A genetic screen for vegetative gene expression in the micronucleus of Tetrahymena thermophila.

The presence of a micronucleus with at least a small portion of the micronuclear genome appears to be indispensable for vegetative viability in the ciliate Tetrahymena thermophila. A genetic screen was devised to detect evidence of expression of essential genes in the vegetative micronucleus by identification of thermosensitive-lethal mutations expressed in the absence of nuclear reorganization. Although control experiments demonstrated the efficacy of the method for induction and recovery of thermosensitive lethal mutations in micronuclear genes, no expressed mutations were recovered in the absence of nuclear reorganization. This finding complements the existing lack of convincing biochemical evidence for gene expression in the vegetative micronucleus and suggests that the essential function may involve genomic DNA sequences for which thermosensitive mutant alleles are not recoverable, or perhaps a non-genomic component of the organelle.     

"Fenestrin" and Conjugation in Tetrahymena thermophila

ABSTRACT Certain monoclonal antibodies interact with proteins of Tetrahymena thermophila found in the conjugation junction as well as around the gametic nuclei (pronuclei) of conjugating cells; they also react with the oral primordium and fission zone of vegetative cells and with the cytoproct and contractile vacuole pores of all cells. One of these (FXIX-3A7) was investigated in detail. Immunogold labelling suggests that the material labelled by the 3A7 monoclonal antibody, which we call “fenestrin,” is located beneath the epiplasm (membrane skeleton). Immunoblots reveal that the major and perhaps sole antigen is a 64 kDa polypeptide, found in two isoelectric variants. Developmental studies implicate fenestrin in two processes involved in conjugation. The first is “tip transformation.” During preliminary starvation (“initiation”), labelling of fenestrin first appeared as a spot at the anterior end of starved mature cells, then after mixing of different mating types (“costimulation”) it extended posteriorly along the anterior suture. After pairing, this region spread to form a widened plate. The second process is pronuclear transfer. Fenestrations representing channels between the conjugating cells began to appear 0.5 to 1 h after the conjugants united, and eventually merged to form a small number of temporary large holes during exchange of the transfer pronuclei. A fenestrin envelope also enclosed both the transfer and resident pronuclei; a strand of fenestrin connected the two. Shortly after pronuclear transfer, both transfer and resident pronuclei were released from fenestrin caps and fused to produce a zygotic nucleus (synkaryon) not associated with fenestrin. Fenestrin thus appears to be intimately involved in the process of pronuclear exchange.     

TIF1 activates the intra-S-phase checkpoint response in the diploid micronucleus and amitotic polyploid macronucleus of Tetrahymena

The ribosomal DNA origin binding protein Tif1p regulates the timing of rDNA replication and is required globally for proper S-phase progression and division of the Tetrahymena thermophila macronucleus. Here, we show that Tif1p safeguards chromosomes from DNA damage in the mitotic micronucleus and amitotic macronucleus. TIF1p localization is dynamically regulated as it moves into the micro- and macronucleus during the respective S phases. TIF1 disruption mutants are hypersensitive to hydroxyurea and methylmethanesulfonate, inducers of DNA damage and intra-S-phase checkpoint arrest in all examined eukaryotes. TIF1 mutants incur double-strand breaks in the absence of exogenous genotoxic stress, destabilizing all five micronuclear chromosomes. Wild-type Tetrahymena elicits an intra-S-phase checkpoint response that is induced by hydroxyurea and suppressed by caffeine, an inhibitor of the apical checkpoint kinase ATR/MEC1. In contrast, hydroxyurea-challenged TIF1 mutants fail to arrest in S phase or exhibit caffeine-sensitive Rad51 overexpression, indicating the involvement of TIF1 in checkpoint activation. Although aberrant micro- and macronuclear division occurs in TIF1 mutants and caffeine-treated wild-type cells, TIF1p bears no similarity to ATR or its substrates. We propose that TIF1 and ATR function in the same epistatic pathway to regulate checkpoint responses in the diploid mitotic micronucleus and polyploid amitotic macronucleus.     

DNA Damage Response in Nucleoli

Molecular Biology - Nucleoli, the largest subnuclear compartments, are formed around arrays of ribosomal gene repeats transcribed by RNA polymerase I. The primary function of nucleoli is ribosome...     

