摘要:

AbstractThe functional interaction of surface cAMP receptors with effector enzymes via G‐proteins was investigated inDictyostelium discoideum.Several experimental conditions were used to investigate signal transduction, such as reduced temperatures, use of down‐regulated cells and of mutants. The results are presented as a model describing the complex interaction between multiple forms of the surface cAMP receptor and different G‐proteins that are responsible for the generation of the second messengers, cAMP, cGMP, InsP3and

ISSN:0192-253X    

DOI:10.1002/dvg.1020090404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractUsing antibodies specific for the 3′, 5′‐cyclic AMP (cAMP) cell surface receptor ofDictyostelium discoideum, we have screened γgtll expression libraries and isolated a series of cDNAs derived from cAMP receptor mRNA during early development. The identity of the cDNA clones was verified by multiple criteria: (1) β‐galactosidase fusion proteins synthesized by isolated cDNA clones stain intensely with cAMP receptor directed antiserum, (2) these fusion proteins affinity purify antibodies specific for the cAMP receptor, (3) the cDNA probes hybridize to a 2 kb mRNA whose change in relative level of abundance during development parallels that of receptor mRNA as assayed by in vitro translation, (4) the 2 kb mRNA size equals that of receptor mRNA as determined by in vitro translation of size fractionated poly (A)+ RNA, and (5) RNA transcribed in vitro from cDNAs containing the entire protein‐coding region produces a polypeptide by in vitro translation with an apparent molecular weight in close agreement with that of nascent cAMP receptor protein produced by in vitro translation of cellular RNA.The DNA sequence predicts an open reading frame of 392 amino acids. The deduced amino acid sequence contains seven domains enriched in hydrophobic residues. A model is proposed in which the cAMP cell‐surface receptor traverses the lipid bilayer seven times in a pattern similar to that of other receptors, such as rhodopsin, which interact with G‐proteins. The structural similarities suggest a gene family of related surface receptors from such evolutionarily diverse species asDictyostelium, yea

ISSN:0192-253X    

DOI:10.1002/dvg.1020090405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractSeveral lines of evidence indicate that cAMP modulates developmental gene activity via cell‐surface receptors. We describe here a novel cAMP receptor, CABP1, whose properties are consistent with the idea that this protein is involved in gene regulation. Firstly, immunological techniques using anti‐CABP1 antibodies as probes showed that this cAMP receptor can be detected on the surface of developing cells. Secondly, there is a steady migration of CABP1 to the nucleus during development. Thirdly, some genetic variants exhibiting an altered pattern of development are found to possess modified CABP1. We also showed that CABP1 co‐purifies with at least seven other polypeptides which share common epitopes with CABP1. Interestingly, four of the CABP1‐related polypeptides can be detected on the cell surface as well as in the

ISSN:0192-253X    

DOI:10.1002/dvg.1020090406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractThe cAMP‐dependent protein kinase (cAK) fromDictyostelium discoideumis an enzyme composed of one catalytic and one regulatory subunit. Upon binding of cAMP, the holoenzyme dissociates to liberate free active catalytic subunits. The cAK is developmentally regulated, ranging from very little activity in vegetative cells to maximal expression in postaggregative cells. Although there is no immunological cross‐reaction between the subunits of cAKs fromDictyosteliumand from other organisms, they share several biochemical properties. A complete cDNA for the regulatory subunit has been cloned and sequenced. Only one copy of the gene for the regulatory subunit is present per haploid genome. On the basis of the comparison of the structure of the cAK fromDictyosteliumwith its counterparts in yeast and higher eukaryotes, we propose a model for the evolution of cyclic‐nucleotide‐binding p

