An intragenic suppressor of a calmodulin mutation in Paramecium: genetic and biochemical characterization.
We describe a suppressor of the calmodulin mutant cam1 in Paramecium tetraurelia. The cam1 mutant, which has a SER----PHE change at residue 101 of the third calcium-binding domain, inhibits the activity of the Ca(2+)-dependent K+ current and causes exaggerated behavioral responses to most stimuli. An enrichment scheme, based on an increased sensitivity to Ba2+ in cam1 cells, was used to isolate suppressors. One such suppressor, designated cam101, restores both the activity of the Ca(2+)-dependent K+ current and behavioral responses of the cells. We show that the cam101 mutant is an intragenic suppressor of cam1, based on genetic and microinjection data. The cam101 calmodulin is shown to be similar to wild-type calmodulin in terms of its ability to stimulate calmodulin-dependent phosphodiesterase at low concentrations of free calcium. However, the cam101 calmodulin has a reduced affinity for a monoclonal antibody to wild-type Paramecium calmodulin, as does the parental cam1 calmodulin, and a different mobility on acid-urea gels relative to both wild-type and cam1 calmodulin. We have been able to demonstrate that the isolation of intragenic suppressors of a calmodulin mutation is possible, which allows for the further genetic analysis of structure-function relationships in the calmodulin molecule.
Hinrichsen
R D
RD
Fred Hutchinson Cancer Research Center, Division of Basic Sciences, Seattle, Washington 98104.
Pollock
M
M
Hennessey
T
T
Russell
C
C
D000818:Animals; D001522:Behavior, Animal / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D009154:Mutation; D010247:Paramecium / Q000235:genetics*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D015221:Potassium Channels / Q000502:physiology; D011485:Protein Binding
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D015221:Potassium Channels; D010727:Phosphoric Diester Hydrolases; D002118:Calcium