The CNA1 histone of the ciliate Tetrahymena thermophila is essential for chromosome segregation in the germline micronucleus

Ciliated protozoans present several features of chromosome segregation that are unique among eukaryotes, including their maintenance of two nuclei: a germline micronucleus, which undergoes conventional mitosis and meiosis, and a somatic macronucleus that divides by an amitotic process. To study ciliate chromosome segregation, we have identified the centromeric histone gene in the Tetrahymena thermophila genome (CNA1). CNA1p specifically localizes to peripheral centromeres in the micronucleus but is absent in the macronucleus during vegetative growth. During meiotic prophase of the micronucleus, when chromosomes are stretched to twice the length of the cell, CNA1p is found localized in punctate spots throughout the length of the chromosomes. As conjugation proceeds, CNA1p appears initially diffuse, but quickly reverts to discrete dots in those nuclei destined to become micronuclei, whereas it remains diffuse and is gradually lost in developing macronuclei. In progeny of germline CNA1 knockouts, we see no defects in macronuclear division or viability of the progeny cells immediately following the knockout. However, within a few divisions, progeny show abnormal mitotic segregation of their micronucleus, with most cells eventually losing their micronucleus entirely. This study reveals a strong dependence of the germline micronucleus on centromeric histones for proper chromosome segregation.     

Nucleoli of cultured human lymphocytes

Summary Nucleolar fusion and nucleolus formation occurred simultaneously, immediately after mitosis, in cultured human lymphocytes. Evidence is presented that in late telophase and post-telophase, the individual nucleolar organising site includes two components, represented in post-telophase by the nucleolus and its attached process; that fusion can be either directly between the nucleoli or via the processes; and that the latter type of fusion persists into metaphase as acrocentric associations, whilst the former is lost.     

Suggestive Structural Evidence for Macronuclear "Subnuclei" in Tetrahymena pyriformis GL

SYNOPSIS Structural changes in the Feulgen-positive material of the Tetrahymena pyriformis GL macronucleus have been observed during the cell cycle. From the finely granulated appearance in the interphase cell it appears as small rods, often arranged in pairs (probably the endomitotic stage) during early morphogenesis and as larger (and fewer) aggregates of granules during the nuclear division. These latter aggregates are also visible in dividing nuclei in the electron microscope where groups of chromation granules are separated by fairly empty nucleoplasm. It is suggested that these Feulgen positive aggregates in dividing nuclei are macronuciear segregation units or "subnuclei." The number per dividing macronucleus may vary from one experiment to another, but the variation seems to be related to cell volume. The distribution of the aggregates among the daughter nuclei is almost equal. The total number per dividing macronucleus is about 80 which is close to the estimated number of "subnuclei" in the T. pyriformis macronucleus (Allen and Nanney, 1958).
Some calculations are made on the polyploidy of the T. pyriformis GL macronucleus. Using published electron micrographs of micronuclei of known age to calculate the total number of chromatin granules per haploid nucleus, the polyploidy of the strain GL macronucleus is about 40. This figure is half of that expected from Allen and Nanney's estimation, since they assumed that the "subnuclei" were diploid; however, it is in agreement with the reported haploid nature of the "subnuclei" as found by Woodard, Gorovsky & Kaneshiro, 1968. Further calculations suggest that each macronuclear "chromosome" is composed of about 40 chromatin granules; an indication of such a chain arrangement of the chromatin granules has been observed in the phase contrast and electron microscope during the earliest macronuclear events, i.e., at the macronuclear "prophase."     

Nucleoli and cytoplasmic male sterility

Summary The paper describes the relation of cytoplasmic male sterility and the differentiation of nucleoli. Defects in the formation of nucleoli of pollen grains of maize with cytoplasmic male sterility were observed whereas the nuclei of control, i.e. fertile maize contained always only a single nucleole. In the nuclei of sterile pollen grains nucleoli of various sizes and numbers are formed.With 8 Figures in the Text     

