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...
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