Efficient transformation of cam2, a behavioral mutant of Paramecium tetraurelia, with the calmodulin gene.
An Ile-136----Thr substitution in calmodulin reduces the Ca(2+)-dependent K+ currents of cam2, a behavioral mutant of Paramecium tetraurelia, and renders it overly susceptible to BaCl2. DNA fragments carrying the wild-type CAM gene injected into cam2 macronuclei reverted these phenotypes in the clonal descendants of the recipients. Tetrahymena telomeric sequences, added in vitro to the fragment termini before injection, enhanced the efficiency and quality of transformation. Five times 10(4) copies of such fragments consistently restored the phenotypes to near normal; even 10(3) or fewer copies could still effect weak transformation. The restored phenotypes were stable for greater than 20 fissions in many clones and were lost after autogamy. We examined the fate of the injected fragments in the transformed clones and discuss the possible application of this efficient transformation in the cloning of other genes of P. tetraurelia.
Kanabrocki
J A
JA
Laboratory of Molecular Biology, University of Wisconsin-Madison 53706.
Saimi
Y
Y
Preston
R R
RR
Haynes
W J
WJ
Kung
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001464:Barium / Q000494:pharmacology; D017609:Barium Compounds; D001483:Base Sequence; D002147:Calmodulin / Q000235:genetics*; D002712:Chlorides; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D008564:Membrane Potentials / Q000187:drug effects; D008845:Microinjections; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010247:Paramecium / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D015221:Potassium Channels / Q000502:physiology; D015183:Restriction Mapping; D014170:Transformation, Genetic
D017609:Barium Compounds; D002147:Calmodulin; D002712:Chlorides; D015221:Potassium Channels; C024986:barium chloride; D001464:Barium; D004247:DNA