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C Kung

Publications and source records attributed to C Kung.

143 records · Page 8Linked to original sources
PubMed1976

A 'TEA+-insensitive' mutant with increased potassium conductance in Paramecium aurelia.

A single-gene mutant of Paramecium aurelia is analysed electrophysiologically. (a) The regenerative Ca-response, triggered by small or moderate current, was smaller and slower in the mutant than in wild type. (b) Input resistance of the mutant membrane is about half of that of wild type bathed in various solutions. This is true for the zero-current input resistance and the chord resistance measured with high depolarizing current. (c) Membrane resistance of the mutant measure with hyperpolarizing currents is smaller than that of wild type only when K+ is the major external cation. (d) Internally applied TEA+ or externally applied Ba+ increases the membrane resistance of the mutant to that of wild type similarly treated. We conclude that the mutant has an increased K conductance.

Action Potentials↗
PubMed1975

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens.

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.

Antigens↗
PubMed1975

Genetic dissection of the excitable membrane of Paramecium.

Paramecium aurelia is chosen as the material for a genetic dissection of the excitable membrane. The simple innate behavior makes the behavioral analyses easy and mutant screening possible. Autogamy, which leads to complete homozygosity, guarantees the expression of recessive mutations. The size of these giant cells allows intracellular recording in electrophysiological studies.--Some two hundred lines of behavioral mutants are isolated. Several of them are studied in detail genetically, behaviorally as well as electrophysiologically. Mutants devoid of proper active electrogenesis are now available. Some of them are shown to be temperature-dependent. They have a slightly aberrant electric pattern when grown at the permissive temperatures and lose their excitability completely when cultured at the restrictive temperatures.--Systematic modifications of the membrane excitation process is demonstrated in this interdisciplinary study. Three unlinked mutations are shown to block the generation of Na-triggered depolarizations at different positions resulting in three different altered forms of electrical activities. The bioelectric profiles of 25 types of single or double mutants are now known. The search for the relevant gene products on the surface membrane is now underway.

Animals↗
PubMed1974

Membrane excitability: made temperature-dependent by mutations.

Three mutants of Paramecium aurelia with genetic lesions at two unlinked loci lost their ability to generate action potentials when grown at high temperatures. Action potentials found at room temperature were slightly aberrant. Kinetics of phenotypic changes after temperature shifts showed that excitation is not immediately sensitive to temperature change in these mutants. The initiation of an action potential must rely on many different gene products (presumably membrane proteins) which are open to modification by conditional as well as unconditional mutations.

Action Potentials↗
PubMed1973

Temperature-sensitive pawns: conditional behavioral mutants of Paramecium aurelia.

"Pawns" are mutants of Paramecium aurelia in which the process of calcium activation during membrane excitation is genetically impaired, with a corresponding loss of avoiding reactions. Mutants are selected that behave normally when grown at 23 degrees C but as pawns at 35 degrees C. The normal excitation can now be disrupted and restored in the same strain at will.

Animals↗
PubMed1973

Calcium-induced ciliary reversal in the extracted models of "Pawn", a behavioral mutant of Paramecium.

"Pawn," a genic mutant of Paramecium aurelia, cannot swim backward as the wild type can upon proper stimulation. In contrast, after membrane disruption by Triton X-100, the adenosine triphosphate-magnesium reactivated models of Pawns swim backward in the presence of calcium as wild-type models do. Thus, the mutant phenotype is due to an impairment in the membrane and not in the calcium-sensitive motile system.

Adenosine Triphosphate↗
PubMed1972

Genetic modification of electric properties in an excitable membrane (paramecium-calcium conductance-electrophysiological measurements-membrane mutant).

A behavioral deficiency produced by a single gene mutation in Paramecium aurelia was traced to impaired electric excitability of the cell membrane. Evidence is presented that the mutant membrane does not exhibit the normal depolarization-activated increase in calcium conductance responsible for regenerative depolarization in the wild type. Other electric properties characteristic of the wild-type membrane remain normal in the mutant.

Alleles↗
PubMedDate not supplied

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.

Amino Acid Sequence↗
PubMedDate not supplied

Efficient expression of the Paramecium calmodulin gene in Escherichia coli after four TAA-to-CAA changes through a series of polymerase chain reactions.

We have expressed the Paramecium calmodulin gene in Escherichia coli by changing the four TAA codons in this gene to CAAs. This was carried out by three polymerase chain reactions (PCRs) and then cloning the product into the expression vector pKK223-3 immediately downstream of its trp-lac hybrid promoter. JM109 strain of E. coli, transformed with the recombinant plasmid harboring the altered Paramecium calmodulin gene, produces a protein judged to be calmodulin. It is recognized by a monoclonal antibody to Paramecium calmodulin; it migrates with the native protein at nearly the same rate in electrophoreses; and it shows a Ca(2+)-dependent shift in electrophoretic pattern. The production of calmodulin is about 170 times as efficient with E. coli as with Paramecium in terms of unit volume of packed cells, and is about 400 times as efficient in unit volume of liquid culture. This method appears useful in site-directed mutageneses and in the heterologous productions of other ciliate proteins. A critique of this method is provided. A calmodulin half-molecule, a by-product of this project, is described.

Amino Acid Sequence↗
PubMedDate not supplied

Induction of antibiotic resistance in Paramecium tetraurelia by the bacterial gene APH-3'-II.

We have generated a transformation marker for Paramecium using a Paramecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'-phosphotransferase-II (APH-3'-II or neor) from the transposon Tn5. The expression vector contained a small multiple cloning site between the 5' and 3' non-coding regions of the calmodulin gene, and Tetrahymena telomere sequences for the stability of the plasmid in Paramecium. After the neor ORF was inserted, the plasmid was referred to as pPXV-NEO. Delivery of approximately 10-20 picoliters of linearized PXV-NEO at > or = 2000 copies/pl into the macronucleus effected 100% transformation. Southern and Northern blot hybridization showed the presence of neor-specific DNA and RNA, respectively, in all of the transformed clones but not in the untransformed clones. The degree of resistance to G-418, and the concentrations of neor-specific DNA and neor-specific RNA in the clones were proportional to the concentration of the vector injected. We have demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon-usage differences.

Animals↗
PubMedDate not supplied

A comparison of internal eliminated sequences in the genes that encode two K(+)-channel isoforms in Paramecium tetraurelia.

We examined both the somatic (macro-) and the germinal (micronuclear) DNAs that encode two K(+)-channel isoforms, PAK1 and PAK11, in Paramecium tetraurelia. The coding regions of these two isoforms are 88% identical in nucleotides and 95% identical in amino acids. Their introns are also highly conserved. Even some of the internal eliminated sequences in PAK1 and PAK11 are clearly related. PAK1 has five IESs; PAK11 has four. The first (5'-most) IESs of the two genes are located at the same site in the coding sequence but differ in size. The 2nd IES in PAK1 (206-bp), the largest among the nine IESs, has no PAK11 counterpart. The 3rd, 4th and 5th IESs in PAK1 have a counterpart in PAK11 that is similar in size and in sequence, and identical in its position in the coding sequence. In addition, the first IES of PAK11 bears some resemblance to the 4th one of PAK1. The similarities and differences between the two sets of IESs are discussed with respect to the origin and divergence of the two K(+)-channel isoforms.

Amino Acid Sequence↗
PubMedDate not supplied

Recent advances in the molecular genetics of Paramecium.

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.

Animals↗