Copine A is expressed in prestalk cells and regulates slug phototaxis and thermotaxis in developing Dictyostelium

Copines are calcium-dependent membrane-binding proteins found in many eukaryotic organisms. We are studying the function of copines using the model organism, Dictyostelium discoideum. When under starvation conditions, Dictyostelium cells aggregate into mounds that become migrating slugs, which can move toward light and heat before culminating into a fruiting body. Previously, we showed that Dictyostelium cells lacking the copine A (cpnA) gene are not able to form fruiting bodies and instead arrest at the slug stage. In this study, we compared the slug behavior of cells lacking the cpnA gene to the slug behavior of wild-type cells. The slugs formed by cpnA- cells were much larger than wild-type slugs and exhibited no phototaxis and negative thermotaxis in the same conditions that wild-type slugs exhibited positive phototaxis and thermotaxis. Mixing as little as 5% wild-type cells with cpnA- cells rescued the phototaxis and thermotaxis defects, suggesting that CpnA plays a specific role in the regulation of the production and/or release of a signaling molecule. Reducing extracellular levels of ammonia also partially rescued the phototaxis and thermotaxis defects of cpnA- slugs, suggesting that CpnA may have a specific role in regulating ammonia signaling. Expressing the lacZ gene under the cpnA promoter in wild-type cells indicated cpnA is preferentially expressed in the prestalk cells found in the anterior part of the slug, which include the cells at the tip of the slug that regulate phototaxis, thermotaxis, and the initiation of culmination into fruiting bodies. Our results suggest that CpnA plays a role in the regulation of the signaling pathways, including ammonia signaling, necessary for sensing and/or orienting toward light and heat in the prestalk cells of the Dictyostelium slug.     

Phototaxis genes on linkage group V in Dictyostelium discoideum

Abstract During the course of mapping and complementation analysis of phototaxis ( pho ) mutations in Dictyostelium discoideum we have assigned to linkage group V three mutant pho alleles belonging to complementation groups phoG and phoK . These are the first genetic markers with an easily recognizable phenotype to be found on this linkage group.     

Mutants of thermotaxis in Dictyostelium discoideum

Amoebae of Dictyostelium discoideum, strain HL50 were mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine, cloned, allowed to form pseudoplasmodia and screened for aberrant positive and negative thermotaxis. Three types of mutants were found. Mutant HO428 exhibits only positive thermotaxis over the entire temperature range (no negative thermotaxis). HO596 and HO813 exhibit weakened positive thermotaxis and normal negative thermotaxis. The weakened positive thermotactic response results in a shift toward warmer temperatures in the transition temperature from negative to positive thermotaxis. Mutant HO209 exhibits weakened positive and negative thermotactic responses and has a transition temperature similar to the ‘wild type’ (HL50). The two types of mutants represented by HO428, HO596 and HO813 support the model that positive and negative thermotaxis have separate pathways for temperature sensing. The type of mutants which contains HO209 suggests that those two pathways converge at some point before the response.     

Light matters: phototaxis and signal transduction in unicellular cyanobacteria

Many photosynthetic microorganisms have evolved the ability to sense light quality and/or quantity and can steer themselves into optimal conditions within the environment. Phototaxis and gliding motility in unicellular cyanobacteria require type IV pili, which are multifunctional cell surface appendages. Screens for cells exhibiting aberrant motility uncovered several non-motile mutants as well as some that had lost positive phototaxis (consequently, they were negatively phototactic). Several negatively phototactic mutants mapped to the tax1 locus, which contains five chemotaxis-like genes. This locus includes a gene that encodes a putative photoreceptor (TaxD1) for positive phototaxis. A second chemotaxis-like cluster (tax3 locus) appears to be involved in pilus biogenesis. The biosynthesis and regulation of type IV pilus-based motility as well as the communication between the pilus motor and photosensory molecules appear to be complex and tightly regulated. Furthermore, the discovery that cyclic AMP and novel gene products are necessary for phototaxis/motility suggests that there might be additional levels of communication and signal processing.     

