Dictyostelium nucleoside diphosphate kinase highly homologous to Nm23 and Awd proteins involved in mammalian tumor metastasis and Drosophila development.

Two complementary DNAs (cDNAs) previously isolated, one by functional screening and the other by immunological screening of a Dictyostelium discoideum expression library, encode two proteins, Gip17 and Guk7.2, sharing 71% homology. In the present study, we found that the expression of their messenger RNAs (mRNAs) is developmentally regulated, with a sharp decrease during the first hours of differentiation. The Gip17 protein was purified to homogeneity from D. discoideum amoebas and from recombinant Escherichia coli and was conclusively identified as a nucleoside diphosphate (NDP) kinase. NDP kinases play a major role in synthesis of nucleoside triphosphates and, in many systems, are found associated with guanosine triphosphate (GTP)-binding proteins. We found the Gip17 protein to be 77% homologous to the human Nm23 protein and 75% homologous to the Drosophila melanogaster Awd protein. The levels of murine and human nm23 mRNA and Nm23 protein are significantly reduced in tumor cells of high metastatic potential, suggesting that Nm23 is involved in suppression of mammalian tumor metastasis, and mutants of the awd gene exhibit widespread development abnormalities, suggesting that Awd is involved in D. melanogaster development. The high percentage of homology of the Gip17 and Guk7.2 proteins with the Nm23 and Awd proteins indicates that Nm23 and Awd also have nucleoside diphosphate kinase activity. Possible modulations in the activity of this metabolic enzyme could be related to the altered metabolism of tumor cells and the control of metastatic potential. Our results point to an unexpected role of NDP kinase in development, growth control, and oncogenic transformation.

Nucleotide sequence of the gene for ribosomal protein S17 from Dictyostelium discoideum.

The nucleotide sequence of the gene for the Dictyostelium homologue of eukaryotic ribosomal protein S17 has been assembled from cDNA and genomic DNA clones. The predicted primary structure of the S17 protein displays a similar level of sequence identity with its counterparts from higher eukaryotes (53%) as other Dictyostelium ribosomal proteins. Although Dictyostelium genes usually are organized in a rather simple manner, the rps17 gene harbors two introns. One of them, located immediately 3' from the ATG initiator codon, appears to be ubiquitously conserved in eukaryotic rps17 genes.

Unconventional mRNA processing in the expression of two calcineurin B isoforms in Dictyostelium.

The genome of Dictyostelium discoideum contains a single gene (cnbA) for the regulatory (B) subunit of the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin (CN). Two mRNA species and two protein products differing in size were found. The apparent molecular masses of the protein isoforms corresponded to translation products starting from the first and second AUG codons of the primary transcript, respectively. The smaller mRNA and protein isoforms accumulated during early differentiation of the cells. Whereas the amount of the higher molecular mass protein isoform remained constant throughout development, the larger mRNA disappeared to virtually undetectable levels during aggregation. 5'RACE amplification of the smaller transcript yielded cDNAs lacking the 5' non-translated region and the first ATG initiator codon. Expression of truncated cDNAs and various chimeric genes encoding CNB-green fluorescent protein fusions in Dictyostelium indicate that the mature cnbA transcript is processed by an unconventional mechanism that leads to truncation of the 5' untranslated region and at least the first AUG initiator codon, and to utilization of the second AUG codon for translation initiation of the small CNB isoform. Determinants for this processing mechanism reside within the coding region of the cnbA gene.

On the role of calcium during chemotactic signalling and differentiation of the cellular slime mould Dictyostelium discoideum.

Transient cytosolic calcium elevations are required for chemotaxis and differentiation of Dictyostelium discoideum since Ca2+ chelating buffers introduced into the cells by scrape loading inhibited motility as well as orientation in a Ca2+ specific manner. Ca2+ changes are provided either by intrinsic cytosolic Ca2+ oscillations that can be determined as periodic Ca2+ efflux, or by receptor-mediated Ca2+ liberation from the InsP3-sensitive store and Ca2+ influx. Cytosolic Ca2+ homeostasis as well as oscillations seem to be regulated by two different Ca2+ stores, the acidosomes and the InsP3-sensitive store, both of which are dependent on Ca2+ pumps and V-type H+ ATPases. Ca2+ transients are sensed by calmodulin-binding proteins. The latter have been detected in Dictyostelium by 125I-calmodulin labeling. A calmodulin-dependent protein phosphatase, calcineurin A, was cloned, sequenced, purified and characterized biochemically. Overproduction of calcineurin A as well as antisense constructs will help to the elucidation of its function in signal transduction. Surprisingly, protein synthesis is also controlled by Ca2+/calmodulin. An integral ribosomal protein of the 60S subunit, L19, proved to be a calmodulin-binding protein and calmodulin antagonists of different classes, inhibited in vitro translation of Dictyostelium and wheat germ extracts.

