RESUMO
Self-organization of cells is central to a variety of biological systems and physical concepts of condensed matter have proven instrumental in deciphering some of their properties. Here we show that microphase separation, long studied in polymeric materials and other inert systems, has a natural counterpart in living cells. When placed below a millimetric film of liquid nutritive medium, a quasi two-dimensional, high-density population of Dictyostelium discoideum cells spontaneously assembles into compact domains. Their typical size of 100 µm is governed by a balance between competing interactions: an adhesion acting as a short-range attraction and promoting aggregation, and an effective long-range repulsion stemming from aerotaxis in near anoxic condition. Experimental data, a simple model and cell-based simulations all support this scenario. Our findings establish a generic mechanism for self-organization of living cells and highlight oxygen regulation as an emergent organizing principle for biological matter.
Assuntos
Dictyostelium , Dictyostelium/fisiologia , Quimiotaxia/fisiologiaRESUMO
Although spatiotemporal changes of oxygen in a microenvironment are known to affect the cellular dynamics of various eukaryotes, the details are not fully understood. Here, we describe the aerotaxis and aerokinesis of Dictyostelium discoideum (Dd), which has long been employed as a model organism for eukaryotic cells. We developed a microfluidic device capable of time-lapse observation of cultured cells while controlling oxygen concentrations in microchannels. Migratory behaviors of Dd were observed and quantitatively evaluated under an oxygen concentration gradient from 0% to 21% O2, as well as in various uniform oxygen conditions. In a hypoxic region within the oxygen concentration gradient, Dd migrated toward regions of higher oxygen concentration at increased velocity, which was independent of cell density. Observed under uniform oxygen concentrations of 1%, 2%, 3%, and 21%, the migration velocity of Dd increased significantly in hypoxic environments of 2% O2 or less. Thus, Dd shows aerotaxis, directed by the oxygen concentration gradient, and simultaneously shows aerokinesis, changing the migration velocity according to the oxygen concentration itself.
Assuntos
Dictyostelium , Linhagem Celular , Células Cultivadas , Quimiotaxia , OxigênioRESUMO
The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.
Assuntos
Dictyostelium/genética , Genoma , Genômica , Comportamento Social , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Composição de Bases , Adesão Celular/genética , Movimento Celular/genética , Centrômero/genética , Sequência Conservada/genética , Elementos de DNA Transponíveis/genética , DNA Ribossômico/genética , Dictyostelium/citologia , Dictyostelium/enzimologia , Dictyostelium/metabolismo , Células Eucarióticas/metabolismo , Duplicação Gênica , Transferência Genética Horizontal/genética , Humanos , Dados de Sequência Molecular , Filogenia , Proteoma , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA de Transferência/genética , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA , Transdução de Sinais/genética , Telômero/genéticaRESUMO
It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA- mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA- PKA-C(over) strain. When developed at high cell densities, acrA- acaA- PKA-C(over) cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high.
Assuntos
Adenilil Ciclases/metabolismo , Dictyostelium/crescimento & desenvolvimento , Adenilil Ciclases/genética , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Genes de ProtozoáriosRESUMO
The gdt1 gene is a negative regulator of the growth-differentiation-transition (GDT) in Dictyostelium. gdt1- cells express the GDT marker discoidin earlier and at higher levels and prematurely enter the differentiation pathway. Protein kinase A is a positive regulator of the GDT and is required for multicellular development. Disruption of the PKA catalytic subunit or overexpression of a constitutively active mutant of the regulatory subunit results in cells which do not form multicellular aggregates and which show strongly reduced levels of discoidin. We have created PKA-/gdt1- double mutants and show that these display high levels of discoidin expression but no aggregation, suggesting that gdt1 may be a downstream target of PKA in a branched signaling cascade initiating differentiation. Data obtained with the PKA inhibitor H89 support these result: in wild type cells H89 inhibits discoidin expression while in gdt1- mutants there is no obvious effect. However, since PKA-/gdt1- cells display less discoidin expression than the single gdt1 mutant, we propose that PKA and gdt1 are in two parallel interacting pathways. To get insight into the mechanism how PKA may block gdt1, we have tested two putative PKA phosphorylation sites in the protein and found that one of them is efficiently phosphorylated by PKA in vitro. A model for the interplay between PKA and gdt1 during the growth-differentiation-transition is discussed.
