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Query: UNIPROT:P06889 (Mol)
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Different Dictyostelium discoideum strains contain between 2 and 200 copies of a retrotransposable element termed DRE (Dictyostelium repetitive element). From the analysis of more than 50 elements, it can be concluded that DRE elements always occur 50 +/- 3 nucleotides upstream of tRNA genes. All analyzed clones contain DRE in a constant orientation relative to the tRNA gene, implying orientation specificity as well as position specificity. DRE contains two open reading frames which are flanked by nonidentical terminal repeats. Long terminal repeats (LTRs) are composed of three distinct modules, called A, B, and C. The tRNA gene-proximal LTR is characterized by one or multiple A modules followed by a single B module (AnB). With respect to the distal LTR, two different subforms of DRE have been isolated. The majority of isolated clones contains a distal LTR composed of a B module followed by a C module (BC), whereas the distal LTR of the other subform contains a consecutive array of a B module, a C module, a slightly altered A module, another B module, and another C module (BC.ABC). Full-length as well as smaller transcripts from DRE elements have been detected, but in comparison with the high copy number in D. discoideum strains derived from the wild-type strain NC4, transcription is rather poor.
Mol Cell Biol 1992 Jan
PMID:Structure of DRE, a retrotransposable element which integrates with position specificity upstream of Dictyostelium discoideum tRNA genes. 130 89

Surface cAMP receptors on Dictyostelium cells are linked to several second messenger systems and mediate multiple physiological responses, including chemotaxis and differentiation. Activation of the receptor also triggers events which desensitize signal transduction. These events include the following: 1) loss of ligand binding without loss of receptor protein; 2) phosphorylation of the receptor protein, which may lead to impaired signal transduction; 3) redistribution and degradation of the receptor protein; and 4) decrease of cyclic AMP (cAMP) receptor mRNA levels. These mechanisms of desensitization were investigated with the use of mutant synag7, with no activation of adenylyl cyclase; fgdC, with no activation of phospholipase C; and fgdA, with defects in both pathways. cAMP-induced receptor phosphorylation and loss of ligand binding activity was normal in all mutants. In contrast, cAMP-induced degradation of the receptor was absent in all mutants. The cAMP-induced decrease of cAMP-receptor mRNA levels was normal in mutant synag7, but absent in mutant fgdC. Finally, the cAMP analogue (Rp)-cAMPS induced loss of ligand binding without inducing second messenger responses or phosphorylation, redistribution, and degradation of the receptor. We conclude that 1) loss of ligand binding can occur in the absence of receptor phosphorylation; 2) loss of ligand binding and receptor phosphorylation do not require the activation of second messenger systems; 3) cAMP-induced degradation of the receptor may require the phosphorylation of the receptor as well as the activation of at least the synag7 and fgdC gene products; and 4) cAMP-induced decrease of receptor mRNA levels requires the activation of the fgdC gene product and not the synag7 gene product. These results imply that desensitization is composed of multiple components that are regulated by different but partly overlapping sensory transduction pathways.
Mol Biol Cell 1992 Jun
PMID:cAMP-induced desensitization of surface cAMP receptors in Dictyostelium: different second messengers mediate receptor phosphorylation, loss of ligand binding, degradation of receptor, and reduction of receptor mRNA levels. 132 48

cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and g alpha 2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in g alpha 2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or g alpha 2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTP gamma S) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and g alpha 2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require G alpha 2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via G alpha 2. 3) Neither cAR1 nor G alpha 2 is required for GTP gamma S-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein.
Mol Biol Cell 1992 Nov
PMID:Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium. 133 42