Prophage lambda libraries for isolating cDNA clones by functional screening.

Isolation of cDNA clones from lambda gt11 phage libraries by functional screening is limited by the low amount of lacZ-cDNA-encoded fusion protein synthesized in an isolated phage plaque. The amount of specific cDNA-encoded protein can be significantly enhanced by expression in bacterial colonies rather than phage plaques. Escherichia coli was lysogenized with a lambda gt11 cDNA expression library from Dictyostelium discoideum. Bacteria were selected for the presence of the lambda gt11 prophage by elimination of nonlysogenic parental cells with a lambda cI phage. The usefulness of the lysogen library was demonstrated by immuno-screening and functional screening with two different radiolabeled ligands. cDNA clones encoding a well-characterized D. discoideum protein, the regulatory subunit of the cAMP-dependent protein kinase, were isolated by screening the lysogen library with antibodies. Clones encoding this protein could also be identified by functional screening with [3H]cAMP, demonstrating that the limit of detection of positive clones by ligand screening is at least an order of magnitude lower for the lysogen library than for the corresponding phage library. We have subsequently used the lysogen library to isolate cDNA clones encoding calmodulin-binding protein(s) from D. discoideum by functional screening with [125I]calmodulin. For these clones, screening of the corresponding phage library had previously been found unsuccessful.

Chloroperoxidase-encoding gene from Pseudomonas pyrrocinia: sequence, expression in heterologous hosts, and purification of the enzyme.

The nucleotide sequence of a 1.5-kb fragment of Pseudomonas pyrrocinia DNA containing the chloroperoxidase(CPO)-encoding gene (cpo) and its flanking regions was determined. The cpo codes for a protein of 278 amino acids (aa). The mature enzyme contains no N-terminal methionine, so that the CPO monomer consists of 277 aa with a calculated M(r) of 30,304. Expression studies showed that the cpo from P. pyrrocinia is functionally expressed in Escherichia coli and Streptomyces lividans. Based on the overproduction of the CPO in E. coli, a novel and simple purification procedure was developed allowing the isolation of about 800-fold more CPO per gram of cells than was originally isolated from P. pyrrocinia. Comparison with the aa sequence of the bromoperoxidase BPO-A2 from S. aureofaciens ATCC10762 revealed an identity of 38%.

Gene isolation by direct in situ cAMP binding.

The regulatory subunit of the cAMP-dependent protein kinase expressed in clones isolated by immunoscreening of a lambda gt11 cDNA library from Dictyostelium discoideum exhibits high affinity for cAMP [Mutzel et al., Proc. Natl. Acad. Sci. USA 84 (1987) 6-10]. Based on this property, we have developed a screening procedure to detect in situ cAMP-binding activity directly on phage plaques transferred to nitrocellulose filters. Highly radioactive cAMP was synthesized using [alpha-32P]ATP at 3000 Ci/mmol as the substrate of purified adenylate cyclase from Bordetella pertussis. Filter replicas of the library plated at 3 X 10(4) pfu/dish, were incubated in the presence of 2 nM [32P]cAMP and then washed thoroughly. Three clones out of 1.2 X 10(5) were detected, all of which coded for the regulatory subunit, as judged by hybridization with a specific DNA probe. The cAMP binding to the purified clones was characterized in situ by displacement with specific analogues. The ability to displace labelled cAMP was in accord with the affinities of the analogues previously reported for the regulatory subunit of the Dictyostelium cAMP-dependent protein kinase. We are able to detect fmol levels of regulatory subunit contained in phage plaques and therefore the method could be used to screen libraries from other organisms for proteins exhibiting high affinities for cyclic nucleotides.

