Mating types in the ciliate Dileptus anser. Genetical instability in the mating type system

In F1 and F2 from a cross between two clones of Dileptus anser isolated from natural sources (MT 1 x x MT III; MT = mating type), along with "normal" clones, many clones were observed demonstrating abnormal phenotype with respect to the MT-character. Irregular features of the latter were as follows: a) a delay in maturation; b) temporary reversion to immature or adolescent state, which means instability of maturity state; c) expression of MT I and MT III, rather than MT II as in properly matured clones; d) changes in MT (i.e., MT instability); e) appearance of totally unexpected MTs in terms of the scheme of genetic control of MTs in D. anser previously suggested by Afon'kin and Yudin (1987)--e.g., of all three MTs in F1 from the initial (analysing!) cross. Amazingly, these abnormal D. anser clones closely resembled some selfer-clones of Tetrahymena pigmentosa, previously reported as an example of genetic instability in the ciliate MT system (Simon, 1980; Simon, Orias, 1987).     

Serotypes of the lower ciliate Dileptus anser

The author's data on the serotype composition in various clones of Dileptus anser are reviewed. This species is a novel subject of choice for studying the serotype problem in ciliates. The results obtained for serotypes of D. anser demonstrated several features shared also by some higher ciliates of the genera Paramecium and Tetrahymena, i.e. the well known classical models for serotype research. At the same time, some particular data obtained for D. anser do not fit in with the generally accepted picture of relevant events. Special attention is paid to a poor knowledge of the sexual process in this ciliate, which obviously hampers the true understanding of many events. Nevertheless, D. anser may be regarded as a suitable model for studying such important problems of general biology as epigenetic variability or inheritance.     

Nuclear differentiation for mating types in the ciliate Dileptus anser: a hypothesis

We have obtained the first data on inheritance and genetic determination of mating types (MTs) in the lower ciliate Dileptus anser (=D. margaritifer). Observation of MT instability in young, just matured exconjugant clones obtained from some (not all) crosses appears to be the key finding in our study. In a clone of this kind, the states of maturity and immaturity (or adolescence) often alternate and/or one MT changes to another, sometimes repeatedly, during the period of several weeks after the clone's maturation. On occasion, all three MTs found in this species can be expressed consecutively. All cells of the culture are synchronously involved in such changes of their sexual state--no spontaneous selfing (intraclonal conjugation) was ever observed in such cultures. These observations suggest that the mat locus in dilepti is a compound integral one; it is inherited as a whole and can specify expression of any one of possible MTs (much as it occurs in Tetrahymena thermophila). Some other mechanisms, supposedly epigenetic ones among them, control what MT will be expressed in a given exconjugant clone in particular. Steady functioning of these mechanisms provides stable, unambiguous differentiation of the compound mat locus to one and only one MT and subsequent Mendelian behavior of the character over sexual generations. If, for some still unknown reasons the control of such differentiation is disturbed, MT expressed in a given exconjugant clone becomes unstable and its Mendelian behavior can be violated.     

The hormone-sensitive adenylyl cyclase system of the ciliate Dileptus anser

The hormone-sensitive adenylyl cyclase system (AC system) was found and characterized for unicellular eukaryotes--the ciliatae Dileptus anser. It has been first shown that hormones of higher eukaryotes--biogenic amines (adrenalin, isoproterenol and serotonin) and peptide glucagon--stimulate in dose-dependent manner the activity of adenylyl cyclase (AC) of D. anser. The enzymatic activity was stimulated also by guanine nucleotides--GTP and their non-hydrolysable analogue Gpp[NH]p. Stimulating effects of hormones and guanine nucleotides strongly depend on the level of AC basal activity, which is relatively easy to reach (1430 to 3900 pmol cAMP/min per 1 mg of protein). The sensitivity of D. anser AC system to hormones and guanine nucleotides shows the presence of receptor or receptor-related molecules, capable of interacting with the hormone and activating AC through heterotrimeric G-proteins, in ciliatae. On the base of obtained data, a conclusion is made about the similarity of the structural-functional organization of AC systems of D. anser and higher eukaryotes.     