Previous studies have demonstrated that the Dictyostelium G alpha subunit G alpha 2 is essential for the cAMP-activation of adenylyl cyclase and guanylyl cyclase and that g alpha 2 null mutants do not aggregate. In this manuscript, we extend the analysis of the function of G alpha 2 in regulating downstream effectors by examining the in vivo developmental and physiological phenotypes of both wild-type and g alpha 2 null cells carrying a series of mutant G alpha 2 subunits expressed from the cloned G alpha 2 promoter. Our results show that wild-type cells expressing G alpha 2 subunits carrying mutations G40V and Q208L in the highly conserved GAGESG (residues 38-43) and GGQRS (residues 206-210) domains, which are expected to reduce the intrinsic GTPase activity, are blocked in multicellular development. Analysis of down-stream effector pathways essential for mediating aggregation indicates that cAMP-mediated activation of guanylyl cyclase and phosphatidylinositol-phospholipase C (PI-PLC) is almost completely inhibited and that there is a substantial reduction of cAMP-mediated activation of adenylyl cyclase. Moreover, neither mutant G alpha 2 subunit can complement g alpha 2 null mutants. Expression of G alpha 2(G43V) and G alpha 2(G207V) have little or no effect on the effector pathways and can partially complement g alpha 2 null cells. Our results suggest a model in which the dominant negative phenotypes resulting from the expression of G alpha 2(G40V) and G alpha 2(Q208L) are due to a constitutive adaptation of the effectors through a G alpha 2-mediated pathway. Analysis of PI-PLC in g alpha 2 null mutants and in cell lines expressing mutant G alpha 2 proteins also strongly suggests that G alpha 2 is the G alpha subunit that directly activates PI-PLC during aggregation. Moreover, overexpression of wild-type G alpha 2 results in the ability to precociously activate guanylyl cyclase by cAMP in vegetative cells, suggesting that G alpha 2 may be rate limiting in the developmental regulation of guanylyl cyclase activation. In agreement with previous results, the activation of adenylyl cyclase, while requiring G alpha 2 function in vivo, does not appear to be directly carried out by the G alpha 2 subunit. Our data are consistent with adenylyl cyclase being directly activated by either another G alpha subunit or by beta gamma subunits released on activation of the G protein containing G alpha 2.
Mol Biol Cell 1992 Jul
PMID:Amino acid substitutions in the Dictyostelium G alpha subunit G alpha 2 produce dominant negative phenotypes and inhibit the activation of adenylyl cyclase, guanylyl cyclase, and phospholipase C. 135 76

A Dictyostelium discoideum cDNA encoding an alpha-type subunit of casein kinase II was isolated, and its cDNA was used to study developmental expression of casein kinase II during the Dictyostelium life cycle. The 1.3-kb cDNA insert contained an open reading frame of 337 amino acids (M(r) 39,900). The deduced amino acid sequence has high homology with those of casein kinase II alpha subunits from other species. Genomic Southern blot analysis suggested that there is a single gene encoding casein kinase II alpha subunit in D. discoideum. Northern (RNA) blot analysis showed that the casein kinase II alpha-subunit gene is expressed constitutively as a 1.9-kb mRNA throughout vegetative growth and multicellular development. Casein kinase purified from normal vegetative cells contained a major protein band of approximately 36 kDa, which was recognized by antisera raised against rat testis casein kinase II. Comparison of the in vitro transcription/translation product of the alpha-subunit cDNA clone and the purified 36-kDa protein by partial proteolysis indicated that the isolated cDNA clone encodes the Dictyostelium casein kinase II alpha subunit. No protein corresponding to a beta subunit was detected in purified casein kinase. Immunoblot analysis using anti-rat casein kinase II sera showed that the alpha subunit of casein kinase II is expressed constitutively like its mRNA during the life cycle of D. discoideum. Casein kinase II activity measured by using a specific peptide substrate paralleled the level of alpha subunit detected by immunoblotting during the life cycle, with a maximum variation of approximately 2-fold. We were unable to obtain disruptants of the casein kinase II alpha gene, suggesting that there is a single casein kinase II alpha gene, which is essential for vegetative growth of D. discoideum.
Mol Cell Biol 1992 Dec
PMID:Molecular cloning of casein kinase II alpha subunit from Dictyostelium discoideum and its expression in the life cycle. 144

The phytochrome gene (phyCer) of the moss Ceratodon purpureus was isolated and characterized. phyCer is composed of three coding exons: exon I of 2035 bp, exon II of 300 bp and exon III of 1574 bp. The deduced polypeptide encoded by exon I and II exhibits substantial sequence homology to the conserved NH2-terminal chromophore domain of known phytochromes. In contrast, the COOH-terminal polypeptide encoded by exon III shows no sequence homology to any phytochrome molecule. phyCer most likely represents a single-copy gene and is expressed in a light-independent manner. From the DNA sequence analysis it can be deduced that the PhyCer polypeptide is composed of 1303 amino acids (including the starting Met) which predicts a molecular mass for PhyCer of 145 kDa. The polypeptide encoded in exon III exhibits striking homology within the 300 carboxy-terminal amino acids to the catalytic domain of protein kinases. The carboxy terminus of PhyCer was found to be most homologous to protein-tyrosine kinases of Dictyostelium discoideum and to the products of retroviral oncogenes which belong to the Raf-Mos serine/threonine kinase family. From the hydropathy profile PhyCer appears to be a soluble protein. The predicted structure suggests that PhyCer represents a soluble light-sensor protein kinase which is linked with a cellular phosphorylating cascade.
Plant Mol Biol 1992 Dec
PMID:Molecular cloning of a novel phytochrome gene of the moss Ceratodon purpureus which encodes a putative light-regulated protein kinase. 146 36

