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.

     

Selection of the germinal micronucleus in Paramecium caudatum: nuclear division and nuclear death

Each cell of Paramecium caudatum has a germinal micronucleus. When a bi-micronucleate state was created artificially by micronuclear transplantation, both micronuclei divided for at least 2 cell cycles after nuclear transplantation. However, this bi-micronucleate state was unstable and reduced to a uni-micronucleate state after several fissions. Although the number of micronuclei was usually 1 during the vegetative phase, 4 presumptive micronuclei differentiated after conjugation. At the first post-conjugational fission, only 1 of the 4 micronuclei divided, indicating that there is tight regulation of micronuclear number in exconjugants. Micronuclei that did not divide at the first post-conjugational fission may persist through the first and second post-conjugational cell cycles. The decision to divide appears to be separate from the decision to degenerate, as evidenced by division of a remaining micronucleus upon removal of the dividing micronucleus at the first division. Degeneration of micronuclei in exconjugants differs from that of haploid nuclei after meiosis. Nutritional state affected micronuclear degeneration. Under well-fed conditions, the micronuclei destined to degenerate lost the ability to divide earlier than after starvation treatment, suggesting that micronuclear degeneration is an "apoptotic" phenomenon, probably under the control of the new macronuclei (macronuclear anlagen).     

Autoradiographic evidence for self-fertilization in Tetrahymena thermophila

Conjugation in Tetrahymena thermophila consists of a sequence of nuclear events, including meiosis and reciprocal cross-fertilization, which result in biparental genetic endowment of the sexual progeny. Genetic evidence was recently provided that the normal exchange of gametic nuclei between conjugating cells can be efficiently blocked by hyperosmotic shock. In this paper we confirm this finding autoradiographically. We also report that the inhibitor of microtubule assembly, vinblastine, also blocks this step, as well as the subsequent fusion of gametic nuclei. The ability of conjugating cells to survive and continue more or less normally after blocks of self-fertilization and pro-nuclear fusion demonstrates a surprisingly high degree of developmental regulation during conjugation. Self-fertilization has proven useful for the isolation of recessive mutants in T. thermophila.     

Repetitive Micronuclear Divisions in the Absence of the Macronucleus During Conjugation of Paramecium caudatum

ABSTRACT. The germinal micronucleus divides six times during conjugation of Paramecium caudatum : this includes two meiotic divisions and one mitosis of haploid nuclei during mating, and three mitoses of a fertilization nucleus (synkaryon). Microsurgical removal of the macronucleus showed that micronuclei were able to divide repeatedly in the absence of the macronucleus, after metaphase of meiosis I of the micronucleus and also after synkaryon formation. When the macronucleus was removed after the first division of synkaryon, in an extreme case the synkaryon divided five times and produced 32 nuclei, compared to three divisions and eight nuclei produced in the presence of the macronucleus. Treatment with actinomycin D (100 μ /ml) inhibited the morphological changes of the macronucleus during conjugation and induced a multimicronucleate state in exconjugants. However, in other cells, it induced production of a few giant micronuclei. We conclude that the micronucleus is able to undergo repeated divisions at any stage of conjugation in the absence of the macronucleus once the factor(s) for induction of the micronuclear division has been produced by the macronucleus. The macronucleus may also produce a regulatory factor required to stop micronucler division.     

ATM controls meiotic DNA double-strand break formation and recombination and affects synaptonemal complex organization in plants