Mode of serotype inheritance in exoconjugant progeny of the ciliate Dileptus anser

Two clones of Dileptus anser, originally isolated from natural reservoirs and referred to below as B and D clones, were found to display different serotypes, when cultured under identical laboratory conditions. On being tested with two different polyclonal rabbit immune sera against each particular clone (the classic immobilization test) these clones showed no cross-reaction. At a standard dilution (1:50) and at a standard exposure time (4 h), either of the two immune sera immobilized 100% or commonly 0% of homologous and heterologous clone cells, respectively. In addition, the difference in serotypes was confirmed by the immunofluorescence analysis. By crossing (conjugation) between B (mating type I) and D (mating type III) cells, exconjugant F1 clones were obtained. Their serotypes were then tested (the same immobilization test) with antisera against both the "parental" clones: some clones were tested before their sexual maturation in ca. one month after conjugation, while others were examined in approximately 4 months after conjugation, i.e. after reaching maturity. Each of the F1 clones could react with both immune sera, which means that they possessed the intermediate, "hybrid" phenotype. Five different F1 clones were selected, and each of them was back-crossed to both "parental" clones, B and D. We succeeded in raising 25 exconjugant F2 (B1, to be more exact) clones from F1 x B crosses and 26 clones from F1 x D crosses. The conventional testing of these clones in 5-10 weeks after conjugation provided quite unexpected results, since among them no segregation for "parental" serotypes was observed. Each of the 51 tested clones demonstrated the "hybrid" serotype--seemingly the same as that of F1 clones. Such a non-Mendelian inheritance of the character is hardly to explain from the standard, canonical assumptions on the genetic control of serotype difference between original "parental" clones (different alleles in one locus? different loci?). Also it does not seem likely that the absence of segregation could result from differential survival of various phenotypes in F2 (although the total viability of exconjugant clones appeared rather low). The above data obviously need further confirmations and experimental analyses. We attempt to discuss the obtained results in terms of the epigene hypothesis (Tchuraev, 1975) and in relation to the epigenetic control of serotype expression in species of the Paramecium aurelia complex and in Tetrahymena thermophila, which are "the chosen few" subjects in ciliate genetics.     

Mating types in the ciliate Dileptus anser. Inheritance and genetic determination

Hybridological analysis of mating types (MTs) has been first made for the lower ciliate Dileptus anser. Clones of an initially unknown genotype belonging to three MTs (MT I, MT II and MT III), characteristic of D. anser, were isolated from natural reservoirs and further used for crosses. In one group crosses, synclonal inheritance and typical Mendelian behaviour of the character were observed over sexual generations of ciliates. The results suggest that MTs in D. anser may be directly controlled by a single mat locus with three alleles showing peck-order dominance (mat1 > mat2 > mat3). In other words, cells with mat1/mat1, mat1/mat2 and mat1/mat3 genotypes belong to MT I, those with mat2/mat2 and mat2/mat3, and the mat3/mat3 belong to MT II and MT III, respectively. Sexually mature exconjugant clones stably retain their MTs corresponding to their genotypes on vegetative reproduction. The progeny of other group crosses showed various deviations from typical Mendelian behaviour of the character. In some cases, standard Mendelian ratios were more or less violated. Most typical was instability of differentiation for MT in maturing exconjugant clones. Shortly after their maturation, the majority of clones change their MT, rather frequently more than once, although the finally established MT is stably inherited afterwards, during vegetative reproduction. When unstable, exconjugant clones can successively express two or even three MTs characteristic of this species, including MTs that should not have been expected on the basis of parental genotypes available in a given cross. It looks likely that the mat locus in D. anser is complex and multipotential; it is inherited as a whole providing for expression of any MT characteristic of the species (in this respect bearing similarity with Tetrahymena thermophila). Other mechanisms, epigenetic in particular (Nanney, 1958), determine the final expression of one of the three MT potentialities by a given exconjugant clone. Stable, persistent functioning of these mechanisms ensures a stable differentiation for MT and Mendelian behaviour of the character in sexual generations and in crosses. Any disturbances in differentiation control may trigger MT instability in maturing exconjugant clones and violation of regular Mendelian behaviour.     