Assuntos
Diferenciação Celular/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Lectinas , Proteínas de Membrana/metabolismo , Animais , Divisão Celular/fisiologia , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Dictyostelium/genética , Discoidinas , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Proteínas de Membrana/genética , Mutação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Discoidin I expression was used as a marker to screen for mutants affected in the growth-differentiation transition (GDT) of Dictyostelium. By REMI mutagenesis we have isolated mutant 2-9, an overexpressor of discoidin I. It displays normal morphogenesis but shows premature entry into the developmental cycle. The disrupted gene was denominated gdt1. The mutant phenotype was reconstructed by disruptions in different parts of the gene, suggesting that all had a complete loss of function. gdt1 was expressed in growing cells; the levels of protein and mRNA appear to increase with cell density and rapidly decrease with the onset of development. gdt1 encodes a 175-kDa protein with four putative transmembrane domains. In the C terminus, the derived amino acid sequence displays some similarity to the catalytic domain of protein kinases. Mixing experiments demonstrate that the gdt1(-) phenotype is cell autonomous. Prestarvation factor is secreted at wild-type levels. The response to folate, a negative regulator of discoidin expression, was not impaired in gdt1 mutants. Cells that lack the G protein alpha2 display a loss of discoidin expression and do not aggregate. gdt1(-)/Galpha2(-) double mutants show no aggregation but strong discoidin expression. This suggests that gdt1 is a negative regulator of the GDT downstream of or in a parallel pathway to Galpha2.
Assuntos
Dictyostelium/citologia , Dictyostelium/genética , Lectinas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Diferenciação Celular/genética , Divisão Celular/genética , Dictyostelium/metabolismo , Discoidinas , Ácido Fólico/metabolismo , Regulação da Expressão Gênica , Técnicas Genéticas , Dados de Sequência Molecular , Mutação , Fenótipo , Análise de Sequência de DNARESUMO
A variety of extracellular signals lead to the accumulation of cAMP which can act as a second message within cells by activating protein kinase A (PKA). Expression of many of the essential developmental genes in Dictyostelium discoideum are known to depend on PKA activity. Cells in which the receptor-coupled adenylyl cyclase gene, acaA, is genetically inactivated grow well but are unable to develop. Surprisingly, acaA(-) mutant cells can be rescued by developing them in mixtures with wild-type cells, suggesting that another adenylyl cyclase is present in developing cells that can provide the internal cAMP necessary to activate PKA. However, the only other known adenylyl cyclase gene in Dictyostelium, acgA, is only expressed during germination of spores and plays no role in the formation of fruiting bodies. By screening morphological mutants generated by Restriction Enzyme Mediated Integration (REMI) we discovered a novel adenylyl cyclase gene, acrA, that is expressed at low levels in growing cells and at more than 25-fold higher levels during development. Growth and development up to the slug stage are unaffected in acrA(-) mutant strains but the cells make almost no viable spores and produce unnaturally long stalks. Adenylyl cyclase activity increases during aggregation, plateaus during the slug stage and then increases considerably during terminal differentiation. The increase in activity following aggregation fails to occur in acrA(-) cells. As long as ACA is fully active, ACR is not required until culmination but then plays a critical role in sporulation and construction of the stalk.
Assuntos
Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Dictyostelium/crescimento & desenvolvimento , Chaperonas Moleculares , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Clonagem Molecular , Dictyostelium/classificação , Dictyostelium/genética , Regulação da Expressão Gênica no Desenvolvimento , Histidina Quinase , Dados de Sequência Molecular , Mutagênese , Mutação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
SDF-2 is a peptide released by prestalk cells during culmination that stimulates prespore cells to encapsulate. Genetic evidence indicates that the response is dependent on the dhkA gene. This gene encodes a member of the histidine kinase family of genes that functions in two-component signal transduction pathways. The sequence of the N-terminal half of DhkA predicts two hydrophobic domains separated by a 310-amino-acid loop that could bind a ligand. By inserting MYC6 epitopes into DhkA, we were able to show that the loop is extracellular while the catalytic domain is cytoplasmic. Cells expressing the MYC epitope in the extracellular domain of DhkA were found to respond only if induced with 100-fold-higher levels of SDF-2 than required to induce dhkA+ cells; however, they could be induced to sporulate by addition of antibodies specific to the MYC epitope. To examine the enzymatic activity of DhkA, we purified the catalytic domain following expression in bacteria and observed incorporation of labelled phosphate from ATP consistent with histidine autophosphorylation. Site-directed mutagenesis of histidine1395 to glutamine in the catalytic domain blocked autophosphorylation. Furthermore, genetic analyses showed that histidine1395 and the relay aspartate2075 of DhkA are both critical to its function but that another histidine kinase, DhkB, can partially compensate for the lack of DhkA activity. Sporulation is drastically reduced in double mutants lacking both DhkA and DhkB. Suppressor studies indicate that the cyclic AMP (cAMP) phosphodiesterase RegA and the cAMP-dependent protein kinase PKA act downstream of DhkA.