Meiosis is a specialized cell division that gives rise to genetically distinct gametic cells. Meiosis relies on the tightly controlled formation of DNA double-strand breaks (DSBs) and their repair via homologous recombination for correct chromosome segregation. Like all forms of DNA damage, meiotic DSBs are potentially harmful and their formation activates an elaborate response to inhibit excessive DNA break formation and ensure successful repair. Previous studies established the protein kinase ATM as a DSB sensor and meiotic regulator in several organisms. Here we show that Arabidopsis ATM acts at multiple steps during DSB formation and processing, as well as crossover (CO) formation and synaptonemal complex (SC) organization, all vital for the successful completion of meiosis. We developed a single-molecule approach to quantify meiotic breaks and determined that ATM is essential to limit the number of meiotic DSBs. Local and genome-wide recombination screens showed that ATM restricts the number of interference-insensitive COs, while super-resolution STED nanoscopy of meiotic chromosomes revealed that the kinase affects chromatin loop size and SC length and width. Our study extends our understanding of how ATM functions during plant meiosis and establishes it as an integral factor of the meiotic program.

Arabidopsis ATM acts at multiple steps during DSB formation and processing, as well as crossover formation and synaptonemal complex organization, all vital for the successful completion of meiosis.     

Behavior of germinal micronuclei under control of the somatic macronucleus during conjugation in Paramecium caudatum.

In conjugating pairs of Paramecium caudatum, the micronuclear events occur synchronously in both members of the pair. To find out whether micronuclear behavior is controlled by the somatic macronucleus or by the germinal micronucleus, and whether or not synchronization of micronuclear behavior is due to intercellular communication between conjugating cells, the behavior of the micronucleus was examined after removal of the macronuclei from either or both cells of a mating pair at various stages of conjugation. When macronuclei were removed from both cells of a pair, micronuclear development was arrested 1 to 1.5 hr after macronuclear removal. When the macronucleus of a micronucleate cell mating with an amicronucleate cell was removed later than 3 to 3.5 hr of conjugation, that is, an early stage of meiotic prophase of the micronucleus, micronuclear events occurred normally in the operated cell. These results suggest that most micronuclear events are under the control of the macronucleus and that the gene products provided by the macronucleus are transferable between mating cells. One such product is required for induction of micronuclear division and is provided just before metaphase of the first meiotic division of the micronucleus. This factor is effective at a lower concentration in the cytoplasm and/or is more transferable between mating cells than the factors required for other stages. This factor, which seems to be present at least until the stage of micronuclear disintegration, is able to induce repeated micronuclear division as long as it remains active. The factor can act on a micronucleus which has not passed through a meiotic prophase. Moreover, the results suggest the existence of a second factor which is provided by the macronucleus after the first meiotic division that inhibits further micronuclear division.     

周期蛋白Cyc2的过量表达及突变抑制嗜热四膜虫有性生殖发育

周期蛋白在细胞增殖过程中呈现周期性表达变化,不同的周期蛋白通过结合周期蛋白激酶(cyclin-dependent kinase,CDKs)及靶向特定蛋白质参与细胞有丝分裂和减数分裂过程的精确调控。嗜热四膜虫有性生殖期特异表达的B3型周期蛋白Cyc2(cyclin 2,Cyc2)对减数分裂的启始是必需的,但Cyc2蛋白的分子调控机制并不清楚。本研究通过0.1μg/mL和0.3μg/mL镉离子诱导突变细胞株OE-CYC2-B2086和OE-CYC2-CU428中CYC2基因在金属硫蛋白1基因(metallothionein gene 1,MTT1)启动子调控下上调表达。实时荧光定量PCR检测突变株OE-CYC2-B2086和OE-CYC2-CU428中CYC2的转录水平分别上调7.8倍和9.8倍。细胞有性生殖发育进程的荧光显微观察发现CYC2的表达上调并不影响有性生殖前期减数分裂的启始,但是干扰四膜虫有性生殖后期中新大核和新小核的正确形成。同时突变株OE-CYC2-B2086和OE-CYC2-CU428交配后,在镉离子诱导下不能产生有性生殖后代,但是该突变株分别和两种不同野生型细胞或CYC2敲除的突变细胞株交配能够恢复产生3%,15%或32%的有性生殖后代,有性生殖发育异常程度与CYC2的表达水平成正相关。进一步突变Cyc2第312位磷酸化位点丝氨酸为丙氨酸,获得CYC2单位点突变细胞株CYC2-S312A-B和CYC2-S312A-C。丝氨酸单位点突变阻止了四膜虫有性生殖期小核减数第1次分裂起始。结果表明周期蛋白2的表达水平和磷酸化修饰影响了不同阶段细胞核的功能,周期蛋白2对四膜虫有性生殖发育的正常进行是必需的。     