Inhibitory action of polycationic peptides on hormonal regulation of adenylyl cyclase of the ciliate Dileptus anser

To analyse molecular mechanisms of regulatory action of different hormones on the activity of the adenylyl cyclase signaling system (ACS) of the ciliate Dileptus anser, we studied the influence on this process of six synthetic polycationic peptides and peptides, corresponding to C-terminal regions of mammalian G-protein 385-394 alphas- and 346-355 alphai2-subunits. As we reported earlier, these peptides block hormonal signal transduction in tissues of the higher eukaryotes. Now it has been found that both polycationic peptides, containing hydrophobic C to-radicals, and branched peptides decrease regulatory effects of peptide hormones (insulin, relaxin) and biogenic amines (serotonin, adrenaline) on adenylyl cyclase (AC) activity and GTP-binding. In regard to the following peptides Cys-epsilonAhx-Trp-Lys-Lys(C10)-Lys2-Lys(C10)-Lys3-Lys(C10)-Tyr-Lys-Lys(C10)-Lys-Lys-amide and [(Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro- Pro-Gly)2-Lys-EAhx-Cys]2 (epsilonAhx - E-aminocaproyl, C10 - caprinoyl group) their dose-dependent inhibitory action is shown. In cell culture of D. anser with a lower basal AC activity, both hydrophobic and branched peptides stimulated AC and GTP-binding without hormones. The data give evidence that these peptides can activate ACS of ciliates in a receptor-independent manner. No influence of peptides 385-394 alphas and 346-355 alphai2 on hormonal signal transduction in D. anser was observed, due, presumably, to some structural differences of G-proteins of the lower and higher eukaryotes. A conclusion was made about an important role of polycationic regions for functional coupling of hormone-activated receptor and G-proteins in the ciliate D. anser.     

Mating types in the ciliate Dileptus anser. Shortening of maturation period after micrurgical bisection of exconjugant cells

In many ciliates, young exconjugant clones demonstrate sexual immaturity: they are not able to conjugate with mature cells of complementary mating types (MTs). After several scores of cell divisions, a short period of adolescence (partial maturity) commonly occurs followed by maturation, after which these cells are able to conjugate with cells of other, i.e. complementary, MTs again. Tavrovskaja (1981) reported a significant reduction in the maturity period in Dileptus anser clones, grown from exconjugant ciliates regenerated from small cell fragments. To verify this, in the present study exconjugant D. anser cells were bisected with glass needle after 3 to 4 cell divisions following conjugation. The same procedure was performed with survived and regenerated cells on the 2nd and 3rd days. The clones thus obtained were cultivated, and their ability to mate with each of the three standard clones of MT I, II and III was tested week by week. Indeed, in 22 F1 clones from cross N 7C (MT I) x N 2 (MT II) the immaturity period was reduced 1.4-3.4-fold (2.18 in average) after a threefold bisection, as compared with that in intact subclones. Similarly, in 27 F1 clones from cross N 20 (MT I) x N 14 (MT II) this period was shortened 1.6-3.0-fold (2.19 in average). 12 of these clones showed a 0.9-2.4-fold (1.53 in average) reduction after a single bisection, and 1.6-2.8 (2.12 in average) after a threefold operation. Thus, micrurgical fragmentation of young exconjugant cells can be used to accelerate maturation in D. anser.     

Fifty years of research on serotypes and mating types in Dileptus anser: A review

The ciliate Dileptus anser is increasingly used as a laboratory model not only in protozoological research sensu stricto, but also in general biology. However, genetic studies of this ciliate have never been carried out, and this species is new to the comparative genetics of ciliates. This review describes the genetic experiments conducted at the Institute of Cytology of the Russian Academy of Sciences for the last 50 years. Two characters that are classical for the genetics of ciliates, serotypes and mating types were selected for analysis. The results presented do not fit into conventional genetic schemes and may have epigenetic nature. Features of this model that were revealed earlier (the simplest possible system of multiple mating types, full serial dominance of the alleles in the mat locus, the excretion of pheromones, etc.) are promising with regard to interesting comparisons of breeding systems in ciliates. The results obtained in studies of mating pheromones in D. anser have demonstrated that this model is a perspective one for further exploration of intercellular recognition in lower eukaryotes and of other related issues.     

Inhibition of functional activity of the adenylyl cyclase signaling system of the ciliate Dileptus anser by colchicine and vinblastine

Cytoskeleton plays a key role in the functioning of hormonal signaling systems in vertebrate animals. However, data on the effect of cytoskeletal components, in particular microtubules, on the functional activity of chemosignaling systems of unicellular organisms are currently lacking. The goal of this work consisted of studying the effects of microtubule-disrupting agents, colchicine and vinblastine, on the adenylyl cyclase system of free living infusoria Dileptus anser. The incubation of D. anser with colchicine and vinblastine (10^?5–10^?6 M) weakly affected the basal activity of adenylyl cyclase (AC), but led to a significant decrease in or complete block of AC stimulation with nonhormonal (GppNHp, sodium fluoride) and hormonal agents (adrenaline, serotonin, glucagon). The basal level of GTP binding in heterotrimeric G proteins decreased and there was observed inhibition of stimulation of G proteins by hormones. Colchicine and vinblastine have been shown to interrupt adrenalin-produced AC stimulation achieved through G_s-protein, but weakly affect its inhibiting AC effect caused by the G_i-protein. Thus, it has been established for the first time that, in unicellular organisms, i.e., infusoria D. anser, microtubules are involved in the regulation of the functional activity of the AC system and their action is realized at the level of G proteins, which is similar to Gs-proteins in vertebrate animals.     

