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A Mendelian mutation affecting mating-type determination also affects developmental genomic rearrangements in Paramecium tetraurelia.

In Paramecium tetraurelia, mating type is determined during the differentiation of the somatic macronucleus from a zygotic nucleus genetically competent for both types, O and E. Determination of the developing macronucleus is controlled by the parental macronucleus through an unknown mechanism resulting in the maternal inheritance of mating types. The pleiotropic mutation mtFE affects macronuclear differentiation. Determination for E is constitutive in mutant homozygotes; a number of unrelated mutant characters are also acquired during development. We have examined the possibility that the mutation causes a defect in the developmental rearrangements of the germ-line genome. We show that the excision of an IES (internal eliminated sequence) interrupting the coding sequence of a surface antigen gene is impaired in the mutant, resulting in an alternative macronuclear version of the gene. Once established, the excision defect is indefinitely transmitted across sexual generations in the cytoplasmic lineage, even in a wild-type genetic context. Thus, the processes of mating-type determination and excision of this IES, in addition to their common sensitivity to the mtFE mutation, show a similar maternal inheritance of developmental alternatives in wild-type cells, suggesting a molecular model for mating-type determination.

Animals↗

Phenotypic and genetic analysis of "Chameleon," a paramecium mutant with an enhanced sensitivity to magnesium.

Three mutant strains of Paramecium tetraurelia with an enhanced sensitivity to magnesium have been isolated. These new "Chameleon" mutants result from partial- or codominant mutations at a single locus, Cha. Whereas the wild type responded to 5 mM Mg2+ by swimming backward for 10-15 sec, Cha mutants responded with approximately 30 sec backward swimming. Electrophysiological analysis suggested that this behavior may be caused by slowing in the rate at which a Mg(2+)-specific ion conductance deactivates following membrane excitation. This would be consistent with an observed increase in the sensitivity of Cha mutants to nickel poisoning, since Ni2+ is also able to enter the cell via this pathway. More extensive behavioral analysis showed that Cha cells also overresponded to Na+, but there was no evidence for a defect in intracellular Ca2+ homeostasis that might account for a simultaneous enhancement of both the Mg2+ and Na+ conductances. The possibility that the Cha locus may encode a specific regulator of the Mg(2+)- and Na(+)-permeabilities is considered.

Animals↗

The cloning and molecular analysis of pawn-B in Paramecium tetraurelia.

Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca(2+) current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca(2+) current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5' neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

Animals↗

ND9P, a novel protein with armadillo-like repeats involved in exocytosis: physiological studies using allelic mutants in paramecium.

In Paramecium, a number of mutants affected in the exocytotic membrane fusion step of the regulated secretory pathway have been obtained. Here, we report the isolation of one of the corresponding genes, ND9, previously suspected to encode a soluble protein interacting with both plasma and trichocyst membranes. Nd9p is a novel polypeptide that contains C-terminal Armadillo-like repeats. Point mutations were found in the first N-terminal quarter of the molecule and in the last putative Armadillo repeat, respectively, for the two thermosensitive mutants, nd9-1 and nd9-2. The different behaviors of these mutants in recovery experiments upon temperature shifts suggest that the N-terminal domain of the molecule may be involved in membrane binding activity, whereas the C-terminal domain is a candidate for protein-protein interactions. The nonsense nd9-3 mutation that produces a short N-terminal peptide has a dominant negative effect on the nd9-1 allele. We show here that, when overexpressed, the dominant negative effect can be produced even on the wild-type allele, suggesting competition for a common target. We suggest that Nd9p could act, like some SNARE proteins, at the membrane-cytosol interface to promote membrane fusion.

Algorithms↗

Isolation and characterization of magbane, a magnesium-lethal mutant of paramecium.

