We introduce a PCR-based procedure for generating a gene disruption construct. This method depends on DNA fragment fusion by the PCR technique and requires only two steps of PCR to obtain a sufficient amount of the gene disruption construct for one transformation experiment. The first step involves three separate PCR syntheses of a selectable marker cassette and the 5'- and 3'-regions of a target gene. Of the four primers used in amplification of the 5'- and 3'-regions of the target gene, two primers placed proximal to the site of the marker cassette are designed to have sequence tags complementary to the 5'- or 3'-side of the marker cassette. The two primers used in PCR synthesis of the marker cassette are complementary to the tagged primers. By fusion PCR, the 5' and 3' PCR products are linked to the marker cassette via the regions of tagged primers that overlap. A sufficient amount of the disruption construct can be directly amplified with the outermost primers. This method is simple, rapid and relatively inexpensive. In addition, there is the freedom of attaching long flanking regions to any selectable marker cassette.

Differentiation is a highly regulated process whereby cells become specialized to perform specific functions and lose the ability to perform others. In contrast, the question of whether dedifferentiation is a genetically determined process, or merely an unregulated loss of the differentiated state, has not been resolved. We show here that dedifferentiation in the social amoeba Dictyostelium discoideum relies on a sequence of events that is independent of the original developmental state and involves the coordinated expression of a specific set of genes. A defect in one of these genes, the histidine kinase dhkA, alters the kinetics of dedifferentiation and uncouples the progression of dedifferentiation events. These observations establish dedifferentiation as a genetically determined process and suggest the existence of a developmental checkpoint that ensures a return path to the undifferentiated state. Dedifferentiation is the progression of cells from a more differentiated to a less differentiated state. It is observed in a variety of processes such as cancer, organ regeneration, and stem cell renewal, but it has been difficult to study because there are few experimentally tractable systems that dedifferentiate (1-4). Dedifferentiation in Dictyostelium is an experimentally tractable process. During development, starving cells aggregate and undergo synchronous morphological transitions until they form fruiting bodies after 24 hr (5). If the multicellular structures are disaggregated and incubated in nutrient medium, the cells dedifferentiate. Dedifferentiation is characterized by a loss of developmental markers and a subsequent gain of proliferative capacity (6, 7, 45), but these characteristics do not prove that dedifferentiation is a regulated process. The most convincing evidence in support of regulation has been the observation that a mutant strain (HI4) was defective in dedifferentiation (8,9).An argument against the idea that dedifferentiation is a regulated process comes from the observation that dedifferentiation occurs at different rates, depending on the developmental stage at which the cells were disaggregated (6, 10-13). This dependence may indicate that each developmental stage has a dedicated dedifferentiation program that is executed at a different rate, or that dedifferentiation is a stochastic event whereby cells lose developmental markers and regain growth markers.The purpose of this work was to test whether dedifferentiation is a regulated process by comparing the molecular progression of cells from different developmental stages. We propose that if we found a common set of molecular changes, which is independent of the initial developmental stage, that finding would support the regulated process hypothesis. We used microarray transcriptional profiling to detect changes in the pattern of global gene expression during dedifferentiation. These transcriptional changes reveal physiological changes without prejudice as to what processes are involved (14-18), making them suitable for testing whether...

Macrocysts in Dictyostelium discoideum possess prototypic features of sexual reproduction and are useful for understanding the basic mechanisms of the reproductive process. Here, we randomly analyzed 1,071 gamete cDNAs, and then constructed a gamete-specific subtraction library, FC-IC. Nucleotide sequences of all 903 FC-IC clones were determined and clustered into 272 independent genes. Expression analysis based on real-time RT-PCR revealed 67 gamete-enriched genes, among which those involved in 'signal transduction' and 'multicellular organization' are prevalent. One of them, FC-IC0003, appeared also to be mating-type specific, and was named gmsA. RNAi-mediated silencing as well as disruption of gmsA reduced the cellular competency for sexual cell fusion, indicating the involvement of this gene in the sexual development of D. discoideum.

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