Elongation in a Dictyostelium in vitro translation system is affected by calmodulin antagonists.

We have previously shown that the Dictyostelium discoideum ribosomal protein L19 specifically binds Ca2+/calmodulin [Sonneman et al. (1991) J. Biol. Chem. 266, 23091-23096]. To investigate the role of calmodulin in the regulation of protein synthesis, we have now established an in vitro protein synthesizing system from Dictyostelium cells which can elongate polypeptide chains with high efficiency. Various calmodulin antagonists affected translation in this system. The inhibitory effects of the antagonists could be partially reversed by addition of calmodulin. A monoclonal antibody against D. discoideum calmodulin also specifically inhibited protein synthesis. Similar effects of calmodulin antagonists were found in a standard wheat germ in vitro translation system.

Long term adaptation of a microbial population to a permanent metabolic constraint: overcoming thymineless death by experimental evolution of Escherichia coli.

BACKGROUND: To maintain populations of microbial cells under controlled conditions of growth and environment for an indefinite duration is a prerequisite for experimentally evolving natural isolates of wild-type species or recombinant strains. This goal is beyond the scope of current continuous culture apparatus because these devices positively select mutants that evade dilution, primarily through attachment to vessel surfaces, resulting in persistent sub-populations of uncontrollable size and growth rate. RESULTS: To overcome this drawback, a device with two growth chambers periodically undergoing transient phases of sterilization was designed. The robustness of this device was assessed by propagating an E. coli strain under permanent thymine starvation for over 880 days, i.e. metabolic conditions notoriously known to lead to cell death and clogging of cultivation vessels. Ten thousand generations were required to obtain a descendant lineage that could resist thymine starvation and had recovered wild-type growth rate. CONCLUSIONS: This approach provides a technological framework for the diversification and improvement of microbial strains by long-term adaptation to inescapable metabolic constraints. An E. coli strain that is totally resistant to thymineless death was selected.

Ca2+/calmodulin-binding proteins in Dictyostelium discoideum.

We have initiated a systematic study of Ca2+/calmodulin-regulated enzymes in the cellular slime mold Dictyostelium discoideum. Using 125I-labelled D. discoideum calmodulin (CaM) as a functional probe, several Ca2+/CaM-binding proteins were detected in crude cell lysates. Proteins with apparent molecular weights of 22 kDa and 78-80 kDa, respectively, were found in the soluble fraction. In addition, membrane-bound high molecular weight CaM-binding proteins were identified. Binding of CaM to all of the proteins required the presence of Ca2+ ions and competed efficiently with nonradioactive CaM from both Dictyostelium and bovine brain. The CaM antagonists melittin, W-7 and R24571 inhibited CaM binding. With a functional cloning approach, we previously obtained cDNA clones by screening a lambda gt11 lysogen expression library; in this paper, we report the analysis of CaM-binding activity by one of the recombinant cDNA clones in Escherichia coli. When rabbit antiserum was raised against it, the antiserum recognized a 78-80-kDa protein in Dictyostelium extracts which comigrated on SDS-polyacrylamide gels with 78-80-kDa CaM-binding activity.

Overexpression, purification and characterization of Dictyostelium calcineurin A.

The catalytic subunit of Ca2+/calmodulin-dependent protein phosphatase (calcineurin A) was overexpressed about 50-fold in Dictyostelium discoideum cells transformed with a vector containing the cDNA for D. discoideum calcineurin A under control of the actin-6 promoter. In crude lysates from the overexpressing cell line, high Ca2+/calmodulin-stimulated phosphatase activity was detected. Calcineurin A was purified by anion exchange chromatography and calmodulin-Sepharose affinity chromatography, and the enzymatic activity of the isolated protein was characterized. Its phosphatase activity was strictly dependent on the addition of divalent metal ions such as Mg2+ or Mn2+. Disulphide-reducing agents increased the activity more than 10-fold. Ca2+/calmodulin stimulated the activity by a factor of 2.5-5. Despite the high extra Ca2+/calmodulin-dependent phosphatase activity, the overexpressing cell line showed no phenotypic aberrations.