Discerning the mechanisms responsible for membrane excitation and ionic control in Paramecium has been facilitated by the availability of genetic mutants that are defective in these pathways. Such mutants typically are selected on the basis of behavioral anomalies or resistance to ions. There have been few attempts to isolate ion-sensitive strains, despite the insights that might be gained from studies of their phenotypes. Here, we report isolation of "magbane," an ion-sensitive strain that is susceptible to Mg2+. Whereas the wild type tolerated the addition of > or =20 mm MgCl2 to the culture medium before growth was slowed and ultimately suppressed (at >40 mm), mgx mutation slowed growth at 10 mm. Genetic analysis indicated that the phenotype resulted from a recessive single-gene mutation that had not been described previously. We additionally noted that a mutant that was well described previously (restless) is also highly sensitive to Mg2+. This mutant is characterized by an inability to control membrane potential when extracellular K+ concentrations are lowered, due to inappropriate regulation of a Ca2+-dependent K+ current. However, comparing the mgx and rst mutant phenotypes suggested that two independent mechanisms might be responsible for their Mg2+ lethality. The possibility that mgx mutation may adversely affect a transporter that is required for maintaining low intracellular Mg2+ is considered.

Animals↗

N-ethylmaleimide-sensitive factor is required to organize functional exocytotic microdomains in paramecium.

In exocytosis, secretory granules contact plasma membrane at sites where microdomains can be observed, which are sometimes marked by intramembranous particle arrays. Such arrays are particularly obvious when membrane fusion is frozen at a subterminal stage, e.g., in neuromuscular junctions and ciliate exocytotic sites. In Paramecium, a genetic approach has shown that the "rosettes" of intramembranous particles are essential for stimulated exocytosis of secretory granules, the trichocysts. The identification of two genes encoding the N-ethylmaleimide-sensitive factor (NSF), a chaperone ATPase involved in organelle docking, prompted us to analyze its potential role in trichocyst exocytosis using a gene-silencing strategy. Here we show that NSF deprivation strongly interferes with rosette assembly but does not disturb the functioning of exocytotic sites already formed. We conclude that rosette organization involves ubiquitous partners of the fusion machinery and discuss where NSF could intervene in this mechanism.

Animals↗

Clonal variation in paramecium. I. Persistent unstable clones.

Clones of Paramecium of identical serotype when cultured in test tubes may differ in their ability to give rise to subclones of this serotype. Characteristically, stable clones yield progeny indistinguishable from their parents, while from unstable clones diverse subclones with new serotypes can be isolated repeatedly. Stable lines are resistant to changes in culture medium and also are unaffected by most sera. In contrast, the numbers and kinds of serotypes displayed among subclones derived from unstable lines are often affected by these same agents. Stable and unstable clones are interconvertible when the medium from individual cultures is repeatedly and frequently replaced by fresh culture fluid. This effect is very likely a result of the removal of the initial exhausted medium with any cell products rather than the addition of fresh nutrient.

Animals↗

Clonal variation in paramecium. II. A comparison of stable and unstable clones of the same serotype.

Paramecium generally expresses only one antigen on its surface from among an array of antigens. This mutual exclusion of antigens now has been shown in certain instances to be illusory. Unstable clones which will give rise to subclones with new serotypes possess several antigens. Unstable clones, even though they manifest only one serotype, continually manufacture an antigen other than the surface antigen characteristic of the serotype.

Animals↗

Genetics of chemical induction of conjugation in Paramecium aurella.

Certain stocks of P. aurelia, syngen 8, could not be induced to conjugate in a solution (KCl + acriflavine + calcium-poor conditions) which was effective in inducing conjugation in other species of Paramecium as well as in other stocks of syngen 8. Both stocks could conjugate by interaction with cells of complementary mating type. Breeding analysis shows that each of the two stocks is homozygous for a recessive gene that blocks induction of conjugation by the KCl-acriflavine solution. These two genes are neither allelic nor linked. Analyses of the phenotypes of the two uninducibles and the wild type were carried out by attempting to induce mating in cells of a single mating type by exposing them to detached mating-reactive cilia from cells of complementary mating type and to the KCl-acriflavine solution, either sequentially or simultaneously. The results confirm the conclusions of others that there is at least one unique step in chemical induction not shared with induction by interaction of complementary mating types. But the results also indicate that there is more than one unique step in chemical induction and that the effects of the two genes described here operate during different periods of the hour required for chemical induction.

Acridines↗

Symposium No. 8: Non-chromosomal inheritance. Genetic control of mitochondria in paramecium.

