Expression of Dictyostelium early gene, dutA, is independent of cAMP pulses but dependent on protein kinase A

Abstract The untranslatable, RNA polymerase II-dependent gene ( dutA ) of Dictyostelium discoideum is induced early in development. However, unlike other early genes, dutA induction was not affected by cAMP pulses and occurred normally in various cAMP-related mutant cells, the results indicating that this induction depended solely on factors other than cAMP. In the knockout strain of the catalytic subunit of protein kinase A, dutA expression was severely blocked and not recovered by cAMP pulses. This demonstrates that even the cAMP-independent gene, dutA , requires protein kinase A for its expression.     

dutA RNA functions as an untranslatable RNA in the development of Dictyostelium discoideum.

dutA is a gene specifically expressed during the development of Dictyostelium discoideum. Toward understanding its possible role in development, we isolated and characterized the gene and its complete cDNA. We found that dutA is encoded by the nuclear genome as a single copy gene without introns. In addition, the following unique and interesting features of dutA RNA (1322 nt) emerged: (1) it has no sustained ORFs (MAX = 126 nt) (2) it is extremely AU-rich (83%) (3) it contains peculiar sequence motifs (large palindromes, long AU-stretches and GC-clusters) (4) it is localized in the cytoplasm but completely absent from ribosomes. These features suggest that dutA RNA functions without being translated into protein. Disruption of the dutA gene did not cause phenotypic changes, suggesting that the function of dutA is redundant.     

Expression of zinc transporter family genes in Dictyostelium

Regulation of the zinc ion concentration is physiologically important to control the activities of a variety of cellular molecules. A BLAST search against a conserved domain of known zinc transporters identified twelve putative zinc transporter family genes in the Dictyostelium genome. Phylogenetic analysis revealed the presence of three zinc transporter subfamilies in Dictyostelium. One subfamily of proteins, consisting of the ZntA-D proteins, has weak homology to the STAT3-inducible LIV-1 protein. In addition, in situ hybridization revealed that the zntA-D genes are expressed in the pstAB cells, this expression being absent in the Dd-STATa null mutant. Thus, Dd-STATa may control stalk cell differentiation through some members of the zinc transporter family genes during Dictyostelium development.     

Glycogen synthase kinase-3 enhances nuclear export of a Dictyostelium STAT protein

Extracellular cAMP stimulates the rapid tyrosine phosphorylation and nuclear translocation of the DICTYOSTELIUM: STAT protein Dd-STATa. Here we show that it also induces serine phosphorylation by GskA, a homologue of glycogen synthase kinase-3 (GSK-3). Tyrosine phosphorylation occurs within 10 s of stimulation, whereas serine phosphorylation takes 5 min, matching the kinetics observed for the cAMP regulation of GskA. Phosphorylation by GskA enhances nuclear export of Dd-STATa. The phosphorylated region, however, is not itself a nuclear export signal and we identify a region elsewhere in the protein that mediates nuclear export. These results suggest a biphasic regulation of Dd-STATa, in which extracellular cAMP initially directs nuclear import and then, via GskA, promotes its subsequent export. It also raises the possibility of an analogous regulation of STAT nuclear export in higher eukaryotes.     

Activation of the prespore and spore cell pathway of Dictyostelium differentiation by cAMP-dependent protein kinase and evidence for its upstream regulation by ammonia.

Expression of a dominant inhibitor of the Dictyostelium cAMP-dependent protein kinase in prespore cells blocks their differentiation into spore cells. The resultant structures comprise a normal stalk supporting a bolus of cells that fail to express a sporulation-specific gene and that show greatly reduced levels of expression of several prespore-specific genes. The latter result suggests that in addition to activating spore formation, the cAMP-dependent protein kinase may play a role in initial prespore cell differentiation. Development of the strain expressing the dominant inhibitor is hypersensitive to the inhibitory effects of ammonia, the molecule that is believed to repress entry into culmination during normal development. This result supports a model whereby a decrease in ambient ammonia concentration at culmination acts to elevate intracellular cAMP and hence induce terminal differentiation.     