The Predatory Behavior of Dileptus anser*

SYNOPSIS. A motion picture study of the feeding behavior of Dileptus anser upon Colpidium campylum revealed that feeding begins shortly before dawn (about 4:30 a.m. Pacific DST in July and August) and continues until bright daylight, terminating sharply between 8:30 and 9:00 a.m. Little or no feeding occurs at any other time during a 24 hr cycle. A “biological clock” phenomenon is suggested, but was not further investigated. Attack by Dileptus often results in “escape” behavior by Colpidium. When struck (usually antero-dorsally), the latter rotates, usually adhering to the microscope slide at the end which was struck, constricting the undamaged part of the body, pulling free, swimming off and regenerating if not again struck by Dileptus. After successive attacks and feeding, the proboscis of Dileptus becomes adhesive and sticks to the microscope slide near its tip. Dileptus jerks the proboscis free, sacrificing the attached portion. Several successive such truncations may occur as Dileptus feeds, so that at the end of a feeding period only a stub of the proboscis may remain. The entire proboscis is regenerated before the next feeding period begins. Ingestion of Colpidium by Dileptus appears to involve 2-way streaming of cytoplasm which results in a retreat of cytoplasm from the region of the cytostome toward the rear, forming an ingestion cavity. The cytostome simultaneously opens widely to accommodate passage of prey, even before the prey reaches the cytostome, and is often opened wider than the diameter of the prey. Ingestion is assumed to involve protoplasmic movements other than those only of the fibrillar organelles in the region of the cytostome.     

Actinomycin D-induced apoptosis involves the potassium channel Kv1.3

Several cytostatic agents are known to induce apoptosis in T-leukemic cells. Although a variety of studies show the central role of apoptosis in cytostatic drug-induced cell death, many molecular details require definition. Here, we demonstrate that cells genetically deficient for the potassium channel Kv1.3 are resistant to apoptosis initiated by the cytostatic drug actinomycin D. Retransfection of Kv1.3 restores sensitivity of the cells to actinomycin D. Cells lacking Kv1.3 fail to respond to actinomycin D with DNA fragmentation, release of cytochrome c, and loss of mitochondrial membrane potential (Delta Psi(m)), while cells functionally expressing Kv1.3 rapidly undergo those changes indicative for apoptosis. The data indicate a central role of the ion channel Kv1.3 in actinomycin D-triggered apoptosis.     

Effect of biologically active agents on the feeding response of Dileptus anser]

A study was made of the effects of pharmacological agents--adrenaline, noradrenaline, serotonine, dibenamine, theophylline, and emidazole--on the phagocytar activity of Dileptus anser. These effects were estimated in terms of food response changes towards chemical food models (CFM) made in cysteine, lecithine or tween-40 solutions. The above pharmacological agents were also tested as phagocytosis inductors. Of these, only adrenalin appeared to be an effective inductor of the food response. The CFM, made in adrenaline, was stimulated by 10(-6) M theophylline, and inhibited with 10(-4)--10(-8) M imidazole. The addition of 10(-3)--10(-12) M adrenaline or 10(-8)--10(-10) M serotonine to the Dileptus-containing medium stimulated phagocytosis of CFM. 5.10(-6) M dibenamin decreased phagocytotic intensity of CFM. 10(-6) M theophylline stimulated, while 10(-4) M inhibited the food response. It is proposed that protozoans have receptors capable of accepting hormones. A possible role of the system of cyclic AMP in transporting hormonal and food stimules in protozoans is discussed.     

Re-expression of various i-antigens in Dileptus anser after temporary transformation of serotype

RNP particles containing 20S prosomes (alpha RNP) isolated from human epidermoid carcinoma cell line A-431 are shown to posses strong and regulated endonuclease activity specific for high-molecular-weight RNA, particularly, specific mRNAs. Furthermore, alpha-RNP destabilize the 3'-untranslated regions of c-myc mRNA, creating a specific cleavage pattern. Cleavage point within Alu sequence in high-molecular-weight RNA has been localized by primer-extension method. This RNase activity is induced under the action of EGF. alpha-RNP involvement in the coordinated control of processing and stability of specific messenger RNA molecules is suggested. The endoribonuclease activity of alpha-RNP can represent a link between EGF signalling pathway and RNA processing and degradation.     