A type II casein kinase has been purified from the soluble fraction of Dictyostelium discoideum vegetative cells. The enzyme has been purified 370 fold and behaves catalytically as casein kinase type II, in the sense that it utilizes GTP as well as ATP as phosphoryl donors, it is inhibited by low heparin concentrations and phosphorylates a specific peptide for CK II. It is a tetramer of 38 kDa-subunits with catalytic activity and ability to autophosphorylate in vitro. The comparison of this activity with the nuclear enzyme previously purified from the same organism indicates that both have the same molecular structure. Both enzymes have antigenic determinants in common with casein kinase II from bovine thymus, suggesting a high degree of conservation during evolution. Studies on the activity of this enzyme during early differentiation, and in the transition from quiescence to proliferation shows an increase in specific activity suggesting a crucial role for the enzyme in this organism.
Mol Cell Biochem 1992 Dec 02
PMID:Purification of a soluble casein kinase II from Dictyostelium discoideum lacking the beta subunit: regulation during proliferation and differentiation. 148 55

The single gene encoding calmodulin in the eukaryotic microorganism Dictyostelium discoideum was cloned and sequenced. The gene was found to contain three introns, one lying immediately after the translation initiation codon. The deduced amino acid sequence indicated that Dictyostelium calmodulin contains 19 amino acid differences from vertebrate calmodulin, including extensions at both termini. Northern blot analysis showed that similar levels of calmodulin mRNA are present throughout growth and development of wild-type cells. A complete copy of the calmodulin cDNA was prepared, and an 87-base pair fragment complementary to the 5'-end of the calmodulin mRNA was subcloned into the Dictyostelium transformation vector pVEII, such that expression of the antisense transcript was driven by the discoidin I gamma promoter. Transformed cells were selected and maintained at low cell density, a condition resulting in minimal activity of the discoidin I promoter. High level expression was induced by allowing the transformants to reach high cell density or by growing them in the presence of medium conditioned by high density cells. Under these conditions, in which calmodulin mRNA and protein levels were reduced about twofold, the calmodulin antisense transformants lost the ability to complete cytokinesis. A contractile ring formed and constricted, but the midbody linking daughter cells failed to break. The resulting cell population contained multinucleated cells and networks of cells connected by cytoplasmic bridges. Normal cell division was restored when the cells were diluted to low density. These observations have identified a new point at which calmodulin may regulate cell cleavage.
Mol Biol Cell 1992 Dec
PMID:Inducible expression of calmodulin antisense RNA in Dictyostelium cells inhibits the completion of cytokinesis. 149 36

We used molecular genetic approaches to delete 521 amino acid residues from the proximal portion of the Dictyostelium myosin II tail. The deletion encompasses approximately 40% of the tail, including the S2-LMM junction, a region that in muscle myosin II has been proposed to be important for contraction. The functions of the mutant myosin II are indistinguishable from the wild-type myosin II in our in vitro assays. It binds to actin in a typical rigor configuration in the absence of ATP and it forms filaments in a normal salt-dependent manner. In an in vitro motility assay, both monomeric and filamentous forms of the mutant myosin II translocate actin filaments at 2.4 microns/s at 30 degrees C, similar to that of wild-type myosin II. The mutant myosin II is also functional in vivo. Cells expressing the mutant myosin II in place of the native myosin II perform myosin II-dependent activities such as cytokinesis and formation of fruiting bodies, albeit inefficiently. Growth of the mutant cells in suspension gives rise to many large multinucleated cells, demonstrating that cytokinesis often fails. The majority of the fruiting bodies are also morphologically abnormal. These results demonstrate that this region of the myosin II tail is not required for motile activities but its presence is necessary for optimum function in vivo.
Mol Biol Cell 1992 Dec
PMID:A Dictyostelium myosin II lacking a proximal 58-kDa portion of the tail is functional in vitro and in vivo. 149 38

We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO1 operator fragment was inserted at three different positions in front of a tRNA(Glu) (Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a beta-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 micrograms/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in beta-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal.
Mol Cell Biol 1992 Sep
PMID:Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene. 150 1


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