Number and Nuclear Localisation of Nucleoli in Mammalian Spermatocytes

In seven mammalian species, including man, the position and number of nucleoli in pachytene spermatocyte nuclei were studied from electron microscope (EM) nuclear sections or bivalent microspreads. The number and position of the nucleolar organiser regions (NORs) in mitotic and meiotic chromosomes were also analysed, using silver staining techniques and in situ hybridisation protocols. The general organisation of pachytene spermatocyte nucleoli was almost the same, with only minor morphological differences between species. The terminal NORs of Thylamys elegans (Didelphoidea, Marsupialia), Dromiciops gliroides (Microbiotheridae, Marsupialia), Phyllotys osgoodi (Rodentia, Muridae) and man, always gave rise to peripheral nucleoli in the spermatocyte nucleus. In turn, the intercalated NORs from Octodon degus, Ctenomys opimus (Rodentia, Octodontidae) and Chinchilla lanigera (Rodentia, Cavidae), gave rise to central nucleoli. In species with a single nucleolar bivalent, just one nucleolus is formed, while in those with multiple nucleolar bivalents a variable number of nucleoli are formed by association of different nucleolar bivalents or NORs that occupy the same nuclear peripheral space (Phyllotis and man). It can be concluded that the position of each nucleolus within the spermatocyte nucleus is mainly dependent upon: (1) the position of the NOR in the nucleolar bivalent synaptonemal complex (SC), (2) the nuclear pathway of the nucleolar bivalent SC, being both telomeric ends attached to the nuclear envelope, and (3) the association between nucleolar bivalents by means of their NOR-nucleolar domains that occupy the same nuclear space. Thus, the distribution of nucleoli within the nuclear space of spermatocytes is non-random and it is consistent with the existence of a species-specific meiotic nuclear architecture.     

Persistent Nucleoli in Early Postimplantation of Rat Embryos

Ultrastructural changes of the nucleolus in mitotic embryonic ectodermal cells of 7 1/2-day and 7 2/3-day rat embryos were examined. It was found that the nucleolus was broken down into small fragments during late prophase and metaphase, and that some of these fragments persisted in the cytoplasm of telophase cell (persistent nucleoli). No interphase embryonic ectodermal cells contained persistent nucleoli. Persistent nucleoli were also found in telophase cells of extraembryonic ectoderm, extraembryonic visceral endoderm and parietal endoderm of the embryos, but they disappeared in interphase cells. Persistent nucleoli in telophase cells tended to decrease in size with embryonic age, and they had almost completely disappeared in neuroectodermal cells of the telencephalon in 14 1/2-day embryos. They were concluded to be remnants of disappearing nucleoli in embryonic cells that were cycling too rapidly to permit their nucleoli to disappear completely.     

Nucleoli formation under inhibited RNA synthesis

Formation and fusion of nucleoli after mitosis were studied in cultures of Chinese hamster cells and meristematic cells of Allium cepa and A. fistulosum under blocked RNA synthesis. To identify precisely which cells had passed through mitosis under actinomycin D blockade, cell cultures with micronuclei (induced by colchicine treatment), and binuclear Allium cells (induced by caffeine treatment), were used. It was found that in cells which have passed through mitosis after inhibition of RNA synthesis, the formation and fusion of nucleoli proceed more slowly than in cells not treated with actinomycin D; however, nucleoli appear and coalesce. Thus, telophase reconstruction of the nucleolus does not require simultaneous RNA synthesis and occurs at the expense of RNA that has been synthesized prior to mitosis.     

Some Chemical Properties of Isolated Pea Nucleoli

Isolated nuclei and nucleoli of ungerminated pea embryos have been analyzed chemically for their content of DNA, RNA, zinc, iron, phosphorus, and protein sulfhydryl groups. The values obtained cannot be considered to represent the whole of the living nucleolar body as an undetermined amount of material is extracted from nucleoli in the course of their isolation. Only negligible amounts of DNA have been found in the isolated nucleoli; most of the DNA released on disruption of nuclei appears in a fraction showing very few structures under the light microscope. RNA is more concentrated in the nucleolus than in the nucleus or cytoplasm, but since nucleolar protein is 6 per cent of nuclear and less than 1 per cent of cytoplasmic protein, the total amount of nucleolar RNA is comparatively small. None of the other components listed occurs in high concentration in either nucleus or nucleolus.     

Nucleoli in vaginal cells of hyperestrogenic and hypoestrogenic women

Nucleoli classified according to their functional morphology were investigated in the vaginal cells of 38 hypoestrogenic and 29 hyperestrogenic women to study the effect of estrogen on these cells. While the cells from hyperestrogenic patients were characterized by the presence of a larger number of compact (active) and ring-shaped (less active/resting) nucleoli, vaginal cells from hypoestrogenic women were characterized by the presence of a larger number of micronucleoli (inactive nucleoli).     

The Isolation of Nucleoli from Ungerminated Pea Embryos

A method is described for the isolation of nuclei and nucleoli from ungerminated pea embryos. Electron micrographs of the nucleolar preparation indicate a high degree of purity but only a partial preservation of composition, because of a loss of material from the nucleoli which occurs during the isolation procedure.