Assuntos
Dictyostelium/fisiologia , Proteínas de Membrana/metabolismo , Peptídeos , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Proteínas de Protozoários , Animais , Domínio Catalítico , Diferenciação Celular , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Dictyostelium/efeitos dos fármacos , Dictyostelium/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Quinases/genética , Proteínas/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The avian retroviral v-myb gene and its cellular homologues throughout the animal and plant kingdoms contain a conserved DNA binding domain. We have isolated an insertional mutant of Dictyostelium unable to switch from slug migration to fruiting body formation i.e. unable to culminate. The gene that is disrupted, mybC, codes for a protein with a myb-like domain that is recognized by an antibody against the v-myb repeat domain. During development of myb+ cells, mybC is expressed only in prestalk cells. When developed together with wild-type cells mybC- cells are able to form both spores and stalk cells very efficiently. Their developmental defect is also bypassed by overexpressing cAMP-dependent protein kinase. However even when their defect is bypassed, mybC null slugs and culminates produce little if any of the intercellular signalling peptides SDF-1 and SDF-2 that are believed to be released by prestalk cells at culmination. We propose that the mybC gene product is required for an intercellular signaling process controlling maturation of stalk cells and spores and that SDF-1 and/or SDF-2 may be implicated in this process.
Assuntos
Proteínas de Caenorhabditis elegans , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dictyostelium/fisiologia , Mutação , Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Núcleo Celular/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Análise Mutacional de DNA , Proteínas de Ligação a DNA/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Hexanonas , Hidrocarbonetos Clorados , Dados de Sequência Molecular , Proteínas Oncogênicas v-myb , Proteínas Oncogênicas de Retroviridae/fisiologia , Homologia de Sequência de AminoácidosRESUMO
Unsaturated fatty acids are essential lipid components of Mucor rouxii. Gamma-linolenic acid (GLA) is synthesized via the desaturase enzymes: delta9-desaturase catalyzes mono-unsaturated fatty acids that are utilized as substrate for GLA biosynthesis. We cloned and characterized a M. rouxii gene highly homologous to delta9-desaturase genes. This sequence encodes for a protein of 452 amino acids and contains two introns of 60 and 61 nucleotides. Delta9-desaturase of M. rouxii is expressed during cell growth when cells are subjected to temperature shifts. At 30 degrees C, the mRNA level of late log phase is about 6.4-fold higher than that of early log phase. A shift from 30 to 15 degrees C induced transcription of delta9-desaturase gene in both early and late log phases. However, the pattern of increased transcription by cold induction varied depending on growth conditions: transcription of late log phase is higher than that of early log phase. These results indicate that cell growth and low temperature influence the expression of delta9-desaturase gene and fatty acid composition of M. rouxii.
Assuntos
Genes Fúngicos , Mucor/enzimologia , Mucor/genética , Estearoil-CoA Dessaturase/genética , Sequência de Aminoácidos , Sequência de Bases , Divisão Celular , Sequência Conservada , Primers do DNA/genética , Ácidos Graxos/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Metabolismo dos Lipídeos , Dados de Sequência Molecular , Mucor/crescimento & desenvolvimento , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Estearoil-CoA Dessaturase/química , TemperaturaRESUMO
SDF-1 and SDF-2 are peptides that promote terminal spore differentiation under submerged conditions. The present study shows that they accumulate differentially and are released during the development of wild-type cells and can promote spore formation in cells disaggregated from wild-type culminants. SDF-1 accumulates during the slug stage and is released in a single burst at the onset of culmination while SDF-2 accumulates during early culmination and is released in a single burst from mid-culminants. The effects of SDF-1 and SDF-2 on stalk cell formation in cell monolayers were investigated. SDF-1 by itself induces stalk cell formation in some strains and also synergizes with the stalk-cell-inducing factor, DIF-1. cAMP has an inhibitory effect on stalk cell formation when either DIF-1 or SDF-1 are present on their own but is almost not inhibitory when both are present. SDF-2 alone does not induce stalk cell formation and appears to inhibit the response to DIF-1. At the same time, it increases the extent of vacuolization of the stalk cells that are produced. We propose that the release of SDF-1 and then of SDF-2 may mark irreversible steps in the developmental programme associated, respectively, with culmination and spore maturation.
