Identification of a DNA sequence element required for efficient expression of a developmentally regulated and cAMP-inducible gene of <i>Dictyostelium discoideum</i>

Pears, Catherine J.; Williams, Jeffrey

doi:10.1002/j.1460-2075.1987.tb04738.x

Cited by 81 publications

(85 citation statements)

References 57 publications

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“…Within this 164-nucleotide minimal region there are several G-rich sequence motifs. They resemble previously i d e n t i fied elements that are known to be important in obtaining efficient expression of developmentally regulated genes (Datta and Firtel, 1987;Pears and Williams, 1987;Ceccarelli et al, 1992) but further analysis will be required before we can identify the element or elements that actually specify expression.…”

Section: Analysis Of the Ecma Promotermentioning

confidence: 87%

Two distinct populations of prestalk cells within the tip of the migratory Dictyostelium slug with differing fates at culmination

Early¹,

Gaskell²,

Traynor³

et al. 1993

Trends in Genetics

141

126

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Section: Analysis Of the Ecma Promotermentioning

confidence: 87%

Two distinct populations of prestalk cells within the tip of the migratory Dictyostelium slug with differing fates at culmination

Early¹,

Gaskell²,

Traynor³

et al. 1993

Trends in Genetics

141

126

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“…Other regulatory elements, in addition to GBRE, were also identified and shown to affect the level of expression and, in some instances, the relative response to developmental or cAMP signals. The residual expression found in the GBRE deletions is still developmentally regulated, suggesting that other elements of the promoter respond to developmental signals Firtel 1987, 1988;Pears and Williams 1987). Insertion of oligonucleotides containing the wild-type GBRE restores high levels of regulated expression, whereas a series of mutated GBRE sequences do not (Hjorth et al 1989; this paper).…”

mentioning

confidence: 79%

“…Promoter analysis of the cAMP-induced, prestalkenriched gene pst-cathepsin (Datta and Firtel 1987), or CP2 (Pears and Williams 1987; henceforth called pstcath/CP2), identified an essential 80-bp cis-acting regulatory region that contains a G-rich element, designated GBRE (G-box regulatory element), the deletion of which results in a 50-to 100-fold reduction in the level of expression. Other regulatory elements, in addition to GBRE, were also identified and shown to affect the level of expression and, in some instances, the relative response to developmental or cAMP signals.…”

mentioning

confidence: 99%

“…The UDPGPP gene is expressed in vegetative cells in early development and, at an increased level, during multicellular stages Ragheb and Dottin 1986;. All three of these genes have a G/C-rich element and, for CP1, this element has been shown to confer cAMP-induced expression on a pst-cath/CP2 promoter construct carrying a deletion of the GBRE (Pears and Williams 1988). In this paper, we describe the relative ability of wild-type and mutant oligonucleotides containing these different G/C-rich elements to complement the pst-cath/CP2 GBRE deletion mutant and to interact with GBF in vitro.…”

mentioning

confidence: 99%

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A developmentally regulated trans-acting factor recognizes dissimilar G/C-rich elements controlling a class of cAMP-inducible Dictyostelium genes.

Hjorth¹,

Pears²,

Williams³

et al. 1990

Genes Dev.

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Transcriptional response elements involved in the cAMP-inducible and developmentally regulated expression of the Dictyostelium aggregate-stage gene pst-cath/CP2 have been shown to include a G/C-rich sequence element [G-box regulatory element {GBRE1]. We have recently identified a trans-acting factor, GBF (GBRE binding factor), that specifically interacts with this sequence and have shown that the binding activity of GBF to GBRE is developmentally regulated and inducible by cAMP. Here, we examine further the possible role of GBF in the regulation of pst-cath/CP2 and three other coordinately regulated, cAMP-inducible aggregate-stage genes. We show that GBF itself {or other closely related factorsl recognizes dissimilar G/C-rich elements present in the 5'-flanking regions of these genes and that the ability of the individual, distinct G/C-rich elements to confer regulated expression on a promoter deletion mutant of the pst-cath/CP2 gene is correlated with the relative affinity for GBF. G/C-rich elements carrying point mutations that prevent in vitro binding of GBF to two of the G/C-rich elements fail to activate expression in vivo. An analysis of major points of contact between the GBF protein and two distinctly different binding sites suggests that binding of GBF to these sequence elements involves a considerable degree of flexibility in DNA-protein interactions. These results suggest that the regulated expression of a class of aggregate-stage cAMP-inducible genes involves the interaction of GBF or homologous factors with dissimilar G/C-rich sequence elements and that induction of GBF activity or that of homologous factors by cAMP may thus be a limiting step in the induction of this temporally coordinate set of genes during Dictyostelium development. Dictyostelium discoideum propagates as a vegetative amoeba in the presence of nutrients and initiates, upon starvation, a multicellular differentiation process that results in the formation of a mature fruiting body containing two distinctly different cell types--stalk cells and spores. Within several hours of starvation, Dictyostelium cells aggregate in response to pulsatile cAMP signals. Binding of cAMP to cell-surface receptors activates adenylate cyclase, and repeated cycles of cAMP synthesis and secretion serve to transmit, or relay, the chemotactic signal outward through the population of aggregating cells (see Janssens and van Haastert 1987;Firtel et al. 1989). After formation of tightly associated multicellular aggregates, a migrating pseudoplasmodium (slug) is formed in which precursor cells to the 3Corresponding author.

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“…This fragment was placed in an antisense orientation in three different Dictyostelium transformation vectors. The promoters controlling expression of the antisense transcripts were the actin 6 promoter in pDNeoII (Witke et al, 1987) and the D19 (Early and Williams, 1989) and CP2 (Pears et al, 1985;Pears and Williams, 1987) promoters in vectors derived from pB1OTP1 (Early and Williams, 1987).…”

Section: Construction Of Vectors Expressing Antisensementioning

confidence: 99%

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

Liu

Williams

Clarke

1992

MBoC

100

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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.

show abstract

Identification of a DNA sequence element required for efficient expression of a developmentally regulated and cAMP-inducible gene of Dictyostelium discoideum

Cited by 81 publications

References 57 publications

Two distinct populations of prestalk cells within the tip of the migratory Dictyostelium slug with differing fates at culmination

Two distinct populations of prestalk cells within the tip of the migratory Dictyostelium slug with differing fates at culmination

A developmentally regulated trans-acting factor recognizes dissimilar G/C-rich elements controlling a class of cAMP-inducible Dictyostelium genes.

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

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