Mitochondrial mutations for resistance to various antibiotics (erythromycin, chloramphenicol, spiramycin, mikamycin) have been obtained in Paramecium aurelia and their properties are reviewed. Using these mitochondrial markers, the interactions between nucleus and mitochondria have been studied in two ways: by microinjection of mitochondria from one stock or species into other stocks and species of P. aurelia and by a genetic study of a nuclear mutation affecting mitochondrial multiplication. Both types of experiments show: (1) that there may exist incompatibility between a given type of mitochondria and the cell into which they are introduced and (2) that through multiplication in the host cell, mitochondrial properties can be modified. The possible basis for incompatibility and host-induced modifications is discussed.

Anti-Bacterial Agents↗

A suppressor gene involved in chemical induction of conjugation in Paramecium aurelia.

A dominant gene Su(kau-2) partially suppresses the effect of an apparently unlinked recessive kau-2. Gene kau-2 blocks chemical induction of conjugation in Paramecium aurelia syngen 8 when solutions of acriflavine + either KCl or MgCl-2 are used. Wild-type cells are induced to conjugate in either solution. When cells homozygous for kau-2 also have gene Su(kau-2), they are still uninducible in the solution containing KCl, but become inducible in the MgCl-2 solution. Analysis of the concentrations of solutions which are effective in induction of conjugation of various genotypes shows that the action of Su(kau-2) is not a simple restoration of wild-type phenotype since certain novel features of the suppressor can be seen. Analysis of the duration of ciliary reversal of various genotypes suggests that one necessary step in chemical induction of conjugation is a certain magnitude of depolarization of the surface membrane of the cell.

Acriflavine↗

Genetic analysis of copper resistance in Paramecium aurelia syngen 4.

Measurement of the median tolerance limit to cupric ion of 28 wild stocks of Paramecium aurelia syngen 4 revealed a phenotypic polymorphism: two stocks showed a much greater resistance than the others. Genetic analysis showed the resistant phenotype was produced by a recessive allele, cur, identical in both resistant wild stocks. No modification of the phenotypic expression of this allele could be detected after backcrossing into different genetic backgrounds. The results are interpreted to support the importance of mutational adaptation in this inbreeding species. Possible patterns of internal genetic organization of species which rely on mutation for adaptation are discussed.

Animals↗

Nonbehavioral selection for pawns, mutants of Paramecium aurelia with decreased excitability.

The reversal response in Paramecium aurelia is mediated by calcium which carries the inward current during excitation. Electrophysiological studies indicate that strontium and barium can also carry the inward current. Exposure to high concentrations of barium rapidly paralyzes and later kills wild-type paramecia. Following mutagenesis with nitrosoguanidine, seven mutants which continued to swim in the ;high-barium' solution were selected. All of the mutants show decreased reversal behavior, with phenotypes ranging from extremely non-reversing (;extreme' pawns) to nearly wild-type reversal behavior (;partial' pawns). The mutations fall into three complementation groups, identical to the pwA, pwB, and pwC genes of Kunget al. (1975). All of the pwA and pwB mutants withstand longer exposure to barium, the pwB mutants surviving longer than the pwA mutants. Among mutants of each gene, survival is correlated with loss of reversal behavior. Double mutants (A-B, A-C, B-C), identified in the exautogamous progeny of crosses between ;partial' mutants, exhibited a more extreme non-reversing phenotype than either of their single-mutant (;partial' pawn) parents.---Inability to reverse could be expected from an alteration in the calcium-activated reversal mechanism or in excitation. A normal calcium-activated structure was demonstrated in all pawns by chlorpromazine treatment. In a separate report (Schein, Bennett and Katz 1976) the results of electrophysiological investigations directly demonstrate decreased excitability in all of the mutants, a decrease due to an altered calcium activation. The studies of the genetics, the survival in barium and the electro-physiology of the pawns demonstrate that the pwA and pwB genes have different effects on calcium activation.

Animals↗

Genetic analyses of "paranoiac" mutants of Paramecium tetraurelia.