The transition from conjugal development to the first vegetative cell division is dependent on RAD51 expression in the ciliate Tetrahymena thermophila

Rad51p, the eukaryotic homolog of the prokaryotic recA protein, catalyzes strand exchange between single- and double-stranded DNA and is involved in both genetic recombination and double-strand break repair in the ciliate Tetrahymena thermophila. We have previously shown that disruption of the Tetrahymena RAD51 somatic macronuclear locus leads to defective germline micronuclear division and that conjugation of two somatic rad51 null strains results in an early meiotic arrest. We have constructed Tetrahymena strains that are capable of RAD51 expression from their parental macronuclei and are homozygous, rad51 nulls in their germline micronuclei. These rad51 null heterokaryons complete all of the early and middle stages of conjugation, including meiosis, haploid nuclear exchange, zygotic fusion, and the programmed chromosome fragmentations, sequence eliminations, and rDNA amplification that occur during macronuclear development. However, the rad51 null progeny fail to initiate the first vegetative cell division following conjugal development. Coincident with the developmental arrest is a disproportionate amplification of rDNA, despite the maintenance of normal total DNA content in the developing macronuclei. Fusion of arrested rad51 null exconjugants to wild-type cells is sufficient to overcome the arrest. Cells rescued by cytoplasmic fusion continue to divide, eventually recapitulating the micronuclear mitotic defects described previously for rad51 somatic nulls.     

Ca2+ homeostasis regulates Xenopus oocyte maturation

In contrast to the well-defined role of Ca2+ signals during mitosis, the contribution of Ca2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca2+ deprivation. These findings argue that Ca2+ homeostasis is important in establishing the oocyte's competence to undergo maturation in preparation for fertilization and embryonic development.     

The requirements for protein synthesis and degradation, and the control of destruction of cyclins A and B in the meiotic and mitotic cell cycles of the clam embryo

Fertilization of clam oocytes initiates a series of cell divisions, of which the first three--meiosis I, meiosis II, and the first mitotic division--are highly synchronous. After fertilization, protein synthesis is required for the successful completion of every division except meiosis I. When protein synthesis is inhibited, entry into meiosis I and the maintenance of M phase for the normal duration of meiosis occur normally, but the chromosomes fail to interact correctly with the spindle in meiosis II metaphase. By contrast, inhibition of protein synthesis immediately after completion of meiosis or mitosis stops cells entering the next mitosis. We describe the behavior of cyclins A and B in relation to these "points of no return." The cyclins are synthesized continuously and are rapidly destroyed shortly before the metaphase-anaphase transition of the mitotic cell cycles, with cyclin A being degraded in advance of cyclin B. Cyclin destruction normally occurs during a 5-min window in mitosis, but in the monopolar mitosis that occurs after parthenogenetic activation of clam oocytes, or when colchicine is added to fertilized eggs about to enter first mitosis, the destruction of cyclin B is strongly delayed, whereas proteolysis of cyclin A is maintained in an activated state for the duration of metaphase arrest. Under either of these abnormal conditions, inhibition of protein synthesis causes a premature return to interphase that correlates with the time when cyclin B disappears.     