Convergent evolution of amino acid usage in archaebacterial and eubacterial lineages adapted to high salt.

Chemical composition and physical properties of the total protein of Haloferax mediterranei, a halophilic archaebacterium requiring high salt concentration for growth, of Halomonas elongata, a halotolerant eubacterium able to grow at any concentration of salt, and of Escherichia coli B, a eubacterium related to H. elongata, unable to grow at high salt concentration, were compared using robust standard biochemical methods. The distribution of amino acid abundancies in the bulk protein from H. elongata was found to be intermediate between that from H. mediterranei and that from E. coli. The two high-salt-adapted organisms displayed an enrichment in aspartic acid and glutamic acid together with an impoverishment in lysine as compared to E. coli. This signature in amino acid usage is reflected in the charge distribution of proteins, as revealed by anion exchange chromatography of crude cell extracts. Since H. elongata diverged from H. mediterranei more than three billion years ago, the resemblance of their amino acid usages can be interpreted as a convergent imprint of their common habitats onto the chemical constitution of their proteins.

A metabolic prototype for eliminating tryptophan from the genetic code.

We set out to reduce the chemical constitution of a living organism to 19 amino acids. A strain was constructed for reassigning the tryptophan codon UGG to histidine and eliminating tryptophan from Escherichia coli. Histidine codons in the gene for an essential enzyme were replaced with tryptophan codons and the restoration of catalytic activity by missense suppressor His-tRNA bearing a CCA anticodon was selected. We used automated cultivation to assess the stability of this genetic construct during evolution. Histidine to tryptophan mutation at codon 30 in the transketolase gene from yeast and its cognate suppressor tRNA were stably propagated in a tktAB deletant of E. coli over 2500 generations. The ratio of histidine misincorporation at tryptophan sites in the proteome increased from 0.0007 to 0.03 over 300 days of continuous culture. This result demonstrated that the genetic code can be forced to evolve by permanent metabolic selection.

Cytosolic nucleoside diphosphate kinase associated with the translation apparatus may provide GTP for protein synthesis.

Elongation of nascent polypeptides in a Dictyostelium discoideum in vitro translation system did not require the addition of ATP and GTP when creatine phosphate and creatine phosphokinase were present. However, depletion of the exogenous energy supply completely abolished incorporation of amino acids. Addition of dTTP, a nucleoside triphosphate that can be utilized by nucleoside diphosphate kinase (NDP kinase) to phosphorylate endogenous ADP and GDP, partially restored protein synthesis. Dictyostelium ribosomes were found to contain NDP kinase activity that could not be released by 1 M KCl. Thermal denaturation studies, specific inhibition with antibodies, and Western blotting identify the activity as cytosolic NDP kinase. These data support the idea that GTP can be fed into the translation machinery efficiently by NDP kinase associated with active ribosomes.

Vectors for expression of truncated coding sequences in Escherichia coli.

We describe the construction of vectors for expressing in Escherichia coli DNA fragments obtained by progressive deletions of DNA inserts in single-stranded sequencing vectors as M13 or pTZ according to the methode of Dale et al. (Plasmid 1985, 13, 31-40). These vectors, pIMS1, pIMS5, and pIMS6, harbor all of the elements required for the regulated expression of any open reading frame flanked by EcoRI restriction sites. The encoded peptides contain only a few vector-derived amino acids. A method is described for direct selection of recombinant clones by in situ RNA hybridization. The properties of the expression vector have been analyzed with a DNA deletion series obtained from the cDNA coding for the regulatory subunit of Dictyostelium discoideum cAMP-dependent protein kinase.

tRNA (adenine-N1)-methyltransferase from Dictyostelium discoideum. Purification, characterization and developmental changes in activity.

An enzyme activity transferring methyl groups from S-adenosylmethionine to endogenous tRNA was detected in the cytosol of aggregative Dictyostelium discoideum amoebae. This enzyme was purified more than 1000-fold and was characterized as a tRNA (adenine-N1-)-methyltransferase. Kinetic analysis yielded a K0.5 for S-adenosylmethionine of 0.27 microM and competitive inhibition by S-adenosylhomocysteine showed an I0.5 of 0.26 microM. The tRNA methyltransferase activity was stimulated by monovalent cations and the pH optimum was 7.3. tRNAs isolated from D. discoideum as well as from other eucaryotic sources could be methylated only to a minor extent. In contrast, Escherichia coli tRNA accepted up to 0.6 mol methyl group/mol tRNA, suggesting that the target nucleotide is unmethylated in procaryotic tRNA, but is commonly methylated in tRNAs from eucaryotic organisms. The activity of the methyltransferase increased 4-6-fold during cell differentiation from the vegetative to the aggregative stage.

