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.     

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.     

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).     

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.     

Actinomycin D-induced change of mating type in the ciliate Dileptus anser

The effects of actinomycin D on the expression and inheritance of mating types (MTs) were studied in mature laboratory clones of the ciliate Dileptus anser. In these ciliates, each mature clone isolated from the natural population belongs to one of three complementary MTs, i.e., MT I, MT II, or MT III. In the course of further cultivation of the clone under laboratory conditions, in a series of vegetative generations, its MT remained unchanged. However, treatment with actinomycin D (15 μg/ml, 3 days) causes these clones to transition to a state that is hereditarily unstable for their MTs. At weekly testing for MT for at least 15 weeks after this treatment (which corresponds to more than 100 cell divisions), many subclones of the treated clone were observed to reversibly exchange their MT for another one; a temporary state of immaturity and/or partial maturity was also revealed. These data confirm our hypothesis about epigenetic MT determination in D. anser. Taking into account that actinomycin D also induces the inheritable destabilization of some characters in amoebas Amoeba proteus, which obviously has an epigenetic nature, this antibiotic might be considered an epimutagen.     

Studies on bacterial conjugational crosses via model populations. II. Genetic studies

Model populations, simulating progenies obtained from Escherichia coli Hfr x F- crosses, are generated. Linkage relations and exchange frequencies are expressed in these populations in complex, in some cases, even paradoxical manner. It is demonstrated that the origin of the complexities is located in the particular allelic configuration of the genotypes, characteristic for all exconjugant and selected populations.     

A new metallothionein gene from the giant keyhole limpet Megathura crenulata

Metallothioneins (MTs) are small soluble proteins ubiquitously expressed in animals and plants. Different isoforms are present in deuterostomes and protostomes. They do not differ greatly in primary structure, but are clearly distinguishable. Here, I present the gene and the complete cDNA of a novel MT from the mollusk Megathura crenulata. This protein is closely related to the Cu-inducible MTs of the vineyard snail Helix pomatia, but has also some minor sequence features typical of Cd-inducible isoforms of H. pomatia and other molluscs. Overall, the deduced primary structure is similar to the known molluscan MTs, but in addition possesses an insertion of 5 amino acids not found in any other molluscan MTs, protostomic or deuterostomic MTs. In addition, a pentapeptide insertion, characteristic of mammalian MT-3 is present but it lacks the functional tetrapeptide CPCP within the beta-region of those MT-3 proteins that are known to suppress neuronal growth processes. The M. crenulata MT is a novel form of MT in comparison to all other known MTs. Possible functional aspects for this new MT are discussed.     

Characterization of mat A-2, mat A-3 and deltamatA mating-type mutants of Neurospora crassa.

The mating-type locus of Neurospora crassa regulates mating identity and entry into the sexual cycle. The mat A idiomorph encodes three genes, mat A-1, mat A-2, and mat A-3. Mutations in mat A-1 result in strains that have lost mating identity and vegetative incompatibility with mat a strains. A strain containing mutations in both mat A-2 and mat A-3 is able to mate, but forms few ascospores. In this study, we describe the isolation and characterization of a mutant deleted for mat (deltamatA), as well as mutants in either mat A-2 or mat A-3. The deltamatA strain is morphologically wild type during vegetative growth, but it is sterile and heterokaryon compatible with both mat A and mat a strains. The mat A-2 and mat A-3 mutants are also normal during vegetative growth, mate as a mat A strain, and produce abundant biparental asci in crosses with mat a, and are thus indistinguishable from a wild-type mat A strain. These data and the fact that the mat A-2 mat A-3 double mutant makes few asci with ascospores indicate that MAT A-2 and MAT A-3 are redundant and may function in the same pathway. Analysis of the expression of two genes (sdv-1 and sdv-4) in the various mat mutants suggests that the mat A polypeptides function in concert to regulate the expression of some sexual development genes.     

Independent metal-binding features of recombinant metallothioneins convergently draw a step gradation between Zn- and Cu-thioneins

Data on the metal-binding behaviour of circa 20 recombinant metallothioneins (MTs) from evolutionary divergent organisms, gathered after years of systematic research, are here comprehensively analyzed. The consideration of four independent in vivo and in vitro metal-binding features reveals a gradation of the metal-binding character of the MTs considered that significantly coincides in a robust new classification: a stepwise gradation between Zn- and Cu-thioneins. The intermediate positions in this list are occupied by a group of polyvalent MTs, exhibiting a merging Zn-/Cu-thionein character that would suit general metal handling purposes. In contrast, the extreme positions are respectively occupied by those MTs that would have evolved to fulfil specialized Zn- or Cu-related physiological roles. Overall, the analyzed trends allow the proposal of a chemically- and biologically-sound new reflection on MT classification criteria.     