Parent Genotype and Environmental Factors Influence Introduction Success of the Critically Endangered Savannas Mint (Dicerandra immaculata var. savannarum)

Species previously unknown to science are continually discovered and some of these species already face extinction at the time of their discovery. Conserving new and rare species in these cases becomes a trial-and-error process and conservationists will attempt to manage them by using knowledge of closely related species, or those that fill the same ecological niche, and then adapting the management program as needed. Savannas Mint (Dicerandra immaculata Lakela var. savannarum Huck) is a perennial plant that was discovered in Florida scrub habitat at two locations in 1995, but is nearly extinct at these locations. We tested whether shade, leaf litter, propagation method, parent genotype, parent collection site, planting date, and absorbent granules influenced survival, reproduction, and recruitment of Savannas Mint in a population of 1,614 plants that we introduced between June 2006 and July 2009 into a state protected site. Survival and reproduction of introduced plants, and recruitment of new plants, was higher in microhabitats in full sun and no leaf litter and lower in partially shaded habitats. The two sites from which parent plants were collected differentially influenced survival and reproduction of introduced plants. These differences in survival and reproduction are likely due to underlying genetic differences. Differential survival of progeny from different parent genotypes further supports the idea that underlying genetics is an important consideration when restoring plant populations. The most successful progeny of parent genotypes had survival rates nearly 12 times higher than the least successful progeny. We speculate that many of these environmental and genetic factors are likely to influence allopatric congeners and other critically endangered gap specialists that grow in Florida scrub and our results can be used to guide their conservation.     

Store-operated calcium entry inactivates at the germinal vesicle breakdown stage of Xenopus meiosis

Store-operated calcium entry (SOCE) is the predominant Ca(2+) influx pathway in non-excitable cells and is activated in response to depletion of intracellular Ca(2+) stores. We have studied SOCE regulation during Xenopus oocyte meiosis. SOCE can be measured readily in stage VI Xenopus oocytes arrested at the G(2)-M transition of the cell cycle, either by Ca(2+) imaging or by recording the SOCE current. However, following meiotic maturation, SOCE can no longer be activated by store depletion. We have characterized the time course of SOCE inactivation during oocyte maturation, and show that SOCE inactivates almost completely, in a very short time period, at the germinal vesicle breakdown stage of meiosis. This acute inactivation offers an opportunity to better understand SOCE regulation.     

Micromere fate maps in leech embryos: lineage-specific differences in rates of cell proliferation

Stereotyped early cleavages in glossiphoniid leech embryos yield 25 micromeres, along with 3 macromeres and 10 teloblasts. The micromeres generate prostomial tissues and also give rise to most of the squamous epithelium of a provisional integument that spreads epibolically from the animal pole, covering the rest of the embryo during germinal plate formation.We systematically injected individual micromeres with fluorescent cell lineage tracers at the time of their birth and quantitatively mapped the contributions of all these cells to the late stage 7 embryo, a time in development that is early in the epibolic expansion. At this time, micromere derivatives comprise two types of cells: squamous epithelial (superficial) cells that cover the germinal bands and the region of the animal cap between the germinal bands; and underlying (deep) cells that are confined to the distal ends of the germinal bands and in the area between their distal ends. We find that individual micromeres contribute clones of deep and/or superficial progeny that are stereotyped with respect to both numbers and types of cells in the clone and the domains that they occupy. The N teloblasts also contribute cells to the squamous epithelium.We find significant differences in the rate of cell proliferation between different micromere clones. These differences appear to reflect lineage-specific traits, since there is little or no regulation of cell number after ablation of individual micromeres.     

The ecology and genetics of fitness in Chlamydomonas. XIII. Fitness of long-term sexual and asexual populations in benign environments

We measured the mean fitness of populations of Chlamydomonas reinhardtii maintained in the laboratory as obligately sexual or asexual populations for about 100 sexual cycles and about 1000 asexual generations. Sexuality (random gamete fusion followed by meiosis) is expected to reduce mutational load and increase mean fitness by combining deleterious mutations from different lines of descent. We found no evidence for this process of mutation clearance: the mean fitness of sexual populations did not exceed that of asexual populations, whether measured through competition or in pure culture. We found instead that sexual progeny suffer an immediate loss in fitness, and that sexual lines maintain genetic variance for fitness. We suggest that sexual populations at equilibrium with selection in a benign environment may be mixtures of several or many epistatic genotypes with nearly equal fitness. Recombination between these genotypes reduces mean fitness and creates genetic variance for fitness. This may provide fuel for continued selection should the environment change.     

Cell cycle mutation in Paramecium tetraurelia discriminates between sexual and vegetative functions

The temperature-sensitive mutation cc1 blocks a number of cell cycle processes in Paramecium including macronuclear DNA synthesis, oral morphogenesis, and the later stages of micronuclear mitosis. Oral morphogenesis and micronuclear mitosis also occur in the sexual pathway. This study shows that cc1 cells can proceed through conjugation or autogamy under restrictive conditions; neither stomatogenesis nor micronuclear mitosis is blocked. Fertilization and macronuclear determination occur normally, but DNA synthesis in macronuclear anlagen is blocked. Therefore, this mutation discriminates between oral replacement during meiosis and vegetative prefission stomatogenesis, and between mitotic spindle elongation during the pregamic and postzygotic divisions and spindle elongation during the vegetative cell cycle. These results point to a fundamental regulatory difference between morphogenesis in the vegetative and sexual pathways. © 1994 Wiley-Liss, Inc.     

Cytogenetic Studies on Five Intermediate Forms of Progeny of Triticum aestivum Agropyron glaucum

A stuty has been made of the chromosome number of root-tip cells and the chro- mosome configuration of PMCs in five intermediate forms of progeny of Triticum aestivum × Agropyron glaucum. The somatic cells in each of these intermediate forms have 28 pairs of chromosomes (2n=56) and the PMCs at diakinesis or metaphase Ⅰ of meiosis have 28 bivalents. The process of meiosis in most PMCs is regular. Lagging chromosomes and fragments at anaphase Ⅰ or telophase Ⅰ were observed only in few PWCs. According to our results, all the five intermediate forms of progeny of T. aestivum × Ag. glaucum are allooctoploids. The application of these allooctoploids is also briefly discussed.     

Expression of female sterility in alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.)

Female sterility associated with the presence of callose in the nucellus at anthesis was studied in an F₁ progeny of two alfalfa plants displaying 5 and 81% ovule sterility. Transgressive segregation was observed and 100% sterile plants were obtained. Two of the sterile plants were used for cytological analyses on sectioned and stain-cleared whole ovules, in comparison to a 100% fertile full sib plant. The first sign of sterility was callose deposition in the nucellus cell walls surrounding the sporogenous cells of the young ovules. At the same stage, no trace of callose was present in ovule primordia of the fertile plant. Megaspore mother cells differentiated in both fertile and sterile ovules and meiosis was initiated, as indicated by chromatin patterning typical of a zygotene stage. However, meiosis was never completed in the sterile plants. In the control, callose was deposited around the meiocyte and as sects between the cells of the dyads and tetrads during meiosis, and disappeared after the completion of meiosis; an embryo sac developed and female fertility was normal. In the sterile ovules, some nucellus cells enlarged and callose accumulation continued forming thick deposits. At anthesis, the sterile ovules lacked an embryo sac and showed massive callose accumulation in the nucellus. Male fertility was normal in female-sterile plants, thus a female-specific arrest of sporogenesis appears to be the cause of sterility. Pistil development was aberrant in some sterile genotypes, even with arrested pistil growth in early flower buds.     

Germline and Somatic Transformation of Mating Tetrahymena Thermophila by Particle Bombardment

Mating Tetrahymena thermophila were bombarded with ribosomal DNA-coated particles at various times in development. Both macronuclear and micronuclear transformants were recovered. Optimal developmental stages for transformation occurred during meiosis for the micronucleus and during anlagen formation for the macronucleus. Evidence is given for transient retention of the introduced plasmid. Genetic and molecular tests confirmed that sexually heritable transformation was associated with integration at the homologous site in the recipient micronuclear chromosome.