BIOCHEMICAL BASIS OF DEVELOPMENT IN DICTYOSTELIUM

盘基骨菌可再生转化技术

Techniques for the reproducible transformation of Dictyostelium cells with cloned sequences have allowed us to determine essential cis-acting sequences in the 5' flanking region of a developmentally controlled gene (actin 15) that are shared by another gene that is co-expressed (actin 6) (Cohen et al 1986). Transcripts of both of these genes appear immediately after the initiation of development and accumulate during the aggregation stage. We want to use a similar approach to determine whether a set of cell-type specific genes, including those that code for the spore coat proteins, also share common control regions that determine the stage in development and cell type in which they are transcribed. Moreover, we would like to analyze the extracellular signals that are monitored to ensure proper transcription of these genes. We have isolated cell-lines that fail to form multicellular aggregates because they lack myosin heavy chain specifically. These cell lines were selected after transformation with a vector that carries a portion of the myosin heavy chain gene such that it is transcribed in the reverse orientation under the control of the actin 6 promotor (Knecht and Loomis, 1987, see Appendix). Surprisingly, the almost complete lack of myosin heavy chain protein is not lethal but results in a block to formation of multicellular aggregates and all subsequent differentiations including the expression of pre-spore specific genes. We will attempt to by-pass this block to late biochemical differentiations by altering the conditions of development and adding back extracts of wild-type developing cells. We have been concentrating on the genes for the major spore coat proteins of Dictyostelium discoideum for several reasons. There are strong advantages to working with cloned sequences whose products are known and can be recognized by both biochemical and immunological techniques. The spore coat proteins, SP60, SP70, and SP96 are coordinately synthesized at the tipped aggregate stage of development (14 hr) and accumulate in prespore but not in prestalk cells. They are stored in prespore vesicles that fuse with the plasma membranes during sporulation to form the extracellular coats around each spore. We have antibodies that recognize these spore coat proteins. We have determined the N-terminal amino acid sequence of SP70 nd SP60 and have characterized cDNA clones that appear to be derived from mRNAs for each of the spore coat proteins. We plan to further characterize these clones by constructing transformation vectors that will disrupt their endogenous genes when integrated by homologous recombination (De Lozanne and Spudich, 1987). We also plan to construct anti-sense transformation vectors and show that they can inactivate the expected endogenous mRNAs. We plan to isolate genome clones corresponding to each of the cDNAs to observe the N-terminal sequence coding regions. By directly determining the essential cis-acting sequences in transformants we hope to recognize share cis-acting sequences that integrate expression of this set of genes.