Effect of metallothionein on cell viability and its interactions with cadmium and zinc in HEK293 cells

Metallothioneins (MTs) are thought to participate in a wide variety of physiological roles, but the mechanisms involved are still unclear. The study was designed to examine the possible factors related to these mechanisms. Methods, including transfection, MTT assay and flow cytometry, were used to investigate the effect of MTs on cell viability and their interactions with cadmium and zinc in HEK293 cells. The results showed that transient overexpression of human MT1A, MT2 and MT3 genes dynamically affected cell viability, and the effect was influenced by zinc and cadmium ions. Overexpressed MTs with added zinc showed a greater inhibitory effect on cell viability. Overexpressed MTs protected cells against low concentrations of cadmium ions (10 microM), but increased cell death in response to high concentrations (20-50 microM). Out of the three MTs, MT1A was more efficient than MT2 and MT3 in its resistance to cadmium (10 microM), and MT3 together with zinc showed more cell growth inhibition than MT1 and MT2. These results indicate that both of the divalent metal ions that could bind MTs, as well as the individual MT isoforms, affect the role of MTs on cell viability, which may explain in part why the comprehensive effect of MTs on the cells was elusive.     

Unequal cell division and chromatin diffrentiation in pollen grain cells

The dynamics of the microtubule (MT) were studied by α-tubulin immunofluorescence methods during the polleng rain ontogeny inTradescantia paludosa. Before the microspore division, interphase nuclei of themicrospore cells were twice displaced from the center to one side (NM-1) and from the side to the center near the inner wall (NM-2). During NM-1, a few MTs appeared around the nucleus, but the movement was not interrupted by colchicine treatment. In NM-2, however, which was essential to the unequal division of microspores, many MTs and MT bundles became organized and shifted in a manner corresponding to the nuclear movement. This movement was inhibited by the colchicine treatment. It was concluded that NM-2 was dependent on the MT cytoskeleton, but NM-1 was independent. Througthout the microspore division, mitotic spindles were organized asymmetrically. From prophase to prometaphase, the spindle began to construct itself in the vegetative pole preceding the generative pole. The half-spindles were asymmetric at the metaphase and the phragmoplast developed curving toward the generative pole at the telophase. No pre-prophase band of MTs was observed throughout the cell cycle. The relationship between the characteristic MT dynamics and the nuclear movement, or unequal cell division, was revealed and is discussed here.     

Dynamic Organization of Plant Microtubules at the Three Distinct Transition Points During the Cell Cycle Progression of Synchronized Tobacco BY-2 Cells

Dynamic changes of microtubule (MT) configuration have been examined during the cell cycle progression in tobacco BY-2 cells, which have been highly synchronized by aphidicolin treatment. Although it has been shown previously that four cell cycle stages display characteristic features of MTs (Hasezawa et al., 1991), distinct changes of MT configuration were observed at the interfaces of G₂/M, M/G₁ and G₁/S, and the frequency of appearance of such distinct structures were quantitatively examined. Among others, it is the first observation that at M/G₁ disintegrating phragmoplasts coexisted with short MTs in the perinuclear envelopes, but the MTs disappeared in the later stage, when cortical MTs were organizing. Thus it is supposed that cortical MTs originate from the transiently observed short MTs in the perinuclear region. This observation offered also an experimental system to analyze the molecular changes of MTs at the three interfaces during cell cycle progression in plant cells, as the mass culture of tobacco BY-2 cells is readily available.     

Immunological distinction between piscine and mammalian metallothioneins

1. Separate antisera to metallothioneins (MT) from rainbow trout and horse were produced in mice and their reactivity with the respective immunogen was confirmed using an ELISA. 2. The ELISA, used in a competitive mode, revealed that the anti-horse MT serum did not cross-react with trout MT. Reciprocally, the anti-trout MT serum did not show any reactivity with horse MT. 3. The anti-rainbow trout MT serum was shown to cross-react totally with MTs from plaice, flounder, turbot, perch, salmon and pike, but exhibited no reactivity towards MTs from human, mouse, rat, worm or crab. Partial reactivity with the proteins isolated from oyster and mussel was demonstrated. 4. The anti-horse MT serum cross-reacted totally with MTs from human, rat and rabbit but no reactivity was demonstrable when MT from either plaice or worm was tested. 5. The behaviour of apo-, holo- and recombinant rainbow trout MTs, in which the metal content was different, indicated that reactivity with anti-MT antibodies was not dependent on the presence or nature of the metals bound in the protein. 6. The patterns of reactivities were analysed in relation to the known amino acid sequences of MT.     

