The extrachromosomal replication of Dictyostelium plasmid Ddp2 requires a cis-acting element and a plasmid-encoded trans-acting factor.

Leiting, Barbara; Lindner, Inka; Noegel, Angelika A.

doi:10.1128/mcb.10.7.3727

Cited by 26 publications

(15 citation statements)

References 43 publications

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“…Ax3-ORF ϩ , the parent strain for the overexpressing S-1-P lyase gene, and the transformation vector pDXA3C were gifts from D. Manstein, National Institute for Medical Health, London, United Kingdom (18,26). The 7.7-kb transformation construct carrying the myc-tagged sglA gene (pDXA3C/sglA) is pJM5.…”

Section: Methodsmentioning

confidence: 99%

Overexpression of Sphingosine-1-Phosphate Lyase or Inhibition of Sphingosine Kinase in Dictyostelium discoideum Results in a Selective Increase in Sensitivity to Platinum-Based Chemotherapy Drugs

et al. 2004

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The efficacy of the chemotherapy drug cisplatin is often limited due to resistance of the tumors to the drug, and increasing the potency of cisplatin without increasing its concentration could prove beneficial. A previously characterized Dictyostelium discoideum mutant with increased resistance to cisplatin was defective in the gene encoding sphingosine-1-phosphate (S-1-P) lyase, which catalyzes the breakdown of S-1-P, an important regulatory molecule in cell function and development and in the regulation of cell fate. We hypothesized that the increased resistance to cisplatin was due to an elevation of S-1-P and predicted that lowering levels of S-1-P should increase sensitivity to the drug. We generated three strains that stably overexpress different levels of the S-1-P lyase. The overexpressor strains have reduced growth rate and, confirming the hypothesis, showed an expression-dependent increase in sensitivity to cisplatin. Consistently, treating the cells with D-erythro-N,N,-dimethylsphingosine, a known inhibitor of sphingosine kinase, increased the sensitivity of mutant and parent cells to cisplatin, while addition of exogenous S-1-P or 8-Br-cyclic AMP made the cells more resistant to cisplatin. The increased sensitivity of the overexpressors to cisplatin was also observed with the cisplatin analog carboplatin. In contrast, the response to doxorubicin, 5-flurouracil, or etoposide was unaffected, indicating that the involvement of the sphingolipid metabolic pathway in modulating the response to cisplatin is not part of a global genotoxic stress response. The augmented sensitivity to cisplatin appears to be the result of an intracellular signaling function of S-1-P, because D. discoideum does not appear to have endothelial differentiation growth (EDG/S1P) receptors. Overall, the results show that modulation of the sphingolipid pathway at multiple points can result in increased sensitivity to cisplatin and has the potential for increasing the clinical usefulness of this important drug.

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Section: Methodsmentioning

confidence: 99%

Overexpression of Sphingosine-1-Phosphate Lyase or Inhibition of Sphingosine Kinase in Dictyostelium discoideum Results in a Selective Increase in Sensitivity to Platinum-Based Chemotherapy Drugs

et al. 2004

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“…Plasmids used for transformation were either derivatives of the integrating vector pB10TP2 (Early & Williams, 1987) or the extrachromosomal vector pDXA-3H. The vector pDXA-3H carries the origin of replication of the Dictyostelium high copy number plasmid Ddp2 Leiting et al, 1990), an expression cassette consisting of the strong, constitutive actin15 promoter, a translational start codon upstream from a multiple cloning site (MCS), and sequences for the addition of a histidine octamer at the carboxy terminus of any protein. Plasmids derived from pDXA-3H were transformed into orf+-cells (P. Morandini, manuscript in preparation).…”

Section: Methodsmentioning

confidence: 99%

“…Plasmids derived from pDXA-3H were transformed into orf+-cells (P. Morandini, manuscript in preparation). These cells carry several integrated copies of the rep gene which is essential in trans for the replication of plasmids that carry the Ddp2 origin (Leiting et al, 1990;Slade et al, 1990). The myosin-0c-actinin fusion constructs were created by linking codon 754 of the Dictyostelium mhcA gene to codon 264 of the Dictyostelium c¢-actinin gene.…”

Section: Methodsmentioning

confidence: 99%

Overexpression of myosin motor domains in Dictyostelium: screening of transformants and purification of the affinity tagged protein

