The regulative capacity of prespore amoebae as demonstrated by fluorescence-activated cell sorting and green fluorescent protein.

The ability of prespore Dictyostelium discoideum amoebae to undergo redifferentiation so as to reestablish normal spore/stalk proportioning has been demonstrated in various ways over the years, beginning with the classic microdissection work of K. Raper. The discovery of anterior-like cells in the slug posterior, however, cast doubt on that ability, and more recent experiments using a cell-specific toxin suggested that prespore redifferentiation may not in fact occur. To reexamine this question, we performed fluorescence-activated cell sorting (FACS) upon amoebae expressing a mutated green fluorescent protein gene (S65T-GFP) under the control of a prespore-specific (PsA) promoter. FACS produced prespore cell populations with purities, measured by GFP expression, as high as 99. 5%. Sorted GFP(+) cells were developmentally competent and produced normally proportioned fruits, indistinguishable from those of "sham-sorted" (permissively gated, mixed GFP(+) and GFP(-)) amoebae. This result confirms the developmental totipotency of prespore amoebae.

Effects of protein synthesis inhibition on the transcription and transcript stability of Dictyostelium prespore genes.

The in vivo accumulation of several prespore transcripts of Dictyostelium discoideum has previously been shown to depend upon concomitant protein synthesis (Ratner, D.I., Pentz, W.H. and Pelletier, D.A. (1989) Biochim. Biophys. Acta 1008, 71-78). Measurements of in vivo mRNA decay and nuclear run-on transcription assays have now been used to learn whether protein synthesis is required primarily for mRNA synthesis or transcript stability. The translational inhibitors cycloheximide and pactamycin stabilized existing prespore transcripts, despite their effect upon mRNA accumulation. Transcriptional assays, performed at intervals throughout the developmental cycle, demonstrated that temporal changes in the abundance of several cell-specific transcripts correlated closely with changes in their rates of synthesis. Finally, blocking protein synthesis strongly inhibited the transcription of the prespore genes examined. These results imply that one or more developmentally regulated, labile proteins are needed for the activation of prespore gene transcription.

Lack of 5-methylcytosine in Dictyostelium discoideum DNA.

We find no evidence for the presence of 5-methylcytosine in the DNA of Dictyostelium discoideum. Methylation was absent from CCGG sites in repetitive DNA and in DNA from the actin multigene family. Nor was 5-methylcytosine detected in total DNA when base composition was determined by means of h.p.l.c.

Developmental protein synthesis is required for the transcription of Dictyostelium prespore genes.

It has been established previously that the maintenance of expression of prespore-specific genes of Dictyostelium discoideum is prevented by the translational inhibitor cycloheximide. The drug had no effect upon the level of transcripts of the other genes examined, prestalk-specific or cell type-nonspecific. However, the interpretation of this result is open to question, because of possible nonspecific effects of cycloheximide. We have now characterized the cellular specificity and temporal profiles of mRNA accumulation of additional Dictyostelium cDNA clones, and have examined other inhibitors of in vivo protein synthesis. Four structurally and mechanistically distinct translational inhibitors each prevented the reaccumulation of prespore transcripts in cyclic AMP-primed, disaggregated amoebae. These results establish the importance of developmental protein synthesis in the accumulation of prespore gene transcripts. Nuclear run-on transcription assays were used to learn whether protein synthesis is required primarily for mRNA synthesis or transcript stability. A transcriptional time course first demonstrated that the abundance of these cell-specific transcripts during development mirrors their rates of synthesis. Significantly, the protein synthesis requirement of the prespore genes examined also occurs at the level of mRNA transcription, implying the existence of one or more developmentally regulated transcriptional activators.

Prespore gene expression in Dictyostelium requires concomitant protein synthesis.

It has been established previously that the maintenance of expression of prespore-specific genes of Dictyostelium discoideum is prevented by the translational inhibitor cycloheximide. The drug had no effect upon the level of transcripts of the other genes examined, prestalk-specific or cell type non-specific (Mehdy, M., Ratner, D. and Firtel, R., (1983) Cell 32, 763-771). We have now characterized the cellular specificity and temporal profiles of mRNA accumulation of additional Dictyostelium cDNA clones. Other inhibitors of in vivo protein synthesis have been examined, with emetine shown to be a particularly effective but reversible agent. Four structurally and mechanistically distinct translational inhibitors each prevented the reaccumulation of prespore transcripts in cyclic AMP-primed disaggregated amoebae. These results establish a role for protein synthesis in the transcription or transcript stability of prespore genes.

Homologous recombination and the repair of double-strand breaks during cotransformation of Dictyostelium discoideum.

We examined the ability of unlinked nonreplicating plasmid molecules to undergo homologous recombination during cotransformation of Dictyostelium amoebae. The transformation vector B10S confers resistance to the antibiotic G418 and was always presented to amoebae as a closed circle. Cotransforming DNA, containing a slime mold cDNA and sequences homologous to the primary vector, was presented either as a closed circle or as a linear molecule after digestion with restriction endonucleases which cut within one of three distinct regions of the plasmid. Remarkably, homologous recombination occurred in every clone examined. Moreover, the products of recombination were identical in all instances, irrespective of the presence or position of linearized ends. The ends of the linear templates were not recombinogenic. Repair of the introduced double-strand break occurred frequently during recombination. The repair could occur intermolecularly or, more likely, intramolecularly, i.e., by recircularization. Many of the recombination events were of a nonreciprocal nature. Despite the startlingly frequent level of homologous recombination, the use of cotransforming DNA which contains no homology to the selected vector established that such recombination was not required for cotransformation.

Linkage analysis in Dictyostelium discoideum using multiply marked tester strains: establishment of linkage group VII and the reassessment of earlier linkage data.

To aid linkage analysis and mapping studies in Dictyostelium discoideum, we have constructed several tester strains with easily scored mutations characterizing the six currently identified linkage groups. Use has been made of conditionally lethal mutants unable to grow upon Bacillus subtilis, and the locus of the mutation involved (bsgA) has been assigned to linkage group III. The mutation cobA1, which confers resistance to cobaltous chloride, has been assigned to a previously unidentified linkage group (VII). The temperature-sensitive growth mutation tsgC7, previously reported to define linkage group V, has been reassigned to group III, leaving linkage group V presently unmarked. The further use of genetic tester strains is described.