A ribosomal calmodulin-binding protein from Dictyostelium.

Using 125I-calmodulin as a probe, we have recently identified specific Ca2+/calmodulin-binding proteins in cell extracts from the cellular slime mold, Dictyostelium discoideum: a major 22-kDa activity, a soluble 78/80-kDa protein, and several membrane-associated high Mr proteins (Winckler, T., Dammann, H., and Mutzel, R. (1991) Res. Microbiol. 142, 509-519). cDNA clones for at least two of these proteins have been isolated by ligand screening of a lambda gt11 prophage expression library. Antibodies directed against the lacZ-cDNA-encoded fusion protein from one of the clones recognized a single 22-kDa component in D. discoideum extracts which comigrated with the endogenous 22-kDa calmodulin-binding protein. The cDNA-derived nucleotide sequence predicts a protein of Mr 21,659 with 56% sequence identity (69% homology) with rat ribosomal protein L19. The endogenous 22-kDa calmodulin-binding activity was associated with ribosomes. It was found to be an integral constituent of the large ribosomal subunit, since it cosedimented with 60 S ribosomal subunits in sucrose density gradients in the presence of 0.5 M NH4Cl. Our observations point to a physiological role for calmodulin in the Ca2+ regulation of eukaryotic protein synthesis. Support for this comes from recent studies showing inhibition of protein synthesis by calmodulin antagonists in Ehrlich ascites tumor cells (Kumar, R. V., Panniers, R., Wolfman, A., and Henshaw, E.C. (1991) Eur. J. Biochem. 195, 313-319).

Primary structure, expression and developmental regulation of a Dictyostelium calcineurin A homologue.

cDNA clones for the catalytic subunit of Ca2+/calmodulin(CaM)-dependent protein phosphatase (calcineurin A, protein phosphatase 2B) from Dictyostelium discoideum were isolated by functional screening of a lambda gt11 lysogen expression library with labeled Dictyostelium CaM. A complete cDNA of 2146 bp predicts a protein of 623 amino acids with homology to calcineurin A from other organisms and a similar molecular architecture. However, the Dictyostelium protein contains N-terminal and C-terminal extra domains causing a significantly higher molecular mass than found in any of its known counterparts. Recombinant Dictyostelium calcineurin A was purified from Escherichia coli cells and shown to display similar enzymatic properties as the enzyme from other sources. On Western blots specific antibodies against the protein recognized a band of approximately 80 kDa that migrated with an endogenous CaM-binding activity. Both the mRNA for calcineurin A and the protein are expressed during the growth phase. During early development the abundance of the protein is reduced and then increases to peak after 10 h of starvation, when tight aggregates have formed.

Ca(2+)/calmodulin-independent activation of calcineurin from Dictyostelium by unsaturated long chain fatty acids.

This study describes a novel mode of activation for the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin. Using purified calcineurin from Dictyostelium discoideum we found a reversible, Ca(2+)/calmodulin-independent activation by the long chain unsaturated fatty acids arachidonic acid, linoleic acid, and oleic acid, which was of the same magnitude as activation by Ca(2+)/calmodulin. Half-maximal stimulation of calcineurin occurred at fatty acid concentrations of approximately 10 microM with either p-nitrophenyl phosphate or RII phosphopeptide as substrates. The methyl ester of arachidonic acid and the saturated fatty acids palmitic acid and arachidic acid did not activate calcineurin. The activation was shown to be independent of the regulatory subunit, calcineurin B. Activation by Ca(2+)/calmodulin and fatty acids was not additive. In binding assays with immobilized calmodulin, arachidonic acid inhibited binding of calcineurin to calmodulin. Therefore fatty acids appear to mimic Ca(2+)/calmodulin action by binding to the calmodulin-binding site.