The cloning by complementation of the pawn-A gene in Paramecium.
The genetic dissection of a simple avoidance reaction behavior in Paramecium tetraurelia has shown that ion channels are a critical molecular element in signal transduction. Pawn mutants, for example, were originally selected for their inability to swim backward, a trait that has since been shown to result from the loss of a voltage-dependent calcium current. The several genes defined by this phenotype were anticipated to be difficult to clone since the 800-ploid somatic macronucleus of P. tetraurelia is a formidable obstacle to cloning by complementation. Nonetheless, when the macronucleus of a pawn mutant (pwA/pwA) was injected with total wild-type DNA or a fractional library of DNA, its clonal descendants all responded to stimuli like the wild type. By sorting a fractional library, we cloned and sequenced a 2.3-kb fragment that restores the Ca2+ current and excitability missing in pawn-A. Data from RNase protection assays, followed by the sequencing of mutant alleles and cDNA clones, established an open reading frame. The conceptually translated product suggests a novel protein that may be glycophosphatidylinositol anchored. We also discuss the general usefulness of this method in cloning other unknown DNA sequences from Paramecium that are functionally responsible for various mutant phenotypes.
Haynes
W J
WJ
Laboratory of Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Vaillant
B
B
Preston
R R
RR
Saimi
Y
Y
Kung
C
C
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames / Q000235:genetics; D016805:Paramecium tetraurelia / Q000235:genetics* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015800:Protozoan Proteins; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000235:genetics; D014170:Transformation, Genetic
D015220:Calcium Channels; D008565:Membrane Proteins; D015800:Protozoan Proteins; C114041:pwA protein, Paramecium tetraurelia; D002118:Calcium