Manstein¹,

Hunt²

1995

J Muscle Res Cell Motil

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The eukaryotic organism Dictyostelium discoideum has become one of the organisms of choice for the overexpression of recombinant myosins and myosin fragments. Here, we describe a protocol that facilitates the screening of cells that have been transformed with myosin expression constructs and allows the rapid purification of recombinant myosins. Depletion of cellular ATP is used to recruit most of the endogenous and recombinant myosin into a rigor-like complex with actin. Following cell lysis the insoluble actomyosin complex is precipitated by centrifugation, washed, and Mg(2+)-ATP is added to extract the recombinant protein from the pellet. More than 90% of the protein in the resulting supernatant corresponds to actin, myosin, and the recombinant myosin fragments. Therefore, it is easy to detect any differences in expression level between individual myosin constructs on SDS-polyacrylamide gels. Additionally, the dependence of expression on external factors, such as cell density, can be readily determined. Furthermore, the presence of a band corresponding to the recombinant protein indicates that the overexpressed protein has at least some of the functional properties that are characteristic for a myosin motor. This rapid and selective extraction protocol can also be utilized to facilitate the purification of recombinant myosin motors on a preparative scale and has proved particularly useful in the purification of myosin head fragments, that are tagged with histidine residues, by Ni(2+)-chelate affinity chromatography.

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“…These were then transformed into the wild-type axenic cell line KAx-3 and the ga2 null mutant strain JH104 (Kumagai et al, 1991), enabling both dominant and recessive phenotypes to be examined (see MATERIALS AND METHODS for details). Extrachromosomal vectors were used to ensure that expression was not affected by potential differences in the chromosomal site of integration in different clones and that cells within the population have a uniform copy number of the vector (Ahern et al, 1988;Leiting and Noegel, 1988;Leiting et al, 1990).…”

Section: Developmental Consequences Of Expression Of Ga2mentioning

confidence: 99%

“…The promoter cassette was combined with the Ga2 coding region, the 2-H3 terminator from the Dictyostelium SP70 gene (Crowley et al, 1985;Haberstroh and Firtel, unpublished data). This construct also contained the origin of replication and the gene for the trans-acting factor required for origin function from pDeAl (a clone from the endogenous plasmid Ddp2 [Leiting and Noegel, 1988;Leiting et al, 1990]) cloned into the plasmid backbone pAT153L (Haberstroh and Firtel, 1990 Na+/K+ phosphate-buffered (pH 6.2) agar or filters (Millipore, Bedford, MA) as previously described (Firtel and Chapman, 1990;Kumagai et al, 1991).…”

mentioning

confidence: 99%

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.

Okaichi

Cubitt

Pitt

et al. 1992

MBoC

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Previous studies have demonstrated that the Dictyostelium Ga subunit Ga2 is essential for the cAMP-activation of adenylyl cyclase and guanylyl cyclase and that ga2 null mutants do not aggregate. In this manuscript, we extend the analysis of the function of Ga2 in regulating downstream effectors by examining the in vivo developmental and physiological phenotypes of both wild-type and ga2 null cells carrying a series of mutant Ga2 subunits expressed from the cloned Ga2 promoter. Our results show that wild-type cells expressing Ga2 subunits carrying mutations G40V and Q208L in the highly conserved GAGESG (residues 38-43) and GGQRS (residues [206][207][208][209][210] domains, which are expected to reduce the intrinsic GTPase activity, are blocked in multicellular development. Analysis of downstream 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 Ga2 subunit can complement ga2 null mutants. Expression of Ga2(G43V) and Ga2(G207V) have little or no effect on the effector pathways and can partially complement ga2 null cells. Our results suggest a model in which the dominant negative phenotypes resulting from the expression of Ga2(G40V) and Ga2(Q208L) are due to a constitutive adaptation of the effectors through a Ga2-mediated pathway. Analysis of PI-PLC in ga2 null mutants and in cell lines expressing mutant Ga2 proteins also strongly suggests that Ga2 is the Ga subunit that directly activates PI-PLC during aggregation. Moreover, overexpression of wild-type Ga2 results in the ability to precociously activate guanylyl cyclase by cAMP in vegetative cells, suggesting that Ga2 may be rate limiting in the developmental regulation of guanylyl cyclase activation. In agreement with previous results, the activation of adenylyl cyclase, while requiring Ga2 function in vivo, does not appear to be directly carried out by the Ga2 subunit. Our data are consistent with adenylyl cyclase being directly activated by either another Ga subunit or by 3y subunits released on activation of the G protein containing Ga2.

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The extrachromosomal replication of Dictyostelium plasmid Ddp2 requires a cis-acting element and a plasmid-encoded trans-acting factor.

Cited by 26 publications

References 43 publications

Overexpression of Sphingosine-1-Phosphate Lyase or Inhibition of Sphingosine Kinase in Dictyostelium discoideum Results in a Selective Increase in Sensitivity to Platinum-Based Chemotherapy Drugs

Overexpression of Sphingosine-1-Phosphate Lyase or Inhibition of Sphingosine Kinase in Dictyostelium discoideum Results in a Selective Increase in Sensitivity to Platinum-Based Chemotherapy Drugs

Overexpression of myosin motor domains in Dictyostelium: screening of transformants and purification of the affinity tagged protein

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.

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