Examining the specific contributions of individual Arabidopsis metallothioneins to copper distribution and metal tolerance

Metallothioneins (MTs) are small cysteine-rich proteins found in various eukaryotes. Plant MTs are classified into four types based on the arrangement of cysteine residues. To determine whether all four types of plant MTs function as metal chelators, six Arabidopsis (Arabidopsis thaliana) MTs (MT1a, MT2a, MT2b, MT3, MT4a, and MT4b) were expressed in the copper (Cu)- and zinc (Zn)-sensitive yeast mutants, Deltacup1 and Deltazrc1 Deltacot1, respectively. All four types of Arabidopsis MTs provided similar levels of Cu tolerance and accumulation to the Deltacup1 mutant. The type-4 MTs (MT4a and MT4b) conferred greater Zn tolerance and higher accumulation of Zn than other MTs to the Deltazrc1 Deltacot1 mutant. To examine the functions of MTs in plants, we studied Arabidopsis plants that lack MT1a and MT2b, two MTs that are expressed in phloem. The lack of MT1a, but not MT2b, led to a 30% decrease in Cu accumulation in roots of plants exposed to 30 mum CuSO(4). Ectopic expression of MT1a RNA in the mt1a-2 mt2b-1 mutant restored Cu accumulation in roots. The mt1a-2 mt2b-1 mutant had normal metal tolerance. However, when MT deficiency was combined with phytochelatin deficiency, growth of the mt1a-2 mt2b-1 cad1-3 triple mutant was more sensitive to Cu and cadmium compared to the cad1-3 mutant. Together these results provide direct evidence for functional contributions of MTs to plant metal homeostasis. MT1a, in particular, plays a role in Cu homeostasis in the roots under elevated Cu. Moreover, MTs and phytochelatins function cooperatively to protect plants from Cu and cadmium toxicity.     

Ultrastructure of the cytoskeleton in freeze-substituted pollen tubes ofNicotiana alata

Summary The ultrastructure of the cytoskeleton inNicotiana alata pollen tubes grownin vitro has been examined after rapid freeze fixation and freeze substitution (RF-FS). Whereas cytoplasmic microtubules (MTs) and especially microfilaments (MFs) are infrequently observed after conventional chemical fixation, they occur in all samples prepared by RF-FS. Cortical MTs are oriented parallel to the long axis of the pollen tube and usually appear evenly spaced around the circumference of the cell. They are always observed with other components in a structural complex that includes the following: 1. a system of MFs, in which individual elements are aligned along the sides of the MTs and crossbridged to them; 2. a system of cooriented tubular endoplasmic reticulum (ER) lying beneath the MTs, and 3. the plasma membrane (PM) to which the MTs appear to be extensively linked. The cortical cytoskeleton is thus structurally complex, and contains elements such as MFs and ER that must be considered together with the MTs in any attempt to elucidate cytoskeletal function. MTs are also observed within the vegetative cytoplasm either singly or in small groups. Observations reveal that some of these may be closely associated with the envelope of the vegetative nucleus. MTs of the generative cell, in contrast to those of the vegetative cytoplasm, occur tightly clustered in bundles and show extensive cross-bridging. These bundles, especially in the distal tail of the generative cell, are markedly undulated. MFs are observed commonly in the cytoplasm of the vegetative cell. They occur in bundles oriented predominantly parallel to the pollen tube axis. Although proof is not provided, we suggest that they are composed of actin and are responsible for generating the vigorous cytoplasmic streaming characteristic of living pollen tubes.Abbreviations EGTA ethylene glycol bis-(-aminoethyl ether), N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - MF microfilament - MT microtubule - PEG polyethylene glycol - PM plasma membrane - RF-FS rapid freeze fixation-freeze substitution     

Propagation of cortical differences in tetrahymena

Progeny clones were derived from crosses arranged so that the number of ciliary meridians (corticotype) was unusually high in one partner, and normal in the other. An analysis of the propagation of corticotypes during maintenance of these clones for up to 1,000 fissions indicated that corticotypes above 21 undergo a rapid downward shift, while corticotypes in the range of 18 to 21 change slowly. Although these observations are consistent with Nanney's earlier deduction of a "stability center" at corticotype 19, there appears to be little if any difference in the stability of perpetuation of corticotypes 18, 19 and 20. Within this "stability range," the inertia of maintenance of pre-existing corticotypes is sufficiently strong that sister clones derived from an exconjugant pair can remain different for 1,000 fissions. These findings are consistent with observations made earlier, and those in the present study, indicating that cells in stock cultures express a substantial range of corticotypes even when maintained with frequent transfer. The results suggest that mechanisms of spatially ordered structural assembly within the cell can show sufficient fidelity to allow long-term vegetative perpetuation of phenotypic differences without artificial selection.     

Evidence of simple genetic control in potato of ability to restrict potato leaf roll virus concentration in leaves

Summary The concentration of potato leaf roll virus (PLRV), measured by quantitative enzyme-linked immunosorbent assay, in foliage of plants of cv Maris Piper and clone G7445(1) with secondary infection was 2,700 ng/g leaf and 120 ng/g leaf, respectively. In experiments to examine the genetic control of their ability to restrict the multiplication of PLRV, reciprocal crosses were made between these two clones. Among 40 genotypes from the progeny of the crosses, about half had a low PLRV concentration in plants with secondary infection and the other half had a high concentration. The possibility of monogenic control of the character that restricts PLRV multiplication in such clones of Solanum tuberosum is discussed.