An analysis of culmination in Dictyostelium using prestalk and stalk-specific cell autonomous markers

The ecmA (pDd63) and ecmB (pDd56) genes encode extracellular matrix proteins of the slime sheath and stalk tube of Dictyostelium discoideum. Using fusion genes containing the promoter of one or other gene coupled to an immunologically detectable reporter, we previously identified two classes of prestalk cells in the tip of the migrating slug; a central core of pstB cells, which express the ecmB gene, surrounded by pstA cells, which express the ecmA gene. PstB cells lie at the position where stalk tube formation is initiated at culmination and we show that they act as its founders. As culmination proceeds, pstA cells transform into pstB cells by activating the ecmB gene as they enter the stalk tube. The prespore region of the slug contains a population of cells, termed anterior-like cells (ALC), which have the characteristics of prestalk cells. We show that the ecmA and ecmB genes are expressed at a low level in ALC during slug migration and that their expression in these cells is greatly elevated during culmination. Previous observations have shown that ALC sort to surround the prespore cells during culmination (Sternfeld and David, 1982 Devl Biol. 93, 111-118) and we find just such a distribution for pstB cells. We believe that the ecmB protein plays a structural role in the stalk tube and its presence, as a cradle around the spore head, suggests that it may play a further function, perhaps in ensuring integrity of the spore mass during elevation. If this interpretation is correct, then a primary role of anterior-like cells may be to form these structures at culmination. We previously identified a third class of prestalk cells, pstO cells, which lie behind pstA cells in the slug anterior and which appeared to express neither the ecmA nor the ecmB gene. Using B-galactosidase fusion constructs, which give more sensitive detection of gene expression, we now find that these cells express the ecmA gene but at a much lower level than pstA cells. We also show that expression of the ecmA gene becomes uniformly high throughout the prestalk zone when slugs are allowed to migrate in the light. Overhead light favours culmination and it may be that increased expression of the ecmA gene in the pst 'O' region is a preparatory step in the process.     

A genetic melanotic neoplasm of Drosophila melanogaster

The construction of mature fruiting bodies occurs during the culmination stage of development of Dictyostelium discoideum. These contain at least two different cell types, spores and stalks, which originate from an initially homogenous population of vegetative amoebas. As an attempt to identify proteins whose synthesis is regulated in each cell type during differentiation, we have analyzed the two-dimensional profiles of proteins synthesized by spore and stalk cells during the culmination stage. We have identified 5 major polypeptides which are specifically synthesized by spore cells during culmination and 9 which are only made by stalk cells. Furthermore, synthesis of about 20 polypeptides appears to be enriched either in the spore or in the stalk cells. We also show that synthesis of actin, a major protein synthesized during Dictyostelium development, is specifically inhibited in the spore cells during culmination. Synthesis of most of the cell type-specific proteins initiates at 19–20 hr, during culmination. Moreover, the proteins whose synthesis is induced after formation of tight aggregates, the time when the major change in gene expression occurs, are not specifically incorporated into spores or stalk cells, and appear to be synthesized by both cell types. We conclude that a new class of genes is expressed during the culmination stage in Dictyostelium, giving rise to specific patterns of protein synthesis in spore and stalk cells.     

Mutual suppression of mukB and seqA phenotypes might arise from their opposing influences on the Escherichia coli nucleoid structure

A strain of Escherichia coli in which both the seqA and mukB genes were inactivated displayed partial suppressions of their individual phenotypes. Temperature sensitivity, anucleate cell production and poor nucleoid folding seen in the mukB strain were suppressed by the seqA null mutation, whereas filamentation, asymmetric septation and compact folding of the nucleoids observed in the seqA strain were suppressed by inactivation of the mukB gene function. However, the asynchronous initiation of chromosome replication in the seqA strain was not reversed in the mukBseqA double mutant. Membrane-associated nucleoids were isolated from the wild-type, mukB, seqA and mukBseqA strains and their sedimentation rates were compared under identical conditions. Whereas the mukB mutation caused unfolding of the nucleoid, the seqA mutation led to a more compact packaging of the chromosome. The mukBseqA double mutant regained the wild-type nucleoid organization as revealed from its rate of sedimentation. Microscopic appearances of the nucleoids were consistent with the sedimentation profiles. The mukB mutant was oversensitive to novobiocin and this susceptibility was suppressed in the mukBseqA strain, suggesting possible roles of MukB and SeqA in maintaining chromosome topology. The mutual phenotypic suppression of mukB and seqA alleles thus suggests that these genes have opposing influences on the organization of the bacterial nucleoid.