DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion.

Nellen, W; Silan, Colleen M.; Firtel, Richard A.

doi:10.1128/mcb.4.12.2890

Cited by 261 publications

(173 citation statements)

References 14 publications

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“…The coding sequences of ␥-tubulin and GFP are separated by a spacer of 4 amino acids (i.e., SRGS). The resulting vector, pB15␥tubGFP, was transformed into AX2 cells by using calcium phosphate (25).…”

Section: Methodsmentioning

confidence: 99%

Centrosome positioning and directionality of cell movements

Ueda¹,

Gräf²,

MacWilliams³

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

132

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In several cell types, an intriguing correlation exists between the position of the centrosome and the direction of cell movement: the centrosome is located behind the leading edge, suggesting that it serves as a steering device for directional movement. A logical extension of this suggestion is that a change in the direction of cell movement is preceded by a reorientation, or shift, of the centrosome in the intended direction of movement. We have used a fusion protein of green f luorescent protein (GFP) and ␥-tubulin to label the centrosome in migrating amoebae of Dictyostelium discoideum, allowing us to determine the relationship of centrosome positioning and the direction of cell movement with high spatial and temporal resolution in living cells. We find that the extension of a new pseudopod in a migrating cell precedes centrosome repositioning. An average of 12 sec elapses between the initiation of pseudopod extension and reorientation of the centrosome. If no reorientation occurs within approximately 30 sec, the pseudopod is retracted. Thus the centrosome does not direct a cell's migration. However, its repositioning stabilizes a chosen direction of movement, most probably by means of the microtubule system.

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Section: Methodsmentioning

confidence: 99%

Centrosome positioning and directionality of cell movements

Ueda¹,

Gräf²,

MacWilliams³

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

132

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show abstract

“…diseoideum strain AX3 was transformed by calcium-phosphate precipitation with vector pA15RM which harbours the actin6-NEO R selection cassette [12] and a gene fusion of the actinl 5 promoter directing expression of PKA-R subunit gene in which both cAMP binding sites where inactivated by site-directed mutagenesis yielding the mutant RM. Control cell lines (Rc) were transformed with vector pA15Rc which carries an additional mutation, that prevents interaction with the PKA-C subunit [5,6].…”

Section: Cell Lines and Culture Conditionsmentioning

confidence: 99%

cAMP‐dependent protein kinase activity is essential for preaggregative gene expression in Dictyostelium

Schulkes

Schaap

1995

FEBS Letters

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“…Dictyostelium transformation, culture and development Transformants of the axenic strain AX-2 were isolated as described previously (Nellen et al, 1984;Early and Williams, 1987) and grown in axenic medium containing G418 at 20 µg/ml. In our experience, the safest method of analysis is to study pooled populations derived from a large number of individual clones.…”

Section: Plasmid Constructs and Sequencingmentioning

confidence: 99%