Six mutants of Paramecium tetraurelia with curious "Paranoiac" phenotypes have been isolated and examined. Instead of the normal transient avoiding reactions in Na+ solution, these mutants show "violent avoidances"--backing continuously for 10 to over 60 sec. This behavior corresponds to prolonged membrane excitation excitation.--Genetic analyses establish five genic loci at which mutations give the "Paranoiac" phenotype. Close linkage between two of these genes occurs. Allelic variants are found for two of the genes. In one case, the two alleles determine very different behavioral phenotypes ("Paranoiac" and "fast-2"). These results show that the mechanism(s) which shuts off excitation in the wild-type membrane is (are) complex, but in the future may be fruitfully pursued in mutants which are defective.

Animals↗

Age-correlated changes in expression of micronuclear damage and repair in Paramecium tetraurelia.

In Paramecium, age is defined as the number of mitotic divisions which have elapsed since the previous cross-fertilization (conjugation) or self-fertilization (autogamy). As the mitotic interval between fertilizations increases, the percentage of nonviable progeny clones increases. In the current study, resolution of conflicting previous reports on the pattern of increase of death and reduced viability in progeny from aging parent cells is found. Some exautogamous clones exhibit a high mortality at young clonal ages, others show no mortality throughout their life span, but most (73%) show an abrupt increase in the percent death and reduced viability in progeny from cells 50-80 fissions old. Ultraviolet-irradiation-induced micronuclear mutations, repairable by photoreactivation, increased with increased clonal age when monitored by percent death and reduced viability of exautogamous progeny of irradiated cells. Loss of dark repair is considered a contributor to the increased expression of micronuclear mutations with increased clonal age.

Cell Division↗

Mutants of sexual maturity in Paramecium caudatum selected by erythromycin resistance.

Cells of Paramecium caudatum, syngen 3 usually become sexually mature about 50 fissions after conjugation. In order to study the genetic mechanisms that control fission-department expression of maturity, an attempt was made to obtain early mature mutants by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. A new cytoplasmic marker, erythromycin resistance, was used to eliminate nonconjugant and macronuclear regeneration clones. Twenty early mature clones were obtained from five different mutagenized cultures. Three of them were genetically analyzed by crosses to wild-type stocks. The results show all three mutants to be controlled by incompletely dominant genes, i.e., the homozygotes became mature 20-25 fissions and the heterozygotes 15 fissions earlier than the wild-type clones. At least two different loci are suggested for the early maturity.

Animals↗

Genetic analysis of the relationships between the cell surface and the nuclei in Paramecium tetraurella.

In Paramecium tetraurelia, a number of mutations have been shown to affect simultaneously cortical organization (attachment of trichocysts to the cortex) and nuclear divisions (Ruiz et al. 1976). In order to analyze the genetic and physiological basis of this correlation, and we have isolated new mutations affecting the properties of the trichocysts and studied their genetic relationships with other previously known mutations. Of 24 to 28 loci controlling the biogenesis and properties of the trichocysts, mutations only in the 16 to 20 loci tht control trichocysts attachment to the cortex result in nuclear defects. Cytological observations show that all these mutants display the same set of nucler abnormalities: in particular, rounded shape of the resting macronucleus mispositioning and defective elongation of the dividing macronucleus and unequal repartition of the marco- and micronuclei. This common syndrome is independent of both the mutagenic origin and the mutated locus. Furthermore, by microinjection, it is possible to localize the site of action of the mutations in either the trichocyst compartment or the nontrichocyst compartment. It was found by this technique that the nuclear syndrome is also independent of the site of action of the mutation. All the genetic and physiological data support the conclusion that the nuclear defects are the consequence of the lack of trichocyst attachement to the cortex: in wild-type cells, trichocyst attachment would induce a membranar or perimembranar state necessary for correct nuclear positioning during cell division. In the absence of trichocyst attachment, the cortical control of nuclear division would be abolished. The possible involvement of cytoskeletal links between surface and nuclei is discussed.

Animals↗

Telomeric site position heterogeneity in macronuclear DNA of Paramecium primaurelia.

In Paramecium primaurelia, the macronuclear gene encoding the G surface protein is located near a telomere. In this study, multiple copies of this telomere have been isolated and the subtelomeric and telomeric regions of some of them have been sequenced. The telomeric sequences consist of tandem repeats of the hexanucleotides C4A2 or C3A3. We show that the location where these repeats are added, which we call the telomeric site, is variable within a 0.6-0.8-kb region. These results are discussed in relation with the formation of macronuclear DNA.

Animals↗