Genetics of phototaxis in a model eukaryote,Dictyostelium discoideum

The life cycle of Dictyostelium discoideum offers a unique opportunity to study signal transduction in eukaryotic cells at both the unicellular and multicellular levels of organization. Adding to the already extensive knowledge of the unicellular stages, classical and molecular genetics have begun to unravel transduction of signals controlling morphogenesis and behaviour (phototaxis and thermotaxis) in the multicellular ‘slug’ stage of the life cycle. Distributed over all seven genetic linkage groups are probably about 20, but possibly as many as 55, genes of importance for slug behaviour. The encoded proteins appear from pharmacological studies and mutant phenotypes to govern transduction pathways involving the intracellular second messengers cyclic AMP, cyclic GMP, IP₃ and Ca²⁺. Pathways from the photo- and thermoreceptors converge first with each other and thence, at the level of the second messengers, with those from extracellular tip activation (cyclic AMP) and inhibition (Slug Turning Factor and/or ammonia and/or adenosine) signals that control slug movement and morphogenesis.     

Thermal selection behaviour in the estuarine goby Gillichthys mirabilis Cooper

The estuarine goby Gillichthys mirabilis behaviorally thermoregulates when placed in a laboratory temperature gradient. Avoidance of temperatures above 23°C is the most evident component of this thermotaxis. Temperature preferences of non-acclimated fish do not vary significantly as a function of season; nor does temperature acclimation alter the preferendum. Varying the temperature range of the gradient apparently modifies thermotaxis. Negative phototaxis evidenced by this species is subjugated by thermal preferences. Temperature preference does not vary diurnally. The heat resistance of this species was determined; there is no apparent relationship between heat resistance and temperature preference.     

DNA methylation profiling of pseudogene-parental gene pairs and two gene families

A substantial proportion of human genes contain tissue-specifically DNA-methylated regions (TDMRs). However, little is known about the evolutionary conservation of differentially methylated loci, how they evolve, and the signals that regulate them. We have studied TDMR conservation in the PLG and TBX gene families and in 32 pseudogene–parental gene pairs. Among the members of the recently evolved PLG gene family, 5′-UTR methylation is conserved and inversely correlated with the cognate gene expression, indicating as well a conserved regulatory role of DNA methylation. Conversely, many genes of the much older TBX family display complementary tissue-specific methylation, suggesting an epigenetic complementation in the evolution of this gene family. Similar to gene families, unprocessed pseudogenes arose from gene duplications and we found TDMR conservation in some pseudogene–parental gene pairs displaying short evolutionary distances. However, for the majority of unprocessed pseudogenes and for all processed pseudogenes examined, we found that tissue-specific methylation arose de novo after gene duplication.     

Characterization of genes required for protein sorting and vacuolar function in the yeast Saccharomyces cerevisiae.

To further our studies of protein sorting and biogenesis of the lysosome-like vacuole in yeast, we have isolated spontaneous mutations in 11 new VPL complementation groups, as well as additional alleles of the eight previously described VPL genes. These mutants were identified by selecting for cells that mislocalize vacuolar proteins to the cell surface. Morphological examination of the vpl mutants indicated that most contain vacuoles of normal appearance; however, some of the mutants generally lack a large vacuole, and instead accumulate smaller organelles. Of the 19 VPL complementation groups, 12 were found to be identical to 12 of 33 VPT complementation groups identified in a separate study. Moreover, the end1 mutant and all of the previously reported pep mutants, with the exception of pep4, were found to exhibit a profound vacuolar protein sorting defect, and complementation tests between the PEP, VPL VPT and END1 groups demonstrated that there are extensive overlaps between these groups. Collectively, mutants in these four collections define 49 complementation groups required to deliver or retain soluble vacuolar enzymes, including carboxypeptidase Y (CPY) and proteinase A. We have also isolated 462 new mutants that lack normal levels of vacuolar CPY activity. Among these latter mutants, only pep4 mutants were found to be specifically defective in vacuolar zymogen activation. We conclude that there is a large number of gene products required for sorting or retention of vacuolar proteins in yeast, and only a single gene, PEP4, that is essential for activation of CPY and other vacuolar zymogens.     

