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The cloning by complementation of the pawn-A gene in Paramecium
W J Haynes1, B Vaillant, R R Preston
1Laboratory of Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Abstract:
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.
Insights
Researchers cloned a gene responsible for a calcium current defect in Paramecium, enabling normal avoidance behavior. This breakthrough in Paramecium genetics utilized DNA injection and sequencing to identify the novel protein involved in signal transduction.
Area of Science:
- Cellular biology
- Genetics
- Neuroscience
Background:
- Ion channels are crucial for signal transduction in behaviors like avoidance.
- Paramecium pawn mutants exhibit defects in voltage-dependent calcium currents, affecting backward swimming.
- Cloning genes in Paramecium is challenging due to its large, polyploid macronucleus.
Purpose of the Study:
- To clone the gene responsible for the pawn mutant phenotype in Paramecium tetraurelia.
- To identify the molecular basis of the defective calcium current and excitability.
- To demonstrate a novel method for gene cloning in Paramecium.
Main Methods:
- Functional screening of a fractional DNA library by injecting it into pawn mutant Paramecium.
- Cloning and sequencing of a DNA fragment that restored wild-type behavior.
- RNase protection assays and sequencing of mutant alleles and cDNA to identify the gene and its product.
Main Results:
- A 2.3-kb DNA fragment was identified that restored calcium current and excitability in pawn mutants.
- The cloned gene encodes a novel protein, potentially glycophosphatidylinositol-anchored.
- The cloned gene successfully rescued the pawn phenotype, restoring normal avoidance behavior.
Conclusions:
- The study successfully identified a gene critical for calcium channel function and excitability in Paramecium.
- The developed DNA injection and screening method offers a viable approach for cloning other genes in Paramecium.
- This research advances our understanding of the molecular mechanisms underlying simple behavioral responses in single-celled organisms.