Intracellular ATP levels influence cell fates in Dictyostelium discoideum differentiation.

Multicellular organisms contain various differentiated cells. Fate determination of these cells remains a fundamental issue. The cellular slime mold Dictyostelium discoideum is a useful model organism for studying differentiation; it proliferates as single cells in nutrient-rich conditions, which aggregate into a multicellular body upon starvation, subsequently differentiating into stalk cells or spores. The fates of these cells can be predicted in the vegetative phase: Cells expressing higher and lower levels of omt12 differentiate into stalk cells and spores, respectively. However, omt12 is merely a marker gene and changes in its expression do not influence the cell fate, and determinant factors remain unknown. In this study, we analyzed cell fate determinants in the stalk-destined and spore-destined cells that were sorted based on omt12 expression. Luciferase assay demonstrated higher levels of intracellular ATP in the stalk-destined cells than in the spore-destined cells. Live-cell observation during development using ATP sensor probes revealed that cells with higher ATP levels differentiated into stalk cells. Furthermore, reducing the ATP level by treating with an inhibitor of ATP production changed the differentiation fates of the stalk-destined cells to spores. These results suggest that intracellular ATP levels influence cell fates in D. discoideum differentiation.

Supramolecular Chemistry-Based One-Pot High-Efficiency Separation of Solubilizer-Free Pure Semiconducting Single-Walled Carbon Nanotubes: Molecular Strategy and Mechanism.

Single-walled carbon nanotubes (SWCNTs) have the potential to revolutionize nanoscale electronics and power sources; however, their low purity and high separation cost limit their use in practical applications. Here we present a supramolecular chemistry-based one-pot, less expensive, scalable, and highly efficient separation of a solubilizer/adsorbent-free pure semiconducting SWCNT (sc-SWCNT) using flavin/isoalloxazine analogues with different substituents. On the basis of both experimental and computational simulations (DFT study), we have revealed the molecular requirements of the solubilizers as well as provided a possible mechanism for such a highly efficient selective sc-SWCNT separation. The present sorting method is very simple (one-pot) and gives a promising sc-SWCNT separation methodology. Thus, the study provides insight for the molecular design of an sc-SWCNT solubilizer with a high (n,m)-chiral selectivity, which benefits many areas including semiconducting nanoelectronics, thermoelectric, bio and energy materials, and devices using solubilizer-free very pure sc-SWCNTs.

A novel, lineage-primed prestalk cell subtype involved in the morphogenesis of D. discoideum.

Dictyostelium morphogenesis requires the tip, which acts as an organizer and conducts orchestrated cell movement and cell differentiation. At the slug stage the tip region contains prestalk A (pstA) cells, which are usually recognized by their expression of reporter constructs that utilize a fragment of the promoter of the ecmA gene. Here, using the promoter region of the o-methyl transferase 12 gene (omt12) to drive reporter expression, we demonstrate the presence, also within the pstA region, of a novel prestalk cell subtype: the pstV(A) cells. Surprisingly, a sub-population of the vegetative cells express a pstV(A): GFP marker and, sort out to the tip, both when developing alone and when co-developed with an excess of unmarked cells. The development of such a purified GFP-marked population is greatly accelerated: by precocious cell aggregation and tip formation with accompanying precocious elevation of developmental gene transcription. We therefore suggest that the tip contains at least two prestalk cell subtypes: the developmentally-specified pstA cells and the lineage-primed pstV(A) cells. It is presumably the pstV(A) cells that play the dominant role in morphogenesis during the earlier stages of development. The basis for the lineage priming is, however, unclear because we can find no correlation between pstV(A) differentiation and nutrient status during growth or cell cycle position at the time of starvation, the two known determinants of probable cell fate.

A new kind of membrane-tethered eukaryotic transcription factor that shares an auto-proteolytic processing mechanism with bacteriophage tail-spike proteins.

MrfA, a transcription factor that regulates Dictyostelium prestalk cell differentiation, is an orthologue of the metazoan myelin gene regulatory factor (MRF) proteins. We show that the MRFs contain a predicted transmembrane domain, suggesting that they are synthesised as membrane-tethered proteins that are then proteolytically released. We confirm this for MrfA but report a radically different mode of processing from that of paradigmatic tethered transcriptional regulators, which are cleaved within the transmembrane domain by a dedicated protease. Instead, an auto-proteolytic cleavage mechanism, previously only described for the intramolecular chaperone domains of bacteriophage tail-spike proteins, processes MrfA and, by implication, the metazoan MRF proteins. We also present evidence that the auto-proteolysis of MrfA occurs rapidly and constitutively in the ER and that its specific role in prestalk cell differentiation is conferred by the regulated nuclear translocation of the liberated fragment.

