Monika Westphal

Complete sequence and transcript regulation of a cell adhesion protein from aggregating Dictyostelium cells.

Three cDNA clones coding for the contact site A (csA) protein, a cell adhesion molecule of Dictyostelium discoideum, were isolated by screening a cDNA library with monoclonal antibodies. Two of these clones contained the complete coding region for the csA protein of 1542 bp including a sequence of 57 bp coding for the leader. The N terminus of the mature protein, as it was published previously, was identified in the amino acid sequence derived from both full‐length cDNA clones. Southern blot analysis suggests the presence of only one csA gene in the haploid genome. Accumulation of the csA‐specific message of 1.9 kb begins during development on nitrocellulose filters at 9 h of starvation, and reaches a maximum at 12 h, the time of cell aggregation. Expression of the csA glycoprotein follows closely accumulation of the transcripts. In the multicellular slug stage following cell aggregation, the amount of csA transcripts rapidly declines to low levels.

Probing an adhesion mutant of Dictyostelium discoideum with cDNA clones and monoclonal antibodies indicates a specific defect in the contact site A glycoprotein

Expression of developmentally regulated membrane proteins of aggregating cells of Dictyostelium discoideum is subject to several control mechanisms. One of them involves periodic cyclic‐AMP pulses as signals for gene expression. To increase the probability of selecting mutants specifically defective in the contact site A (csA) glycoprotein, one of the characteristic proteins of aggregating cells, we have bypassed the requirement for both cyclic‐AMP pulses and another control element by two runs of mutagenesis. A ‘double bypass’ mutant, HG592, was obtained which aggregated in nutrient medium where wild‐type did not develop. Mutants defective in expression of the csA‐glycoprotein were selected from HG592 by fluorescence‐activated cell sorting and colony immunoblotting using a monoclonal antibody specific for that protein. One among 51 csA‐negative mutants, HG693, specifically lacked the capability of forming EDTA‐stable intercellular contacts. It acquired chemotactic responsiveness and developed into fruiting bodies. Expression of the transcripts for eight developmentally regulated proteins was determined in HG693. Seven of the RNA species were normally expressed; they were recognized by cDNA clones which had been produced from poly(A)+ RNA isolated from membrane‐bound polysomes. The single RNA species which was not substantially expressed in HG693 was recognized by a cDNA clone that was obtained by screening a λgt11 library with an antibody specific for the csA‐glycoprotein. When probing RNA from wild‐type cells, this clone hybridized with a single developmentally regulated RNA species of 1.9 kb whose expression was strongly enhanced by cyclic‐AMP pulses. Appearance of this RNA coincided with the expression of the csA‐glycoprotein.

Complete cDNA sequence of a Dictyostelium ubiquitin with a carboxy-terminal tail and identification of the protein using an anti-peptide antibody

The complete sequence of a Dictyostelium discoideum cDNA is presented that codes for monoubiquitin extended at its C‐terminus by a 52 amino acid tail. The sequence of both the ubiquitin portion and the tail is highly homologous to the one of Saccharomyces cerevisiae and to a partial mouse sequence. The highly basic tail sequence contains a putative metal and nucleic acid‐binding motif. The gene encoding the 0.6 kb mRNA of the C‐terminally extended ubiquitin is represented only once in the haploid genome. The 0.6 kb mRNA as well as its translation product, a 15 kDa protein, is expressed in exponentially growing cells and remains present for at least 5 h of development. Using antibodies against a synthetic peptide that corresponds to the C‐terminal amino acid sequence, a 15 kDa protein containing the extension was also detected in yeast.

Monoclonal antibodies against contact sites a of dictyostelium discoideum: detection of modifications of the glycoprotein in tunicamycin-treated cells.

Tunicamycin acts on cell aggregation in Dictyostelium discoideum by changing cell movement and by inhibiting the EDTA‐stable type of intercellular adhesion. Tunicamycin‐treated cells show unco‐ordinated pseudopodial activity such that pseudopods are simultaneously extended from all parts of the cell surface, and the cells are unable to move in straight paths. Concurrent with the inhibition of formation of EDTA‐stable contacts, N‐glycosylation of a glycoprotein specific for aggregation‐competent cells is inhibited. This glycoprotein, previously called contact site A, has an apparent mol. wt. of 80 kilodaltons (kd). In membranes of tunicamycin‐treated cells, two components are detected that react with certain monoclonal antibodies against contact sites A: one component of 66 kd, the other of 53 kd apparent mol. wt. Another group of monoclonal antibodies reacts only with the 80‐kd glycoprotein and the 66‐kd component. These results are in accord with the assumption that the glycoprotein carries two carbohydrate chains, and that the antibodies differ in their requirement for glycosylation of the antigen. Despite the coincidence between blockage of EDTA‐stable cell adhesion and inhibited glycosylation of contact sites A, direct involvement of the carbohydrate moieties of this glycoprotein in intercellular adhesion seems questionable. EDTA‐stable cell adhesion has not been blocked by Fab fragments from antibodies that specifically react with the glycosylated protein.

Cell differentiation in the absence of intracellular and extracellular cyclic AMP pulses in Dictyostelium discoideum

Abstract The mutant HSB1 was obtained by colony immunoblotting of mutagenized Dictyostelium discoideum cells with a monoclonal antibody against contact site A, a membrane glycoprotein. HSB1 cells undergo early development in the absence of intracellular and extracellular cAMP pulses. The receptor-mediated activation of adenylate cyclase is defective, but the basal activity increases normally during the first hours of development. Other preaggregative gene products, such as the inhibitor of phosphodiesterase, the contact site A glycoprotein, EDTA-stable contacts, and four developmentally regulated gene transcripts are also expressed in the mutant. Postaggregative gene transcripts fail to accumulate, even after pulsatile addition of cAMP. However, complete development of mutant cells is obtained by complementation with cells of the parent strain AX2.

A developmentally regulated gene product from Dictyostelium discoideum shows high homology to human α-L-fucosidase

A cDNA library of poly(A+)‐RNA has been prepared from membrane‐bound polysomes of Dictyostelium discoideum and screened for clones hybridizing to mRNA species that encode developmentally regulated proteins. The clone investigated in this paper recognizes a 1.8 kb transcript that accumulates strongly between the growth phase and aggregation stage. Stimulation of cells with pulses of cAMP enhances the accumulation. The amino acid sequence derived from a complete cDNA and from a genomic clone displays extensive sequence identity to human liver α‐L‐fucosidase. The D. discoideum DNA sequence encodes a 50.5 kDa polypeptide with a hydrophobic signal peptide at the N‐terminus. Antibodies against a synthetic peptide corresponding to amino acids 262–275 of the deduced protein sequence recognize a developmentally regulated 50 kDa protein in D. discoideum that is recovered in the particulate fraction.