Cell, Vol. 28, 793-800, April 1982, Copyright 0 1982 by MIT Differential Expression of the Members of the Discoidin I Multigene Family during Growth and Development of Dictyostelium discoideum Joan M. Devine, Adrian S. Tsang* and Jeffrey G. Williams Imperial Cancer Research Fund Mill Hill Laboratories Buttonhole Lane London NW7 1 AD, England Summary Discoidin I and II are lectins synthesized during the aggregation of Dictyostelium discoideum amoebae which may play a role in cellular cohesion. Discoidin I was thought to consist of two major polypeptides, but we show that there are three. The N-terminal amino acid sequence of the polypeptides has been predicted by determining part of the nucleotide sequence of their respective mRNAs. We obtained the nucleotide sequences by reverse transcription of the mRNAs, using as primers, fragments derived from the coding region of two cloned discoidin I sequences, and utilizing cross hybridization to the various mRNA species and differences in the length of their 5’ noncoding regions to isolate fragments for DNA sequencing. We used primer extension to measure the relative concentration of the three ma- jor discoidin I mRNA sequences. We show that during development changes in the abundance of all three mRNA sequences occur coordinately. In cells growing in nutrient broth, however, only two of the three major discoidin I mRNA sequences accumulate, and if such cells are grown to a very high density, both sequences disappear. These re- sults indicate that the coordination of discoidin I gene expression is not obligatory and that the mem- bers of this multigene family may differ in the mode of their induction during normal development. Introduction In response to starvation, amoebae of the cellular slime mold Dictyostelium discoideum aggregate to form a multicellular mass in which cells differentiate to yield stalk cells and spore cells. During differentia- tion, numerous new proteins are synthesized, and the carbohydrate-binding proteins discoidin I and discoi- din II are among the most abundant of these new products. The discoidin proteins are virtually unde- tectable in cells growing on bacteria,yet together they constitute over 1% of the cellular protein in aggre- gated cells (Rosen et al., 1973). The precise function of these lectins is unknown, but the time course of their synthesis, their sugar-binding properties, the presence of a fraction of the discoidin proteins at the cell surface (Siu et al., 1976) and the aggregation- defective phenotype of a supposed structural mutant * Present address: Department of Biology, York University, Toronto, Ontario M3J 1 P3, Canada. in a discoidin I gene (Ray et al., 1979) suggest that they may be involved in cell cohesion. During development, the ratio of discoidin I to dis- coidin II changes such that discoidin II constitutes 35% of the total discoidin protein during early devel- opment but only 10% at a later stage (Frazier et al., 1975). This difference is reflected in the relative amounts of translatable mRNA (Ma and Firtel, 1978). The mRNA sequences encoding discoidin I have been quantitated by use of cloned molecular hybridization probes (Williams et al., 1979; Rowekamp et al., 1980). Discoidin I mRNA is virtually undetectable in bacte- rially grown cells (Rowekamp et al., 19801, but it is present at a low level in axenically grown cells (Wil- liams et al., 1979). During early development, the discoidin I mRNA concentration rises rapidly such that, in axenically grown cells, the mRNA constitutes almost 2% of the poly(A)+ RNA population at 4 hr of development. The amount of discoidin I mRNA then drops rapidly such that by 8 to 9 hr of development it is virtually undetectable. The rate of discoidin I gene transcription mirrors this pattern of mRNA accumula- tion, suggesting that the primary control of mRNA concentration occurs at the level of transcription (Wil- liams et al., 1979). Discoidin I was thought to be a single polypeptide, but lshiguro and Weeks (1978) showed that on high percentage acrylamide-SDS gels it could be resolved into two polypeptides. This was confirmed by two- dimensional gel electrophoresis (Tsang et al., 19811, and this same study showed that there might be three discoidin I polypeptides, since one discoidin I gel spot sometimes resolved into two. The discoidin I genes have been analyzed by restriction enzyme analysis of genomic DNA with cloned discoidin I probes, and it has been estimated that there are four or possibly five different genes (Rowekamp et al., 1980). Cells growing in axenic medium contain discoidin I protein (Rosen et al., 19731, and biochemical evi- dence suggests that this protein is indistinguishable from that present in cells during development (Simp- son et al., 1974). However, we show that this is not the case, since one of the three discoidin I polypep- tides and the mRNA encoding it are absent from axenically growing cells. Results Three Different Major Discoidin I mRNA Sequences Are Synthesized during Normal Development When purified discoidin protein isolated from Dictyo- stelium cells at 9 hr of development is analyzed by two-dimensional gel electrophoresis under the condi- tions described in Figure 1, three discoidin I polypep- tides, which we term discoidin la, lb and Ic, are re- solved. The separation of the lb and Ic polypeptides is small but reproducible and is also seen when dis- coidin I mRNA is translated in vitro and similarly ana-