Steely enzymes are involved in prestalk and prespore pattern formation.

4-Methyl-5-pentylbenzene-1,3-diol (MPBD), a product of SteelyA enzyme, controls Dictyostelium spore maturation. Since the expression of stlA split the in early and terminal stages, we cannot exclude the possibility that MPBD regulates spore differentiation from the early stage by creating a bias between the cells. 1-(3,5-Dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-on (DIF-1), a product of SteelyB, was identified as the major stalk cell inducer by in vitro assay, but in vivo assay revealed that DIF-1 induces only prestalkB (pstB) and prestalkO (pstO) cells and, that the major prestalkA (pstA) cells differentiated without DIF-1. In order to determine mechanism of polyketide regulated pattern formation, we examined the spatial expression patterns of prestalk and prespore markers in stlA and stlB knockout mutants. We found that MPBD regulates spore maturation at the culmination stage. We also found that the stlA and stlB double-knockout mutant lost pstA marker gene expression.

Protein kinase B gene homologue pkbR1 performs one of its roles at first finger stage of Dictyostelium.

Dictyostelium discoideum has protein kinases AKT/PKBA and PKBR1 that belong to the AGC family of kinases. The protein kinase B-related kinase (PKBR1) has been studied with emphasis on its role in chemotaxis, but its roles in late development remained obscure. The pkbR1 null mutant stays in the first finger stage for about 16 h or longer. Only a few aggregates continue to the migrating slug stage; however, the slugs immediately go back probably to the previous first finger stage and stay there for approximately 37 h. Finally, the mutant fingers diversify into various multicellular bodies. The expression of the pkbR1 finger protein probably is required for development to the slug stage and to express ecmB, which is first observed in migrating slugs. The mutant also showed no ST-lacZ expression, which is of the earliest step in differentiation to one of the stalk cell subtypes. The pkbR1 null mutant forms a small number of aberrant fruiting bodies, but in the presence of 10% of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form nonviable stalk cells. These results suggest that the mutant has defects in a system that changes the physiological dynamics in the prestalk cell region of a finger. We suggest that the arrest of its development is due to the loss of the second wave of expression of a protein kinase A catalytic subunit gene (pkaC) only in the prestalk region of the pkbR1 null mutant.

Phenotypes of pain behavior in phospholipase C-related but catalytically inactive protein type 1 knockout mice.

Phospholipase C-related inactive protein (PRIP) plays important roles in trafficking to the plasma membrane of GABA(A) receptor, which is involved in the dominant inhibitory neurotransmission in the spinal cord and plays an important role in nociceptive transmission. However, the role of PRIP in pain sensation remains unknown. In this study, we investigated the phenotypes of pain behaviors in PRIP type 1 knockout (PRIP-1 (-/-)) mice. The mutant mice showed hyperalgesic responses in the second phase of the formalin test and the von Frey test as compared with those in wild-type mice. In situ hybridization studies of GABA(A) receptors revealed significantly decreased expression of γ2 subunit mRNA in the dorsal and ventral horns of the spinal cord in PRIP-1 (-/-) mice, but no difference in α1 subunit mRNA expression. ß2 subunit mRNA expression was significantly higher in PRIP-1 (-/-) mice than in wild-type mice in all areas of the spinal cord. On the other hand, the slow decay time constant for the spontaneous inhibitory current was significantly increased by treatment with diazepam in wild-type mice, but not in PRIP-1 (-/-) mice. These results suggest that PRIP-1 (-/-) mice exhibit the changes of the function and subunits expression of GABA(A) receptor in the spinal cord, which may be responsible for abnormal pain sensation in these mice.

Control of prestalk-cell differentiation by transcription factors.

Transcriptional control of developmental genes is important for cell differentiation and pattern formation. Developing Dictyostelium discoideum cells form a multicellular structure in which individual cells differentiate into either stalk cells or spores. This simplicity makes the organism an attractive model for studying fundamental problems in developmental biology. However, the morphogenetic process of forming a stalked fruiting body conceals a certain degree of complexity. This is reflected in the presence of multiple prestalk subtypes that have individual roles to generate the fruiting body. This review describes recent advances in understanding the molecular mechanisms, mediated by transcription factors that generate prestalk-cell heterogeneity.

