Extrachromosomal replication of shuttle vectors in Dictyostelium discoideum.

Abstract

We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddpl, found in several wild-type and laboratory strains of Dictyostelium discoideum into pBR322. The cloned plasmids have been used to cotransform D. discoideum cells with B1OS, a transformation vector carrying a gene fusion conferring resistance to G418. Whereas BIOS DNA alone appears to integrate in a tandem array, the cloned Ddpl plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell. The Ddpl-derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells. Preliminary deletion analysis indicates that not all regions of Ddpl are necessary for stable replication in D. discoideum. Several recombinant vectors which replicate extrachromosomally in D. discoideum were also isolated. One contains the Act6-neor gene fusion from BiOS recombined into one of the cloned derivatives of Ddpl and can be used to directly transform D. discoideum amoebae, selecting for G418 resistance. Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddpl hybrid plasmid. An analysis of the vector DNAs present in clones derived from single D. discoideum transformants is also described.Dictyosteliium discoideium grows as single-celled amoebae. Upon starvation, the cells initiate a multicellular developmental program in which approximately 105 cells form an aggregate which then differentiates into a fruiting body containing predominantly two cell types: spores and stalk cells (7). We and others have been interested in understanding the mechanisms by which cell-type differentiation is regulated in this relatively simple multicellular system. To this end, genes which are expressed at specific developmental stages were cloned and used to examine the biochemical and physiological factors which regulate their expression. These genes include those expressed during vegetative growth and repressed during early development, genes expressed under various conditions during preaggregation, and the cell-type-specific genes that are preferentially expressed in either prestalk or prespore cells (1, 9; for a review, see reference 4). Several factors have been identified which are required for the expression of sets of coordinately regulated genes, including cAMP (3, 5, 8-10; S. Mann, R. A. Firtel, and J. Brandis, manuscript in preparation).We have begun by examining the cis-acting DNA sequences that are involved in the proper developmental regulation of the expression of these genes and have established that there is a DNA-mediated transformation system in which cloned genes can be transformed into stably maintained and expressed D. discoidelum (12, 13 Firtel; Proc. Natl. Acad. Sci. USA, in press; W. Nellen and R. A. Firtel, Gene, in press). For several types of analyses, it would be advantageous to have a shuttle vector which would replicate extrachromosomally in D. discoideutm cells and which could be easily recovered by transforming...

Molecular complementation of a genetic marker in Dictyostelium using a genomic DNA library.

“…If we have isolated a suppressor, our library or our method may not be as universally applicable as our experiments suggest. We see an -5-fold overexpression of the Thy+ mRNA in our transformants, but this is a function ofthe Dictyostelium transformation system in which extrachromosomal vectors are present in multiple copies in the cell (23,24). Moreover, integrating vectors produce multiple-copy tandem arrays (26).…”

“…Recently, a series of extrachromosomal shuttle vectors for D. discoideum has been constructed in this laboratory (23,24). They contain a Dictyostelium actin promoter-bacterial neomycin gene (neo) fusion for selection in Dictyostelium with G418, a bacterial plasmid backbone for selection and propagation in E. coli, and a region from the endogenous plasmid Ddpl that confers extrachromosomal replication in Dictyostelium.…”

Dictyostelium discoideum cobB mutants show reduced heavy metal accumulation associated with gene amplification

“…In contrast, the mobility of circular molecules of known size varied enormously at different switch times in comparison to the linear Lambda concatemer size markers (data not shown). For example, a plasmid of 18 kb (pBMW4; Firtel et al 1985) had an apparent size of 95 kb at 10 s switch time and 250 kb at 40 s switch time, while a cosmid of about cobB mutants show reduced nickel accumulation Short-term experiments to examine nickel uptake rates in wild-type and amplification-bearing strains using Ni> and axenic cultures were unsuccessful. No measurements above background were obtained over periods up to 6 h. Therefore cells were grown for 15-20 generations on solid medium containing 18.5 kBq/ml (0.5 Ci/ml) Ni> (0.16 nM) and varying amounts of nonradioactive NiCl (0.004-0.8 mM).…”

Mutagenesis and gene identification in Dictyostelium by shotgun antisense.

“…To isolate the antisense cDNA contained in the pV18neo vector, 2 x 104 logarithmically growing transformed cells were collected by centrifugation for 1 min at 1000 x g. The cell pellet was resuspended in 40 ,ll of lx PCR reaction buffer containing gelatin (Perkin-Elmer) and treated with 4 ,ul of PreTaq (GIBCO/BRL) for 5 min at 75°C. The reaction was stopped by the addition of 4 ,ul of 20 mM EGTA and incubated at 75°C for 10 min, followed by 6 min at 100°C. A 100-,l quick start PCR reaction (Perkin-Elmer) contained 2 (18).…”

“…However, both extrachromosomal and integrating transformation vectors exist for Dictyostelium (3)(4)(5)(6)(7)(8), and gene expression can be repressed by both antisense RNA and homologous recombination-mediated gene disruption (9)(10)(11)(12)(13)(14)(15)(16). Mutants can also be generated by random insertion of transformation plasmids into the genome (17,18).…”