Assuntos
Proteínas de Caenorhabditis elegans , Dictyostelium/fisiologia , Proteínas , Proteínas de Protozoários/metabolismo , Animais , Proteínas de Transporte/farmacologia , Agregação Celular , Diferenciação Celular , Linhagem Celular , AMP Cíclico/farmacologia , Dictyostelium/citologia , Dictyostelium/metabolismo , Relação Dose-Resposta a Droga , Proteínas de Helminto/farmacologia , Hexanonas , Hidrocarbonetos Clorados , Estágios do Ciclo de Vida , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/farmacologia , Esporos/citologia , Esporos/crescimento & desenvolvimento , Esporos/metabolismo , Fatores de TempoRESUMO
The catalytic subunit of the cAMP-dependent protein kinase (PKA) from Dictyostelium discoideum contains several domains, including an unusually long N-terminal extension preceding a highly conserved catalytic core. We transformed the aggregationless PkaC-null strain with several deletion constructs of both domains. Strains transformed with genes expressing catalytically-inactive polypeptides could not rescue development. Cotransformation with constructs encoding the N-terminal extension and the catalytic core, both unable to rescue development by themselves, yielded transformants able to proceed to late development. A 27-amino acid long hydrophobic region, immediately upstream of the catalytic core, was found indispensable for PKA function. A putative role of this sequence in the acquisition of the active conformation of the protein is discussed.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dictyostelium/enzimologia , Sequência de Aminoácidos , Animais , Catálise , Proteínas Quinases Dependentes de AMP Cíclico/genética , Dictyostelium/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Cells that overexpress PKA as a consequence of carrying multiple copies of the gene encoding the catalytic subunit can be induced to sporulate when developing as single cells. A peptide phosphorylated by PKA, termed SDF-1, has recently been shown to stimulate this process (Anjard et al., 1997). Several genes have been implicated in a signal transduction pathway by which prestalk cells induce encapsulation of prespore cells during terminal differentiation including a prestalk-specific putative membrane protease (TagC) and a two-component system consisting of a receptor-histidine kinase (DhkA) and a response regulator with cAMP phosphodiesterase activity (RegA). To determine whether SDF-1 uses this pathway, strains carrying null mutations in the pertinent genes were transformed with a pkaC plasmid such that they can overexpress PKA. Since these mutant strains all sporulated efficiently when SDF-1 was added, it appears that other gene products mediate the response. However, we found that regA- mutant cells release a distinct factor, SDF-2, that rapidly induces encapsulation of test cells overexpressing pkaC. Since cells in which tagC is disrupted do not form SDF-2 and cells in which dhkA is disrupted do not respond to SDF-2, this peptide appears to use the two-component system that regulates PKA activity. SDF-2 is a small peptide released by prestalk cells in a manner dependent on TagC. It appears to act on prespore cells through the DhkA receptor to inhibit the cAMP phosphodiesterase of RegA, thereby activating PKA via cAMP. The process of induction by SDF-2 can be shown to be distinct from that by SDF-1. SDF-2 appears to stimulate prestalk cells to release additional SDF-2 by acting through a signal transduction pathway that also involves DhkA, RegA, and PKA. Based on these results we present a model for the signal transduction cascade regulating spore differentiation.