Prespore cell inducing factor, psi factor, controls both prestalk and prespore gene expression in Dictyostelium development.

Prespore cell-inducing (psi, psi) factor (PsiA), encoded by the psiA gene of Dictyostelium, is a secreted signal glycoprotein that induces prespore cell differentiation when added to monolayer cultures. In situ hybridization during normal development showed that the psiA gene is highly expressed in scattered cells at the mound stage and in prespore cells at the onset of culmination. The conventional prespore-cell marker genes, cotC and pspA, were expressed normally in psiA(-) and psiA overexpressing strains. Expressions of rnrB and cudA are repressed in the prestalk cells of a wild type slug to render prespore specific pattern. However, a promoter-reporter fusion gene, rnrB:lacZ, showed an ectopic expression in the prestalk cells of the psiA(-) strain while cudA(psp):lacZ did so in those of the psiA overexpressing strain. Overexpression of psiA delayed expression of the prestalk specific gene, ecmB, during development, while knocking out psiA promoted its expression. In addition, overexpression inhibited DIF-1-induced stalk formation in monolayer cultures. Together with the known prespore inducing activity, the results indicate that PsiA regulates both prespore and prestalk/stalk cell differentiation. These results indicate that PsiA is also involved in prestalk cell differentiation.

Pathomechanism of nocturnal paroxysmal dystonia in autosomal dominant sleep-related hypermotor epilepsy with S284L-mutant α4 subunit of nicotinic ACh receptor.

To study the pathomechanism and pathophysiology of nocturnal paroxysmal dystonia of autosomal dominant sleep-related hypermotor epilepsy (ADSHE), this study determined functional abnormalities in thalamic hyperdirect pathway from reticular thalamic nucleus (RTN), motor thalamic nuclei (MoTN), subthalamic nucleus (STN) to substantia nigra pars reticulata (SNr) of transgenic rats (S286L-TG) bearing S286 L missense mutation of rat Chrna4 gene, which corresponds to the S284 L mutation in the human CHRNA4 gene. The activation of α4ß2-nAChR in the RTN increased GABA release in MoTN resulting in reduced glutamatergic transmission in thalamic hyperdirect pathway of wild-type. Contrary to wild-type, activation of S286L-mutant α4ß2-nAChR (loss-of-function) in the RTN relatively enhanced glutamatergic transmission in thalamic hyperdirect pathway of S286L-TG via impaired GABAergic inhibition in intra-thalamic (RTN-MoTN) pathway. These functional abnormalities in glutamatergic transmission in hyperdirect pathway contribute to the pathomechanism of electrophysiologically negative nocturnal paroxysmal dystonia of S286L-TG. Therapeutic-relevant concentration of zonisamide (ZNS) inhibited the glutamatergic transmission in the hyperdirect pathway via activation of group II metabotropic glutamate receptor (II-mGluR) in MoTN and STN. The present results suggest that S286L-mutant α4ß2-nAChR induces GABAergic disinhibition in intra-thalamic (RTN-MoTN) pathway and hyperactivation of glutamatergic transmission in thalamic hyperdirect pathway (MoTN-STN-SNr), possibly contributing to the pathomechanism of nocturnal paroxysmal dystonia of ADSHE patients with S284L mutant CHRNA4. Inhibition of glutamatergic transmission in thalamic hyperdirect pathway induced by ZNS via activation of II-mGluR may be involved, at least partially, in ZNS-sensitive nocturnal paroxysmal dystonia of ADSHE patients with S284L mutation.

Upregulated Connexin 43 Induced by Loss-of-Functional S284L-Mutant α4 Subunit of Nicotinic ACh Receptor Contributes to Pathomechanisms of Autosomal Dominant Sleep-Related Hypermotor Epilepsy.