Regulatory calcium effect on adenylyl cyclase functional activity in the infusorian Dileptus anser

Earlier we have shown that some non-hormonal activators of adenylyl cyclase (AC) and hormones of higher vertebrate animals are able to affect functional activity of the AC system in the infusorian Dileptus anser. In the present work, sensitivity of this infusorian AC to Ca2+ was studied and it was found that calcium cations at concentrations of 0.5-10 microM stimulated significantly the enzyme activity in D. anser partially purified membranes. An increase of Ca2+ concentrations to 100 microM and higher led to the complete block of their stimulatory effect. In the EDTA-treated membranes the enzyme activity was reduced markedly, but it was restored significantly by addition of Ca2+. Calmodulin antagonists--chlorpromazine, W-7, and W-5--caused a dose-dependent decrease of the enzyme activity stimulated by 5 microM Ca2+ with IC50 values of 35, 137, and 174 microM, respectively. The AC-stimulating effects of biogenic amines (serotonin and octopamine) were completely retained in the presence of 2.5 and 100 microM Ca2+, whereas effects of peptide hormones (relaxine and EGF) were hardly changed in the presence of 2.5 microM calcium ions, but were markedly inhibited by 100 microM Ca2+. In the EDTA-treated membranes, the AC effects of biogenic amines were reduced, while the effects of peptide hormones were not revealed. On addition of Ca2+, the AC effects of biogenic amines were completely restored, whereas the effects of peptide hormones were not detected or were restored to a non-significant degree. Calmodulin antagonists slightly affected the AC effects of peptide hormones at concentrations efficient in the case of vertebrate AC, but decreased them markedly at higher concentrations. The AC effects of biogenic amines were little sensitive even to high antagonist concentrations. The obtained data show that targets of action of peptide hormones in the infusorian D. anser cell culture are the AC forms whose activity does not D. depends on calcium cations and possibly is regulated by Ca2+/calmodulin, whereas targets of action of biogenic amines are calcium-independent enzyme forms.     

Preparatory changes and the development of the conjugation junction in a ciliate,Dileptus

Summary The present observations concern changes in the cortical structure of the ciliateDileptus prior to and during conjugation. The results can be summarized as follows: (1) Mating cells join each other in heteropolar configuration, i.e., the distal part of the oral apparatus of one partner faces the proximal part of the oral apparatus of another partner. (2) The structural changes prior to conjugation occur in the area of the oral apparatus. (3) The bonding area is situated within the oral apparatus, while the oral ciliature that encircles the oral apparatus remains outside the bonding area. (4) The fusion area is formed within the bonding area as a newly formed zone, without cortical organelles typically associated with the cell membrane. Two aspects of the formation of cell-to-cell union inDileptus are discussed: firstly, the heteropolar configuration of mating cells and its possible bearing upon an ability to form double cells when partners separation is inhibited. Secondly, the bonding area ofDileptus is compared to bonding areas of peniculine and hymenostome ciliates. A striking likeness in structural changes was found within the bonding areas of all three ciliates, in spite of the fact that the changes in question are localized within the oral apparatus ofDileptus, while inParamecium andTetrahymena the changed territory is situated between the oral apparatus and the anterior pole of the cell.     

Actinomycin D-induced phenocopies in Drosophila melanogaster and their relevance to the time of gene action

Drosophila melanogaster larvae of a wild-type and a mutant stock, cultured in an axenic, chemically defined medium, were treated for one day with different concentrations of actinomycin D at different stages of development. Phenocopies affecting various organs of the adult occurred in different frequencies and in different patterns depending on the age at treatment. Assuming that the induced phenocopies were due primarily to the inhibition of DNA-dependent RNA synthesis by actinomycin D, the differential phenocopy effect indicates that: (1) Many genes which affect the differentiation of imaginal discs are activated in the third larval instar. (2) The developmental timing of gene activation in the third instar differs for various genes within a imaginal disc and in different imaginal discs.Submitted in partial fulfillment of the requirements for the Ph. D. degree. Supported by U.S. Public Health Service Grant GM1 1537 to I. H. Herskowitz.