Assuntos
Quimiocinas CXC , Dictyostelium/crescimento & desenvolvimento , Peptídeos , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas/biossíntese , Quimiocinas/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Dictyostelium/enzimologia , Dictyostelium/genética , Endopeptidases/fisiologia , Proteínas Fúngicas/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Membrana/metabolismo , Mutação , Proteínas Quinases/fisiologia , Proteínas/fisiologia , Transdução de Sinais/genética , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
Upon starvation, Dictyostelium discoideum unicellular amoebae form a multicellular organism leading to the development of a fruiting body containing spores. Single cells of sporogenous mutants, unlike wild type cells, are able to differentiate into spores under specific conditions. We show in this report that overexpression of the catalytic subunit of the cAMP dependent protein kinase (PKA), not only renders the cells sporogenous, but is also accompanied by the production/release of a diffusible spore differentiation factor (SDF). SDF is a small, thermostable phospho-polypeptide. In vitro dephosphorylation reduces SDF spore differentiation capacity, which can be regained in vitro by PKA phosphorylation. These results indicate that SDF is a PKA substrate and might be activated in vivo by this protein kinase. Since spore differentiation requires PKA catalytic subunit activation, we conclude that the response of prespore cells to SDF involves an intracellular pathway dependent on PKA.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dictyostelium/metabolismo , Proteínas Fúngicas/metabolismo , Esporos Fúngicos/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Dictyostelium/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/farmacologia , Peso Molecular , Fosforilação , Esporos Fúngicos/efeitos dos fármacosRESUMO
The C subunit of Dictyostelium cAMP-dependent protein kinase (PKA) is unusually large (73 kDa) due to the presence of 330 amino acids N-terminal to the conserved catalytic core. The sequence following the core, including a C-terminal -Phe-Xaa-Xaa-Phe-COOH motif, is highly conserved. We have characterized the catalytic activity and stability of C subunits mutated in sequences outside the catalytic core and we have analyzed their ability to interact with the R subunit and with the heat-stable protein-kinase inhibitor PKI. Mutants carrying deletions in the N-terminal domain displayed little difference in their kinetic properties and retained their capacity to be inhibited by R subunit and by PKI. In contrast, the mutation of one or both of the phenylalanine residues in the C-terminal motif resulted in a decrease of catalytic activity and stability of the proteins. Inhibition by the R subunit or by PKI were however unaffected. Sequence-comparison analysis of other protein kinases revealed that a -Phe-Xaa-Xaa-Phe- motif is present in many Ser/Thr protein kinases, although its location at the very end of the polypeptide is a particular feature of the PKA family. We propose that the presence of this motif may serve to identify isoforms of protein kinases.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dictyostelium/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Temperatura Alta , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Desnaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , UreiaRESUMO
A single open reading frame (ORF) encoding cytochrome c oxidase subunit I and II (cox1/2) was identified in the mitochondrial genome of the slime mold Dictyostelium discoideum. The cox1 coding region shares intron positions with its counterparts in fungi and algae. Northern blot analysis, using exon and intron-specific probes, suggests that the cox1/2 gene is transcribed as part of a large, efficiently processed, polycistronic RNA.
Assuntos
Dictyostelium/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/genética , RNA/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Complexo IV da Cadeia de Transporte de Elétrons/química , Dados de Sequência Molecular , Transcrição GênicaRESUMO
The cAMP-dependent protein kinase (cAPK) plays an essential role during differentiation and fruit morphogenesis in Dictyostelium discoideum. The presence of an open reading frame on the gene, pkaC (previously named either Dd PK2 or Dd PK3 by different groups), predicts a 73-kDa polypeptide with 54% similarity to the catalytic subunits of cAPKs from other organisms. Using anti-peptide antibodies, we show that the pkaC gene product, PkaC, is a 73-kDa polypeptide. Despite the fact that PkaC is about twice the size of its mammalian counterparts, it possesses all of the properties required of a catalytic subunit. It is physically associated with the regulatory subunit, and this association results in an inhibition of the catalytic activity which is reverted by cAMP. PkaC copurifies with cAPK activity, and an increased cAPK activity is observed in cells overexpressing PkaC. We conclude that PkaC is a catalytic subunit of the Dictyostelium discoideum cAPK and discuss the unusual features of this protein with the highest molecular weight of known cAPKs.
