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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023822-13
Application #
3271875
Study Section
Genetics Study Section (GEN)
Project Start
1979-04-01
Project End
1994-07-31
Budget Start
1992-08-01
Budget End
1994-07-31
Support Year
13
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Insall, R; Kuspa, A; Lilly, P J et al. (1994) CRAC, a cytosolic protein containing a pleckstrin homology domain, is required for receptor and G protein-mediated activation of adenylyl cyclase in Dictyostelium. J Cell Biol 126:1537-45
Richardson, D L; Loomis, W F; Kimmel, A R (1994) Progression of an inductive signal activates sporulation in Dictyostelium discoideum. Development 120:2891-900
Fosnaugh, K L; Loomis, W F (1993) Enhancer regions responsible for temporal and cell-type-specific expression of a spore coat gene in Dictyostelium. Dev Biol 157:38-48
Richardson, D L; Loomis, W F (1992) Disruption of the sporulation-specific gene spiA in Dictyostelium discoideum leads to spore instability. Genes Dev 6:1058-70
Kuspa, A; Loomis, W F (1992) Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. Proc Natl Acad Sci U S A 89:8803-7
Loomis, W F; Fuller, D L (1991) Antisense RNA inhibition of expression of a pair of tandemly repeated genes results in a delay in cell-cell adhesion in Dictyostelium. Antisense Res Dev 1:255-60
Fosnaugh, K L; Loomis, W F (1991) Coordinate regulation of the spore coat genes in Dictyostelium discoideum. Dev Genet 12:123-32
Richardson, D L; Hong, C B; Loomis, W F (1991) A prespore gene, Dd31, expressed during culmination of Dictyostelium discoideum. Dev Biol 144:269-80
Fosnaugh, K L; Loomis, W F (1989) Spore coat genes SP60 and SP70 of Dictyostelium discoideum. Mol Cell Biol 9:5215-8
Peters, D J; Knecht, D A; Loomis, W F et al. (1988) Signal transduction, chemotaxis, and cell aggregation in Dictyostelium discoideum cells without myosin heavy chain. Dev Biol 128:158-63

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