To study the pathomechanism and pathophysiology of autosomal dominant sleep-related hypermotor epilepsy (ADSHE), this study determined functional abnormalities of glutamatergic transmission in the thalamocortical motor pathway, from the reticular thalamic nucleus (RTN), motor thalamic nuclei (MoTN) tosecondary motor cortex (M2C) associated with the S286L-mutant α4ß2-nicotinic acetylcholine receptor (nAChR) and the connexin43 (Cx43) hemichannel of transgenic rats bearing the rat S286L-mutant Chrna4 gene (S286L-TG), which corresponds to the human S284L-mutant CHRNA4 gene using multiprobe microdialysis, primary cultured astrocytes and a Simple Western system. Expression of Cx43 in the M2C plasma membrane fraction of S286L-TG was upregulated compared with wild-type rats. Subchronic nicotine administration decreased Cx43 expression of wild-type, but did not affect that of S286L-TG; however, zonisamide (ZNS) decreased Cx43 in both wild-type and S286L-TG. Primary cultured astrocytes of wild-type were not affected by subchronic administration of nicotine but was decreased by ZNS. Upregulated Cx43 enhanced glutamatergic transmission during both resting and hyperexcitable stages in S286L-TG. Furthermore, activation of glutamatergic transmission associated with upregulated Cx43 reinforced the prolonged Cx43 hemichannel activation. Subchronic administration of therapeutic-relevant doses of ZNS compensated the upregulation of Cx43 and prolonged reinforced activation of Cx43 hemichannel induced by physiological hyperexcitability during the non-rapid eye movement phase of sleep. The present results support the primary pathomechanisms and secondary pathophysiology of ADSHE seizures of patients with S284L-mutation.

Upregulated and Hyperactivated Thalamic Connexin 43 Plays Important Roles in Pathomechanisms of Cognitive Impairment and Seizure of Autosomal Dominant Sleep-Related Hypermotor Epilepsy with S284L-Mutant α4 Subunit of Nicotinic ACh Receptor.

To understand the pathomechanism and pathophysiology of autosomal dominant sleep-related hypermotor epilepsy (ADSHE), we studied functional abnormalities of glutamatergic transmission in thalamocortical pathway from reticular thalamic nucleus (RTN), mediodorsal thalamic nucleus (MDTN) to orbitofrontal cortex (OFC) associated with S286L-mutant α4ß2-nicotinic acetylcholine receptor (nAChR), and connexin43 (Cx43) hemichannel of transgenic rats bearing rat S286L-mutant Chrna4 gene (S286L-TG), corresponding to the human S284L-mutant CHRNA4 gene using simple Western analysis and multiprobe microdialysis. Cx43 expression in the thalamic plasma membrane fraction of S286L-TG was upregulated compared with that of wild-type. Subchronic administrations of therapeutic-relevant doses of zonisamide (ZNS) and carbamazepine (CBZ) decreased and did not affect Cx43 expression of S286L-TG, respectively. Upregulated Cx43 enhanced glutamatergic transmission during both resting and hyperexcitable stages in S286L-TG. Furthermore, activation of GABAergic transmission RTN-MDTN pathway conversely enhanced, but not inhibited, l-glutamate release in the MDTN via upregulated/activated Cx43. Local administration of therapeutic-relevant concentration of ZNS and CBZ acutely supressed and did not affect glutamatergic transmission in the thalamocortical pathway, respectively. These results suggest that pathomechanisms of ADSHE seizure and its cognitive deficit comorbidity, as well as pathophysiology of CBZ-resistant/ZNS-sensitive ADSHE seizures of patients with S284L-mutation.

Pathogenesis and pathophysiology of autosomal dominant sleep-related hypermotor epilepsy with S284L-mutant α4 subunit of nicotinic ACh receptor.