Assuntos
Dictyostelium/enzimologia , Proteínas Fúngicas/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/imunologia , Proteínas Fúngicas/isolamento & purificação , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/imunologia , Proteínas Quinases/isolamento & purificaçãoRESUMO
Expression of the catalytic (C) subunit of the cAMP-dependent protein kinase (PKA) of Dictyostelium under the control of heterologous, cell-type-specific promoters causes ectopic terminal differentiation. When expressed under the control of a prespore-specific promoter, development is accelerated, to yield highly aberrant fruiting bodies that contain a basal mass of spore cells surrounding a central stalk-like structure. When expressed under the control of a prestalk-specific promoter, development arrests much earlier, at the tight mound stage. Prestalk cells move to the apices of these mounds, apparently normally, but no tip is formed. Most of the prestalk cells remain arrested in their development but there are a few isolated stalk cells scattered within such mounds. We show that extracellular cAMP represses stalk cell-specific gene expression in cells where the kinase is constitutively active, suggesting that inhibition of stalk cell differentiation by cAMP in normal cells (Berks and Kay, 1988) occurs because of an effect of extracellular cAMP on an intracellular signalling pathway independent of PKA. We propose a scheme whereby two separate events, a rise in intracellular cAMP levels and a fall in extracellular cAMP concentration, are required to induce stalk cell differentiation.
Assuntos
Dictyostelium/citologia , Animais , AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico , Dictyostelium/efeitos dos fármacos , Dictyostelium/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Recombinantes de Fusão/genética , beta-Galactosidase/metabolismoRESUMO
The Dd PK2 gene codes for a putative protein of 648 amino acids with a C-terminal half sharing high homology with protein kinase A catalytic subunits from other organisms. In order to find out more about the physiological role of the Dd PK2 kinase, its gene, and a version having a frame shift mutation in the middle of the catalytic region, were overexpressed in developing Dictyostelium cells. Both the intact gene (K-) and the frame shift mutant (Kdel-) caused rapid development with spores formed in 16-18 hours compared to the 24 hours required by their parent. This result was confirmed by the pattern of expression of some developmentally regulated genes. Other rapid developing strains (rde) are activated in the cAMP second messenger system. Both K- and Kdel-containing strains have lower cAMP levels than the parental strain during late development, thus resembling rdeC mutants. K-cells (but not Kdel-cells) produced bizarre fruiting bodies with many prostrate forms. The parallel with rde mutants was confirmed by demonstrating that K-cells are able to form spores in submerged monolayer culture. Furthermore, K-cells have about four times more protein kinase A (cAPK) activity than wild-type cells. These results indicate that the N-terminal domain of Dd PK2 is sufficient to influence cAMP levels and to provoke rapid development, whereas kinase activity seems to be required for the sporogenous phenotype. The association between elevated cAPK and Dd PK2 overexpression phenotype further indicates a role for cAPK in the formation of spores.
Assuntos
AMP Cíclico/metabolismo , Dictyostelium/metabolismo , Genes/genética , Proteínas Quinases/genética , Animais , Dictyostelium/crescimento & desenvolvimento , Expressão Gênica , EsporosRESUMO
Cells of the monocyte lineage act as a major reservoir for HIV, and ways of enhancing the resistance of mononuclear phagocytes to HIV replication would be useful for delaying the onset of AIDS in infected individuals. Seif et al. (J. Virol. 65:664, 1991) have recently shown the possibility of obtaining stable antiviral expression (SAVE), directed against three nonretroviral RNA viruses, and normal cell viability in a significant percentage of murine BALB/c 3T3 cells transformed with an IFN-beta expression plasmid under the control of the 0.6-kb XhoII-NruI promoter region of the murine H-2Kb MHC gene. In the present paper, we show that it is possible to establish SAVE in human promonocytic cells. Cells of the human promonocytic U937 line were stably transfected with a human IFN-beta expression plasmid carrying the neo- and human IFN-beta-coding sequences under the control of the H-2Kb promoter fragment previously used in murine cells. After selection with G418, two transformed clones were isolated that released small amounts of human IFN-beta into the culture medium, without affecting the expression of CD4 and leucocyte function-associated Ag-1 differentiation Ag. The presence of construct-derived IFN-beta mRNA was demonstrated by polymerase chain reaction amplification of cDNA, and the level of 2-5A synthetase, one of the major IFN-induced antiviral proteins, was shown to be constitutively increased. These clones were less permissive for HIV-1 than control clones transformed with the neo gene only. The antiviral state could be modulated by anti-IFN-beta antibodies, in that the continuous presence of antibodies in the culture medium abolished the enhanced resistance to HIV-1 replication, whereas the withdrawal of the antiserum restored the antiviral state, indicating that it did indeed result from the constitutive synthesis of human IFN-beta. These results demonstrate the possibility of restricting HIV-1 replication in human promonocytic cells by establishing SAVE. Further exploration of this method as a possible approach to somatic cell gene therapy of HIV infection appears worthwhile.