Inducible expression of calmodulin antisense RNA in Dictyostelium cells inhibits the completion of cytokinesis.

Abstract

The single gene encoding calmodulin in the eukaryotic microorganism Dictyostelium discoideum was cloned and sequenced. The gene was found to contain three introns, one lying immediately after the translation initiation codon. The deduced amino acid sequence indicated that Dictyostelium calmodulin contains 19 amino acid differences from vertebrate calmodulin, including extensions at both termini. Northern blot analysis showed that similar levels of calmodulin mRNA are present throughout growth and development of wild-type cells. A complete copy of the calmodulin cDNA was prepared, and an 87-base pair fragment complementary to the 5'-end of the calmodulin mRNA was subcloned into the Dictyostelium transformation vector pVEII, such that expression of the antisense transcript was driven by the discoidin I gamma promoter. Transformed cells were selected and maintained at low cell density, a condition resulting in minimal activity of the discoidin I promoter. High level expression was induced by allowing the transformants to reach high cell density or by growing them in the presence of medium conditioned by high density cells. Under these conditions, in which calmodulin mRNA and protein levels were reduced about twofold, the calmodulin antisense transformants lost the ability to complete cytokinesis. A contractile ring formed and constricted, but the midbody linking daughter cells failed to break. The resulting cell population contained multinucleated cells and networks of cells connected by cytoplasmic bridges. Normal cell division was restored when the cells were diluted to low density. These observations have identified a new point at which calmodulin may regulate cell cleavage.

Biochemical Characterization of Two Analogues of the Apoptosis-linked Gene 2 Protein in Dictyostelium discoideumand Interaction with a Physiological Partner in Mammals, Murine Alix

“…Simple and double knock-outs of Dd-alg-2a/2b produced no obvious developmental phenotype, thus excluding an indispensable role for Dd-ALG-2a/2b in the calcium response during differentiation, as also reported for other calciproteins in D. discoideum (21)(22)(23)(24)(25)(26)(27). Nevertheless, an alix-null mutant has a strong defect in development 2 and Dd-ALG-2a, by binding Alix, may participate in the overall regulation of that pathway.…”

“…Besides the calciproteins with 4 EF-hands identified in D. discoideum (21)(22)(23)(24)(25)(26)(27), we describe two new Ca 2ϩ -binding proteins with 5 putative EF-hands, Dd-ALG-2a and -2b. During this work, these proteins have been independently identified by others (28).…”

“…Even though a number of calciumbinding proteins have been characterized in this organism, among which are CBP1-4, CAF1, CaM, and CalB (21)(22)(23)(24)(25)(26)(27), none so far has been assigned a specific role as a mediator of * This work was supported in part by the Commissariat à l'Energie Atomique, CNRS, INSERM, the University Joseph Fourier Grenoble, and grants from the Association pour la Recherche sur le Cancer. A preliminary report of this work was presented at the Dictyostelium International Meetings in Dundee, United Kingdom, July 30 -August 4, 2000 and San Diego, CA, July [22][23][24][25][26]2001. The costs of publication of this article were defrayed in part by the payment of page charges.…”

Dictyostelium discoideum contains a family of calmodulin‐related EF‐hand proteins that are developmentally regulated

“…During a scareh for small cytoskeletal proteins we isolated a cDNA clone whose derived amino acid sequence showed similarities to EFhands but was different from the corresponding legions in Dictyostelium a-actinin [11] and calmodulin [12]. By using this DNA fragment as a probe for screening a Dictyostelium eDNA library [17] we isolated a 455 bp internal fragment that spanned the coding region between nucleotides 86-705 (Fig.…”

“…This regulation is mediated by two perfect EF-hand regions in the C-terminus of the subunits [4,11]. The concentrations of the small acidic Ca2+.binding proteins c, almodulin [12] and calfumirin [13] are also essentially unaltered during growth and development. *Con'eslmnding author.…”