Suppression of the growth/differentiation transition in Dictyostelium development by transient expression of a novel gene, dia1.

In Dictyostelium discoideum Ax-2 cells, a specific checkpoint (PS point) from which cells enter the differentiation phase in response to starvation has been specified in the cell cycle. Using the differential display method, we isolated a novel gene, dia1 (differentiation-associated gene 1), that is specifically expressed in cells differentiating from the PS point. The dia1 mRNA has an open reading frame of 1,368 bp and is deduced to code for a 48.6 kDa protein (DIA1). The DIA1 protein is highly serine-rich and the serine residues are predominantly located in the C-terminal region. After the PSORT II search, the protein is predicted to be GPI-anchored at the plasma membrane. Unexpectedly, dia1 overexpression rather impaired the progression of differentiation, possibly coupled with the reduced expression of early genes such as cAMP receptor1 (car1). The inhibitory effect of dia1 expression on early differentiation was almost completely nullified by externally applied cAMP pulses. In contrast to dia1 overexpression, antisense RNA-mediated dia1 inactivation was found to enhance the initial step of cell differentiation, as exemplified by precocious expression of car1 and other early genes. We discuss the unique structure and function of DIA1 in relation to the cooperative development of cells during the establishment of multicellular organization.

Transient expression of a mitochondrial gene cluster including rps4 is essential for the phase-shift of Dictyostelium cells from growth to differentiation.

Using synchronized Dictyostelium discoideum Ax-2 cells and the differential display method, a mitochondrial gene cluster (referred to as differentiation-associated gene 3; dia3) was isolated as one of the genes expressed specifically during the transition of Ax-2 cells from growth to differentiation. The dia3 gene encodes for a mitochondrial protein cluster (NADH dehydrogenase (NAD) subunit 11, 5, ribosomal protein S4 (RPS4), RPS2, and NAD4L). Northern blot analysis using nonsynchronized Ax-2 cells has shown that the dia3 RNA of about 8 kb is scarcely expressed during the vegetative growth phase, and the maximal expression was attained at 2 h after starvation. To analyze the gene function of dia3, we tried inactivation of rps4 by means of homologous recombination and obtained several transformed clones showing mitochondrial DNA heteroplasmy. The transformed cells grew normally in nutrient medium, but their development after starvation was greatly impaired, thus resulting in the failure of many cells to differentiate. In this connection, the cAMP receptor 1 (car1) expression, which is one of the earliest markers of differentiation, was found to be markedly reduced in the rps4-inactivated cells.

Underexpression of a novel gene, dia2, impairs the transition of Dictyostelium cells from growth to differentiation.

In Dictyostelium discoideum Ax-2 cells, a specific point (PS-point) in the cell cycle from which they initiate differentiation in response to starvation has been specified. Using synchronized Ax-2 cells and the differential display method, a novel gene (differentiation-associated gene 2; dia2) was isolated as one of the genes expressed specifically during the shift of Ax-2 cells from growth to differentiation. The dia2 gene codes a lysine- and leucine-rich protein with a predicted molecular mass of 16.9 kDa. Northern blot analysis has shown that the dia2 mRNA, of 0.7 kb, accumulates in differentiating cells starved just before the PS-point, while there is no detectable expression in vegetatively growing cells. Antisense-mediated gene inactivation of dia2 greatly inhibited the progress of differentiation, presumably through the reduced expression of cAMP receptor 1 (car1). Thus, the DIA2 expression was suggested to have an essential role in the initiation of differentiation, closely relating to the cAMP signaling system.