Regulation of the phosphate regulon in Escherichia coli K-12: regulation of the negative regulatory gene phoU and identification of the gene product.

The phoU gene is one of the negative regulatory genes of the pho regulon of Escherichia coli. The DNA fragment carrying phoU has been cloned on pBR322 (Amemura et al., J. Bacteriol. 152:692-701, 1982). Further subcloning, Tn1000 insertion inactivation, and complementation tests localized the phoU gene within a 1.1-kilobase region on the cloned DNA fragment. The gene product of phoU was identified by the maxicell method as a protein with an approximate molecular weight of 27,000. A hybrid plasmid that contains a phoU'-lac'Z fused gene was constructed in vitro. This plasmid enabled us to study phoU gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the pho regulon, and phoU gene expression in these strains was studied under limited and excess phosphate conditions. It was found that phoU is expressed at a higher level when the cells are cultured under the excess phosphate condition. The higher phoU expression was observed in a phoB mutant and a phoR-phoM double mutant. The implications of these findings for the regulation of pho genes are discussed.     

Genetic analysis of the Rhizobium meliloti bacA gene: functional interchangeability with the Escherichia coli sbmA gene and phenotypes of mutants.

The Rhizobium meliloti bacA gene encodes a function that is essential for bacterial differentiation into bacteroids within plant cells in the symbiosis between R. meliloti and alfalfa. An Escherichia coli homolog of BacA, SbmA, is implicated in the uptake of microcin B17, microcin J25 (formerly microcin 25), and bleomycin. When expressed in E. coli with the lacZ promoter, the R. meliloti bacA gene was found to suppress all the known defects of E. coli sbmA mutants, namely, increased resistance to microcin B17, microcin J25, and bleomycin, demonstrating the functional similarity between the two proteins. The R. meliloti bacA386::Tn(pho)A mutant, as well as a newly constructed bacA deletion mutant, was found to show increased resistance to bleomycin. However, it also showed increased resistance to certain aminoglycosides and increased sensitivity to ethanol and detergents, suggesting that the loss of bacA function causes some defect in membrane integrity. The E. coli sbmA gene suppressed all these bacA mutant phenotypes as well as the Fix- phenotype when placed under control of the bacA promoter. Taken together, these results strongly suggest that the BacA and SbmA proteins are functionally similar and thus provide support for our previous hypothesis that BacA may be required for uptake of some compound that plays an important role in bacteroid development. However, the additional phenotypes of bacA mutants identified in this study suggest the alternative possibility that BacA may be needed for membrane integrity, which is likely to be critically important during the early stages of bacterial differentiation within plant cells.     

The sll0886 gene, controlling light-activated heterotrophic growth, is involved in regulating phototaxis in cyanobacterium Synechocystis sp. PCC 6893

The sll0886 gene, controlling light-activated heterotrophic growth (LAHG), was tested for the role in regulating phototaxis in cyanobacterium Synechocystis sp. PCC 6803. Insertional inactivation of the gene in the genome of a wildtype strain did not affect positive (toward light) or negative (away from high light) phototaxis. However, cells lost motility when sll0886 inactivation was combined with the prqRL17Q mutation, which determined negative phototaxis at low light. Immotile cells with the prqRL17Q mutation and the inactivated sll0886 gene did not display any defect in the formation of type IV pili, essential for phototaxis. Hence, the function, rather than biogenesis, of pili was affected. It was concluded that the sll0886 gene, coding for a TPR family protein, is involved in controlling negative phototaxis of cyanobacteria at the level of photoreception and signal transduction and that its role is mediated by the unidentified redundant gene whose function is suppressed by the prqRL17Q mutation.     