ISSN:0192-253X    

DOI:10.1002/dvg.1020090407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractWe have identified a two‐member gene family in theDictyosteliumgenome and have isolated corresponding cDNA or genomic DNA recombinant clones. Analyses of these DNA sequences predicted encoded proteins of ∼︁200 amino acids with ∼︁90% sequence identity to each other. TheseDictyosteliumproteins also share amino acids identity within the GTP‐binding domains in the family of G‐regulatory proteins involved in cellular regulation and transmembrane signalling. Additional structural similarities are seen with members of therassupergene family, such asras, ral, andrho.They are similar in size (usually ∼︁200 amino acids), possess four conserved domains involved in GTP interaction and are believed to be anchored in the membrane by fatty acid modification of a cysteine residue near the carboxy terminus. More extensive identity is observed withYPT1andSEC4, two other members of this family of genes that are essential in yeast. The amino‐terminal half of bothDictyosteliumproteins is 70% identical in amino acid sequence to theYPT1andSEC4yeast proteins with less identity continuing through the remainder of the proteins. In addition these proteins terminate in two cysteine residues that are thought to be required for membrane anchorage.The two genes within thisDictyosteliumfamily are organized differently in the genome and are differentially regulated during development. One gene is colinear in sequence with its mRNA in the protein coding region, whereas the other gene encodes a spliced mRNA. The intron‐containing gene is associated with a developmentally regulated (AAC)‐repeat sequence. Finally, we have shown that the expression of one of the genes is induced during development with kinetics similar to that of other (AAC)n‐associated genes; conversely, the expression of the second gene is repressed at a si

ISSN:0192-253X    

DOI:10.1002/dvg.1020090408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractThe cyclic nucleotide phosphodiesterase (phosphodiesterase) ofDictyostelium discoideumplays an essential role in development by hydrolyzing the cAMP used as a chemoattractant by aggregating cells. We have studied the biochemistry of the phosphodiesterase and a functionally related protein, the phosphodiesterase inhibitor protein, and have cloned the cognate genes. A 1.8‐kb and a 2.2‐kb mRNA are transcribed from the singlephosphodiesterase gene. The 2.2‐kb mRNA comprises the majority of the phosphodiesterase mRNA found in differentiating cells and is transcribed only during development from a promoter at least 2.5 kb upstream of the translational start site. The 1.8‐kb phosphodiesterase mRNA is detected at all stages of growth and development, is present at lower levels than the developmentally induced mRNA, and is transcribed from a site proximal to the protein‐coding region. The phosphodiesterase gene contains a minimum of three exons, and a 2.3‐kb intron, the longest yet reported for this organism. We have shown that thepdsA. gene and fourfgdgenes affect, the accumulation of the phosphodiesterase mRNAs, and we believe that these loci represent a significant portion of the genes regulating expression of the phosphodiesterase. The phosphodiesterase gene was introduced into cells by transformation and used as a tool to explore the effects of cAMP on the terminal stages of development. In cells expressing high levels of phosphodiesterase activity, final morphogenesis cannot be completed, and differentiated spore and stalk cells do not form. We interpret these results to support the hypothesis that cAMP plays an essential role in organizing cell movements in late development as well as in controlling the aggregation of cells in the initial phase of the developmen

ISSN:0192-253X    

DOI:10.1002/dvg.1020090409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractcAMP and cell‐cell contact are involved in the coordination of differentiation and morphogenesis inDictyostelium discoideum.The experiments described in this paper establish a relationship between cAMP and cell‐cell contact. Contact betweenEnterobacter aerogenesand aggregation‐competentDictyosteliumamoebae and contact betweenDictyosteliumamoebae themselves results in the transient secretion of cAMP and an alteration in the amount of cAMP secreted in response to subsequent stimulation by cAMP, i.e., an alteration in magnitude of a cAMP relay response. The subsequent cAMP relay response can be enhanced or diminished depending upon the number of contacts formed and the concentration of cAMP present at the time of contact. Latex beads are capable of evoking cAMP secretion. However, the bead/amoebal contact is unable to alter the magnitude of a subsequent response to cAMP. This suggests that a nonspecific interaction via cell‐cell contactelicits transient cAMP secretion in aggregation‐competentDictyosteliumamoebae.The two responses to cell‐cell contact are distinct from each other and distinct from the cAMP relay response. (1) The dose‐response curves for the responses toEnterobactercontact are clearly different. (2) Contact with latex beads can elicit cAMP secretion but not alter the magnitude of a subsequent cAMP relay response. (3) The temperature dependences of the contact‐induced responses and the cAMP relay response show that only the contact‐induced cAMP secretion is inhibited at 12 and 15°C, while only the cAMP relay response is inhibited at 28°C.A 4‐second application of cAMP at the time that contact is initiated enhances both contact‐induced responses. Whether the relationship between these two developmental regulators is important for the regulation ofDictyosteliumdevelopment ha

