie within the region 338 to 122 nucleotides upstream of the start site of transcription but that flanking sequences seem to be necessary for optimal expression.

Structural characterization of Dictyostelium discoideum prespore-specific gene D19 and of its product, cell surface glycoprotein PsA.

The Dictyostelium discoideum cell surface antigen PsA is a glycoprotein which first appears in the multicellular stage soon after tip formation and is selectively expressed on prespore cells. The D19 gene encodes an mRNA sequence which is highly enriched in prespore over prestalk cells in the slug stage. We have determined 81 amino acid residues of N-terminal sequence from immunoaffinity-purified PsA protein and shown this sequence to be identical to the predicted sequence of the D19 gene. There are several short repeat elements close to the C terminus, and unequal crossing-over within these is proposed to account for the size polymorphism observed in PsA protein isolated from different D. discoideum strains. The repeats are proline rich and show similarity to the C-terminal region of the D. discoideum cell adhesion molecule, contact sites A. The extreme C terminus, which is also homologous to contact sites A, is characteristic of proteins attached to the plasma membrane via a glycosyl-phosphatidylinositol link. We have marked the PsA gene by insertion of an oligonucleotide encoding an epitope of the human c-myc protein. A construct containing this gene and 990 base pairs of 5'-flanking region directed correct temporal and spatial mRNA accumulation. We found the marked PsA protein, detected with the human c-myc antibody, to be correctly localized on the surface of cells.

A Dictyostelium prespore-specific gene is transcriptionally repressed by DIF in vitro.

One important role of DIF, the stalk cell-specific inducer of Dictyostelium, may be to divert cells from the spore cell pathway of differentiation. The D19 gene encodes an mRNA which is highly enriched in prespore over prestalk cells in the migratory slug. We show, using a mutant defective in DIF accumulation, that the concentration of D19, and several other prespore mRNA sequences, decreases in the presence of exogenous DIF. There is evidence that both transcriptional and post-transcriptional controls operate to regulate expression of these genes. We have performed in vitro nuclear transcription and mRNA half-life analyses, and find that DIF acts at the transcriptional level to repress the accumulation of the D19 mRNA.

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

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.