BACKGROUND AND PURPOSE: The mechanisms causing spontaneous epileptic seizures, including carbamazepine-resistant/zonisamide -sensitive seizures and comorbidity in autosomal dominant sleep-related hypermotor epilepsy (ADSHE) are unclear. This study investigated functional abnormalities in thalamocortical transmission in transgenic rats bearing rat S286L-mutant Chrna4 (S286L-TG) of α4 subunit of the nicotinic ACh receptor (nAChR) that corresponds to the human S284L-mutant CHRNA4. EXPERIMENTAL APPROACH: Effects of carbamazepine and zonisamide on epileptic discharges of S286L-TG rat were measured using telemetry electrocorticogram. Transmission abnormalities of L-glutamate and GABA in thalamocortical pathway of S286L-TG rats were investigated using multiprobe microdialysis and ultra-high-performance liquid-chromatography. KEY RESULTS: Epileptic discharges in S286L-TG rats were reduced by zonisamide but not by carbamazepine, similar to that of S284L-ADSHE patients. Carbamazepine unaffected functional abnormality in transmission of S286L-TG rats. However, zonisamide was able to compensate for the attenuated S286L-mutant nAChR induced GABA release in frontal-cortex, without affecting attenuated thalamocortical glutamatergic transmission. Excitatory effects of S286L-mutant nAChR on thalamocortical transmission were attenuated compared with those of wild-type nAChR. Loss-of-function of S286L-nAChR enhanced transmission in thalamocortical motor pathway by predominantly attenuating GABAergic transmission. However, it attenuated transmission in thalamocortical cognitive pathway by reducing inhibitory GABAergic and excitatory glutamatergic transmission. CONCLUSION AND IMPLICATIONS: Our results suggest that functional abnormalities of S286L-nAChR are associated with intra-frontal and thalamocortical transmission, possibly contributing to the pathogenesis of ADSHE-seizure and comorbidity of S284L-ADSHE. Selective compensation of impaired GABAergic transmission by zonisamide (but not by carbamazepine) in frontal cortex may be involved, at least partially, in carbamazepine-resistant ADSHE-seizure of S284L-ADSHE patients.

Differentiation inducing factors in Dictyostelium discoideum: a novel low molecular factor functions at an early stage(s) of differentiation.

There are reports that secreted factor(s) are involved in prespore cell differentiation in Dictyostelium discoideum, but the structures and functions of the various factors have not been elucidated. Previously, we described two prespore cell-inducing factors in conditioned medium; one was a glycoprotein named prespore cell-inducing factor (psi factor, or PSI-1), and the other, a heat stable dialyzable factor(s). In the present paper, we purified and characterized the most potent prespore cell-inducing activity in dialysates. The factor began to be secreted after the onset of starvation and stopped being secreted once the cells had aggregated, which was earlier than the onset of the psi factor gene expression. In addition, unlike psi factor, its secretion did not appear to depend on activation of protein kinase A. Interestingly, the purified factor not only induced prespore cell specific genes such as pspA and cotC but also a prestalk-cell specific gene, ecmB in vitro. The purified factor is tentatively designated polyketide-like factor (PLF), because it seems to be a novel polyketide with 208 Da. Half maximal induction of prespore cell was obtained with 26 nmol/L of PLF. We propose that PLF plays a key role in the acquisition of differentiation commitment, before the psi factor induces specifically prespore cell differentiation.

Element analysis of the Polysphondylium pallidum gp64 promoter.

gp64 mRNA in Polysphondylium pallidum is expressed extensively during vegetative growth, and begins to rapidly decrease at the onset of development. To examine this unique regulation, 5' deletion analysis of the gp64 promoter was undertaken, and two growth-phase activated elements have been found: a food-dependent, upstream regulatory region (FUR, -222 to -170) and a vegetatively activated, downstream region (VAD, -110 to -63). Here we concentrate our analysis on an A1 and A2 sequences in the FUR region: A1 consists of a GATTTTTTTA sequence called a corresponding sequence and A2 consists of the direct repeat TTTGTTGTG. The cells carrying a combined construct of A1 and A2 acted synergistically in a reporter activity. A point mutation analysis in A1 indicates that a G residue is required for the activation of A1. From analyses of promoter regulation in a liquid or a solid medium, the promoter activity of the cells fed on bacteria in A-medium (axenic medium for Polysphondylium) or grown in A-medium alone was only one fourth of that of the cells fed on bacteria. By the gel retardation, we detected a protein bound to the A1 sequence.

OSBPa, a predicted oxysterol binding protein of Dictyostelium, is required for regulated entry into culmination.

The oxysterol binding proteins (OSBPs) are believed to control cholesterol homeostasis but their precise mechanism of action is not well understood. The Dictyostelium osbA gene encodes a predicted OSBP, OSBPa, which lacks the PH domain that in most other OSBPs directs targetting to the Golgi. OSBPa instead localises selectively to the cell periphery and also, in some cells, to the perinuclear region. OSBPa null strains form normal fruiting bodies but are defective in the regulation of the transition from slug migration to culmination. Thus a plasma membrane-enriched OSBP family member is essential for correct regulation of the slug-fruiting body switch.

Spatiotemporal Patterning of discoidin I and II during Development of Dictyostelium discoideum: (cellular slime mold/Dictyostelium discoideum/lectin/discoidin/cell type-specific localization).