ISSN:0192-253X    

DOI:10.1002/dvg.1020090410

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractWe have analyzed the effects of the cAMP relay inhibitor, caffeine, and the receptor antagonist, adenosine, on the regulation of the cell‐surface cAMP receptor in suspensionstarvedDictyostelium discoideumcells by measuring ammonium sulfate‐stabilized binding of [3‐H]cAMP to intact cells. When cells were starved in fast (230 r.p.m.) shaken suspension in 10 mM Na+/5 mM K+phosphate buffer, pH 6.5, plus 1 mM CaCl2and 2.5 mM MgCl2, and assayed for specific cAMP binding, receptor accumulation peaked at approximately 6 hours, reaching a maximum of 1.5 pmol cAMP bound/107cells (saturation binding). Neither caffeine nor adenosine inhibited the accumulation of cAMP receptors. Similar results were obtained in caffeine‐treated, slow shaken (90 r.p.m.) suspension cultures. These results suggest that starvation alone is sufficient stimulus to induce the cAMP receptor. We have also tested the effects of different buffer ionic compositions on the accumulation of cAMP receptors. Elevation of the monovalent ion concentration to 30‐40 mM was found to significantly inhibit the induction of cAMP

ISSN:0192-253X    

DOI:10.1002/dvg.1020090411

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractSimilar to other stages inDictyosteliumdevelopment, spore germination is a particularly suitable model for studying the regulation of gene expression, because developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. Spores are constitutively dormant and must be activated to germinate. Under the proper environmental conditions, spores germinate in a highly synchronous manner to give rise to individual amoebae that can then enter the vegetative growth phase. Protein synthesis is developmentally regulated during this process. Because protein synthesis is transcriptionally controlled during spore germination, the respective genes must be developmentally transcribed, and these can be isolated and analyzed. Three cDNA clones specific for mRNA developmentally regulated during spore germination have been characterized and used as probes to study mRNA accumulation and decay during spore germination. Because we are interested in defining the sequences of developmentally regulated genes that may relate to their regulation of transcription, we have sequenced the cDNAs and have isolated and sequenced their respective genomic clones. The sequences of the three gene families, their genomic organization, and their special structural features are described.

ISSN:0192-253X    

DOI:10.1002/dvg.1020090412

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractWe have previously shown that growing cells ofDictyostelium discoideum(strains NC4 and AX3) produce a soluble substance that accumulates in the medium in proportion to cell density; this substance regulates the production of certain proteins previously thought to be induced by starvation [Clarkeet al., 1987]. We suggest the name PSF (prestarvation factor) for this substance. During growth,Dictyosteliumcells monitor the relative concentrations of PSF and food bacteria. When PSF reaches a sufficiently high level relative to the concentration of bacteria, synthesis of PSF‐regulated proteins is induced. We propose the name prestarvation response for this induction, which takes place in exponentially growing cells several generations before the food bacteria are depleted. We have explored the mechanism by which the food bacteria inhibit the response ofDictyosteliumcells to PSF. We find that the bacteria do not inactivate PSF or inhibit its production; instead, they affect the ability of NC4 cells to detect PSF, possibly by binding to the same cell surface receptor. In the absence of bacteria, as during axenic growth of AX3 cells, the prestarvation response occurs at much lower cell densities, probably accounting for the presence of certain developmentally regulated mRNAs and proteins in axenic culture

ISSN:0192-253X    

DOI:10.1002/dvg.1020090413

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY