It is known from previous work that macrocysts are formed when the two strains of Dictyostelium discoideum (NC4 and V12) are mixed. By culturing NC4 and V12 amoebae together in various ratios and at several densities, and in experiments using vitally stained cells, we have shown that the number of macrocysts formed is directly related to the number of V12 (but not NC4) cells present and that macrocysts may occur without sexual fusion.A starved population of Dictyostelium discoideum amoebae can either develop into asexual fruiting bodies or form macrocysts, depending upon a number of conditions including the appropriate environmental cues (Blaskovics and Raper, '59; Nickerson and Raper, '73), and whether or not amoebae of the opposite mating type are present (Clark et al., '73; Erdos et al., '73). The morphological s e quence leading to macrocyst formation in D. discoideum has been described in some detail by Wallace ('77) and O'Day and Lewis ('81). Briefly, hundreds of amoebae aggregate to form tight cell clusters. Within an aggregate resides the cytophagic giant cell, the putative zygote. A primary wall is secreted around the aggregate transforming it into a precyst. The giant cell proceeds to engulf all of the amoebae in the precyst. In addition to the primary wall, secondary and tertiary walls, rich in cellulose, are formed around the macrocyst thus producing a well-protected, resistant cyst (Erdos et al., '72; Filosa and Dengler, '72; Nickerson and Raper, '73).Because of the obvious importance of genetic studies, it is desirable to know if the two strains, NC4 and V12, of D . discoideum, which produce macrocysts when brought together, are producing zygotes or reproducing asexually possibly by selfing. The extremely low germination efficiency of D. discoideum macrocysts ( < 2%; Wallace and Raper, '79) does not allow one to show that all macrocysts are the results of zygote formation. In this study it is shown that there is macrocyst production without cell fusion of the two mating types. The question of whether there also may be some sexual zygote formation must wait for further analysis. MATERIALS AND METHODS Growth and maintenance of stock culturesStock cultures of D. discoideum strains NC4 and V12M2 were maintained on Escherichia coli Blr as a food source on nutrient agar plates (per liter distilled H,O: 10 g peptone, 10 g dextrose, 0.381 g Na,HPO,, 0.45 g KH,PO,, 20 g difco agar) at 22°C. Stocks were recultured weekly. For long-term storage, spores were lyophilized. Formation of giant cells and macrocystsSpores of NC4 and V12 were washed and heatshocked according to the method of Cotter and Raper ('68) and plated separately on nutrient agar with E. coli Blr. Synchronously developing amoebae could be obtained by harvesting such growth plates 12-18 h after inoculation and separating amoebae from E. coli Blr by differential centrifugation (Bonner, '47). Such NC4 and V12 amoebae, when cultured together on nonnutrient agar and incubated in the dark at 24 k 1 "C, would produce giant cells in approxima...

Experiments were performed to determine effects of buffered solutions (0.01 m) on vegetative development and fertilization of gametophytes of Pteridium aquilinum. Buffered solutions were used to simulate exposures to acidic precipitation up to 3.5 h. Flagellar movement of sperm was reduced at buffer pH levels below 5.8. Specifically, longevity of motility was reduced so that no movements were observed 8–10 and 5–7 min after exposure to pH 5.6 and 5.2, respectively. Addition of sulfate (86 μm) depressed the percent motile sperm by 50%, 2–4 min after exposure at all pH values tested. Longevity of flagellar movement was most affected by pH and additions of sulfate. The effect on fertilization was assayed directly by determinations of sporophyte production. Although gametophyte survival and development were not affected by solutions of pH 5.8 to 2.2, fertilization was reduced after exposure to buffers below pH 4.2. Sporophyte production was not altered at pH 5.2 but was reduced 50% at pH levels of 4.2 and 3.2 compared with exposures to pH 5.8. Addition of sulfate (86 μm) decreased fertilization at least 50% at all pH levels observed. The results suggest that only limited fertilization, and therefore limited genetic recombinations could occur in Pteridium aquilinum under conditions of acidic precipitation (pH and sulfate levels) that prevail in the northeastern United States.

We have previously presented evidence that cell-cell contact is the normal developmental signal to deactivate discoidin I gene expression in D discoideum [Berger EA, Clark JM: Proc Natl Acad Sci USA 80:4983, 1983]. Here we provide genetic evidence to support this hypothesis by examining gene expression in a cohesion-defective mutant, strain EB-21, which enters the developmental program but is blocked at the loose mound stage. When this strain was developed in suspension, the cells remained almost entirely as single amoebae, unlike the wild type, which formed large multicellular aggregates. In both strains, discoidin I mRNA levels were low in vegetative cells but rose sharply during the first few hours of development. However, the peak level reached at 8 hr in EB-21 exceeded that observed in wild type, and while the level declined markedly over the next few hours in wild type, it remained highly elevated in the mutant. Thus, there was a correlation between the inability of EB-21 to form normal cell-cell contacts and its deficiency in inactivating discoidin I gene expression. Previous studies from several laboratories, including this one, have demonstrated that exogenously added cAMP can block or reverse the changes in gene expression normally seen upon cell disaggregation. This has led us to propose that cAMP serves as a second messenger regulating the expression of contact-regulated genes. Here we provide additional support for this hypothesis. Intracellular cAMP levels rapidly dropped several-fold when wild type tight cell aggregates were disaggregated and remained low as the cells were cultured in the disaggregated state. Furthermore, overexpression of discoidin I mRNA late in development in EB-21 was corrected by addition of high concentrations of cAMP. These results are consistent with a second messenger function for cAMP in the contact-mediated regulatory response, and they indicate that the cAMP response machinery for discoidin I gene expression is capable of functioning in the cohesion-defective EB-21 strain.