Kit-Yin Ling

Mutations in paramecium calmodulin indicate functional differences between the C-terminal and N-terminal lobes in vivo

We examined calmodulin and its gene from the wild-type and viable mutants of P. tetraurelia. The mutants, selected for their behavioral aberrations, have little or no defects in growth rates, secretion, excretion, or motility. They can be grouped according to whether they underreact or overreact behaviorally to certain stimuli, reflecting their respective loss of either a Ca2(+)-dependent Na+ current or a Ca2(+)-dependent K+ current. Sequence analyses showed that all three underreactors have amino acid substitutions in the N-terminal lobe of the calmodulin dumbbell, whereas all three overreactors have substitutions in the C-terminal lobe. No mutations fell in the central helix connecting the two lobes. These results may indicate that the sites defined by these mutations are important in membrane excitation but not in other biological functions. They also suggest that the two lobes of calmodulin may be used differentially for the activation of different Ca2(+)-dependent channels.

In vivo Paramecium mutants show that calmodulin orchestrates membrane responses to stimuli

Paramecium generates a Ca2+ action potential and can be considered a one-cell animal. Rises in internal [Ca2+] open membrane channels that specifically pass K+, or Na+. Mutational and patch-clamp studies showed that these channels, like enzymes, are activated by Ca(2+)-calmodulin. Viable CaM mutants of Paramecium have altered transmembrane currents and easily recognizable eccentricities in their swimming behavior, i.e. in their responses to ionic, chemical, heat, or touch stimuli. Their CaMs have amino-acid substitutions in either C- or N-terminal lobes but not the central helix. Surprisingly, these mutations naturally fall into two classes: C-lobe mutants (S101F, I136T, M145V) have little or no Ca(2+)-dependent K+ currents and thus over-react to stimuli. N-lobe mutants (E54K, G40E+D50N, V35I+D50N) have little or no Ca(2+)-dependent Na+ current and thus under-react to certain stimuli. Each mutation also has pleiotropic effects on other ion currents. These results suggest a bipartite separation of CaM functions, a separation consistent with the recent studies of Ca(2+)-ATPase by Kosk-Kosicka et al. [41, 55]. It appears that a major function of Ca(2+)-calmodulin in vivo is to orchestrate enzymes and channels, at or near the plasma membrane. The orchestrated actions of these effectors are not for vegetative growth at steady state but for transient responses to stimuli epitomized by those of electrically excitable cells.

Toward Cloning Genes by Complementation in Paramecium

Conventional methods of gene cloning by complementing mutant defects is made difficult by the 800 ploidy of the Paramecium macronucleus. However, this nucleus is some 30 microns in diameter and readily propagates exogenous DNA fragments as cells divide. These attributes allow for massive injection of engineered DNA fragments and their maintenance in the transformed descendant. If a genomic DNA fraction injected into a mutant macronucleus effects complementation, it should be possible to sort a fractional library to isolate the complementing gene. Here, we investigated four aspects of establishing this method for general use. First, using the cloned CAM gene as a test case, we further investigated transformation by macronuclear injection and showed that phenotypic reversion is directly correlated with the copy number of the transgene, even when it is of a recessive allele, cam2, which has a missense mutation but produces a partially functional protein. Second, we examined the copy number of the transgene established in cells of older clonal age and discussed the likely dilution of the transgene in younger descendants of the injected cell. Third, we showed that the degree of phenotypic reversion is correlated with the transgene product, the cam2 calmodulin protein in the cell. Fourth, we extended the investigation to very recessive mutants whose genes are to be cloned. We showed that size fractions of wild-type genomic DNA digests effect strong phenotypic reversions in several pawn mutants, setting the stage for cloning these Ca(2+)-channel related genes. The general usefulness of this method in cloning genes that complement recessive alleles and current limitations of this method in dealing with dominant alleles are assessed and discussed.

PAK Paradox: Paramecium Appears To Have More K+-Channel Genes than Humans

ABSTRACT K+-selective ion channels (K+ channels) have been found in bacteria, archaea, eucarya, and viruses. In Paramecium and other ciliates, K+ currents play an essential role in cilia-based motility. We have retrieved and sequenced seven closely related Paramecium K+-channel gene (PAK) sequences by using previously reported fragments. An additional eight unique K+-channel sequences were retrieved from an indexed library recently used in a pilot genome sequencing project. Alignments of these protein translations indicate that while these 15 genes have diverged at different times, they all maintain many characteristics associated with just one subclass of metazoan K+ channels (CNG/ERG type). Our results indicate that most of the genes are expressed, because all predicted frameshifts and several gaps in the homolog alignments contain Paramecium intron sequences deleted from reverse transcription-PCR products. Some of the variations in the 15 genomic nucleotide sequences involve an absence of introns, even between very closely related sequences, suggesting a potential occurrence of reverse transcription in the past. Extrapolation from the available genome sequence indicates that Paramecium harbors as many as several hundred of this one type of K+-channel gene. This quantity is far more numerous than those of K+-channel genes of all types known in any metazoan (e.g., ∼80 in humans, ∼30 in flies, and ∼15 in Arabidopsis). In an effort to understand this plurality, we discuss several possible reasons for their maintenance, including variations in expression levels in response to changes in the freshwater environment, like that seen with other major plasma membrane proteins in Paramecium.

Recent Advances in the Molecular Genetics of Paramecium1

Abstract Paramecium continues to be used to study motility, behavior, exocytosis, and the relationship between the germ and the somatic nuclei. Recent progress in molecular genetics is described. Toward cloning genes that correspond to mutant phenotypes, a method combining complementation with microinjected DNA and library sorting has been used successfully in cloning several novel genes crucial in membrane excitation and in trichocyst discharge. Paramecium transformation en masse has now been shown by using electroporation or bioballistics. Gene silencing has also been discovered in Paramecium, recently. Some 200 Paramecium genes, full length or partial, have already been cloned largely by homology. Generalizing the use of gene silencing and related reverse-genetic techniques would allow us to correlate these genes with their function in vivo.