A steep thermal gradient thermotaxis assay for the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans with its well-described nervous system is one of the multicellular organisms of choice to study thermotaxis. The neuronal circuitry for thermosensation has been analyzed at the level of individual cells. Two methods have previously been described to study the behavior of C. elegans with respect to temperature: 1) isothermal tracking assays and 2) linear thermal gradients (Hedgecock and Russell, 1975). Here we present a short linear thermal gradient assay which is faster and which allows statistical evaluation of different populations using a thermotaxis index. Thin agar plates are used on which a temperature gradient from about 10 degrees to 30 degrees is induced over the distance of about 5 cm. The short linear thermal gradient uses inexpensive materials so that multiple tests can be performed in parallel in a short period of time.     

Interactive effects of phosphate deficiency, sucrose and light/dark conditions on gene expression of UDP-glucose pyrophosphorylase in Arabidopsis

The effects of inorganic phosphate (Pi) status, light/dark and sucrose on expression of UDP-glucose pyrophosphorylase (UGPase) gene (Ugp), which is involved in sucrose/ polysaccharides metabolism, were investigated using Arabidopsis wild-type (wt) plants and mutants impaired in Pi and carbohydrate status. Generally, P-deficiency resulted in increased Ugp expression and enhanced UGPase activity and protein content, as found for wt plants grown on P-deficient and complete nutrient solution, as well as for pho1 (P-deficient) mutants. Ugp was highly expressed in darkened leaves of pho1, but not wt plants; daily light exposure enhanced Ugp expression both in wt and pho mutants. The pho1 and pho2 (Pi-accumulating) mutations had little or no effect on leaf contents of glucose and fructose, regardless of light/dark conditions, whereas pho1 plants had much higher levels of sucrose and starch in the dark than pho2 and wt plants. The Ugp was up-regulated when leaves were fed with sucrose in wt plants, but the expression in pho2 background was much less sensitive to sucrose supply than in wt and pho1 plants. Expression of Ugp in pgm1 and sex1 mutants (impaired in starch/sugar content) was not dependent on starch content, and not tightly correlated with soluble sugar status. Okadaic acid (OKA) effectively blocked the P-starvation and sucrose-dependent expression of Ugp in excised leaves, whereas staurosporine (STA) had only a small effect on both processes (especially in -P leaves), suggesting that P-starvation and sucrose effects on Ugp are transmitted by pathways that may share similar components with respect to their (in) sensitivity to OKA and STA. The results of this study suggest that Ugp expression is modulated by an interaction of signals derived from P-deficiency status, sucrose content and dark/light conditions, and that light/sucrose and P-deficiency may have additive effects on Ugp expression.     

Mutations in the PHO80 Gene Confer Permeability to 5''-Mononucleotides in SACCHAROMYCES CEREVISIAE

Yeast mutants permeable to dTMP (tup) were selected and two new complementation groups (tup5 and tup7) were identified. Assay of the levels of both acid and alkaline phosphatase in cells grown under either repressing (5 mM PO4(-3) or derepressing (0.03 mM PO4(-3) conditions indicated that, in general, tup mutations cause cells to be defective in their regulation of phosphatase synthesis. In addition, three of the tup mutations (tup1, tup4 and tup7) displayed markedly elevated rates of inorganic phosphate transport. The tup7 locus was found to be tightly centromere-linked on the right arm of chromosome XV, and was shown to be allelic with the pho80 regulatory locus on the basis of both genetic and biochemical criteria. Analysis of other mutations known to affect phosphatase levels (pho) indicated that some also conferred permeability to dTMP. Possible allelic relationships between tup genes and certain of these pho mutations are discussed. Regardless of the culture conditions, wild-type strains were not permeable to dTMP; in contrast, it was found in the course of this work that normal yeast cells were permeable to dUMP and that dUMP permeability was regulated by the concentration of inorganic phosphate present in the medium used to grow the cells. Thus, permeability to 5'-mononucleotides appears to be under coordinate control with phosphatase synthesis.