We have examined the distribution of Dictyostelium lectins (discoidin I and II) during development by means of a sample preparation method of a whole mount. Monoclonal antibodies which were bound to discoidins revealed unique patterns of discoidin distribution. Discoidin I was localized mainly at the periphery of the aggregates, while the base of the aggregates was devoid of discoidin I staining. Discoidin I was not prominent in the body of the aggregates but when a migrating slug culminated, discoidin I staining appeared in the prestalk region, this suggested that prestalk cells begin to express discoidin I at the onset of culmination. During fruit formation we observed discoidin I staining at the foremost anterior prestalk region of the culminant, which implies a heterogeneity of discoidin I expression among prestalk cells; such a heterogenous pattern has also been found in other prestalk-specific proteins. In addition, anterior-like cells (ALC), which were sorted at the apex and basal parts of a spore mass during culmination, were also strongly stained with anti-discoidin I mAb; interestingly, we observed the staining of ALC from the slug stage through fruit formation. No discoidin II was observed in a migrating slug that had already accumulated prespore antigen ligands for discoidin II; it appeared in prespore cells after the onset of culmination. The present results indicate that, in addition to the early expression of discoidin I, both discoidin I and II are expressed during culmination, and these lectins also seem to be involved in the late development of Dictyostelium.

An orthologue of the Myelin-gene Regulatory Transcription Factor regulates Dictyostelium prestalk differentiation.

The prestalk region of the Dictyostelium slug is comprised of an anterior population of pstA cells and a posterior population of pstO cells. They are distinguished by their ability to utilize different parts of the promoter of the ecmA gene. We identify, by mutational analysis and DNA transformation, CA-rich sequence elements within the ecmA promoter that are essential for pstA-specific expression and sufficient to direct pstA-specific expression when multimerised. The CA-rich region was used in affinity chromatography with nuclear extracts and bound proteins were identified by mass spectrometry. The CA-rich elements purify MrfA, a protein with extensive sequence similarity to animal Myelin-gene Regulatory Factor (MRF)-like proteins. The MRF-like proteins and MrfA also display more spatially limited but significant sequence similarity with the DNA binding domain of the yeast Ndt80 sporulation-specific transcription factor. Furthermore, the ecmA CA-rich elements show sequence similarity to the core consensus Ndt80 binding site (the MSE) and point mutation of highly conserved arginine residues in MrfA, that in Ndt80 make critical contacts with the MSE, ablate binding of MrfA to its sites within the ecmA promoter. MrfA null strains are delayed in multicellular development and highly defective in pstA-specific gene expression. These results provide a first insight into the intracellular signaling pathway that directs pstA differentiation and identify a non-metazoan orthologue of a family of molecularly uncharacterised transcription factors.

A new family of transcription factors.

CudA, a nuclear protein required for Dictyostelium prespore-specific gene expression, binds in vivo to the promoter of the cotC prespore gene. A 14 nucleotide region of the cotC promoter binds CudA in vitro and ECudA, an Entamoeba CudA homologue, also binds to this site. The CudA and ECudA DNA-binding sites contain a dyad and, consistent with a symmetrical binding site, CudA forms a homodimer in the yeast two-hybrid system. Mutation of CudA binding sites within the cotC promoter reduces expression from cotC in prespore cells. The CudA and ECudA proteins share a 120 amino acid core of homology, and clustered point mutations introduced into two highly conserved motifs within the ECudA core region decrease its specific DNA binding in vitro. This region, the presumptive DNA-binding domain, is similar in sequence to domains in two Arabidopsis proteins and one Oryza protein. Significantly, these are the only proteins in the two plant species that contain an SH2 domain. Such a structure, with a DNA-binding domain located upstream of an SH2 domain, suggests that the plant proteins are orthologous to metazoan STATs. Consistent with this notion, the DNA sequence of the CudA half site, GAA, is identical to metazoan STAT half sites, although the relative positions of the two halves of the dyad are reversed. These results define a hitherto unrecognised class of transcription factors and suggest a model for the evolution of STATs and their DNA-binding sites.

Dictyostelium Myb transcription factors function at culmination as activators of ancillary stalk differentiation.

ecmB and mrrA are expressed in the cups that cradle Dictyostelium spore heads, and MybE is necessary for their expression in lower but not upper cup cells. A Myb site within the mrrA promoter is necessary for expression in both cups. Thus, multiple Myb proteins are required for ancillary stalk differentiation.