Identification of ZFR, an ancient and highly conserved murine chromosome-associated zinc finger protein

In a screen for RNA binding proteins expressed during murine spermatogenesis, we cloned a novel, ancient zinc finger protein possessing a region common to a small class of RNA binding proteins. Zfr (zinc finger RNA binding) encodes a protein of 1052 amino acids with three widely spaced Cys2His2 zinc fingers. Outside of the zinc fingers, ZFR shares a region that is highly conserved between several RNA binding proteins containing copies of the double-stranded RNA binding motif. By northern blotting, Zfr is expressed at highest levels within the testis, ovary and brain. Immunohistochemistry and confocal microscopy were used to show that ZFR is highly expressed during meiosis I in males and females and is chromosome associated. Zfr is also expressed in Sertoli cells in the testis and granulosa cells in the ovary where it is localized to the nucleus. Using fluorescent in situ hybridization we mapped Zfr to chromosome 15 region A. ZFR appears to be an ancient protein, as apparent homologs exist in invertebrates (D. melanogaster) nematodes (C. elegans) and humans (H. sapiens).     

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.     

Internuclear control of DNA synthesis in exconjugant cells of Paramecium caudatum

An exconjugant cell of Paramecium caudatum has two kinds of macronuclei, fragmented prezygotic macronuclei and postzygotic new macronuclei (anlagen). Although the DNA synthesis in the fragmented prezygotic macronucleus continues until the third cell cycle after conjugation, selective suppression of the DNA synthesis in the prezygotic macronucleus takes place at the fourth cell cycle. The inhibition of DNA synthesis in prezygotic fragmented macronuclei is due to the presence of a postzygotic macronucleus (anlage) in the same cytoplasm because the inhibition does not occur when the postzygotic macronucleus (anlage) is removed by micromanipulation during the third or fourth cell cycle. Well-developed postzygotic macronuclei (anlagen) with full ability to divide have the ability to depress the DNA synthesis of prezygotic macronuclear fragments. The suppression of DNA synthesis in prezygotic macronuclear fragments seems to be irreversible. Competition for the limited amount of DNA precursors also plays an important role in the onset of the selective suppression of the DNA synthesis.     

Cytogenetics of triplet conjugation in Tetrahymena: Origin of haploid and triploid clones

Triplet conjugation occurs frequently when three different mating types of T. thermophila are mixed under appropriate conditions. Genetic studies show that fertilization is usually tripolar, with each conjugant contributing a pronucleus to one mate and receiving a pronucleus from the other. Triplets are less common and less stable when only two mating types are mixed, and nuclear exchange is not always reciprocal. Some conjugants contribute a pronucleus but do not receive one in return. Others receive two pronuclei instead of one. Such triplets chracteristically yield one haploid, one diploid and one triploid exconjugant, all of which are able to establish vigorous clones.     

Localization and functional analysis of HmgB3p,a novel protein containing high-mobility-group-box domain from Tetrahymena thermophila

The high-mobility-group (HMG)-box domain represents a very versatile protein domain that mediates the DNA-binding of non-sequence-specific and sequence-specific proteins. HMG-box proteins are involved in various nuclear functions, including modulating chromatin structure and genomic stability. In this study, we identified the gene HMGB3 in Tetrahymena thermophila. The predicted HmgB3p contained a single HMG-box, an SK-rich-repeat domain and a neutral phosphorylated C-terminal. HMGB3 was expressed in the growth and starvation stages. Furthermore, HMGB3 showed a higher expression levels during the conjugation stage. HMGB3 knockout strains showed no obvious cytological defects, although initiation of HMGB3 knockout strain mating was delayed and maximum mating was decreased. HA-HmgB3p localized on the micronucleus (MIC) during the vegetative growth and starvation stages. Furthermore, HA-HmgB3p specially decorated the meiotic and mitotic functional MIC during the conjugation stage. Truncated HMGB3 lacking the HMG box domain disappeared from MICs and diffused in the cytoplasm. Overexpressed HmgB3p was abnormally maintained in newly developing macronuclei and affected the viability of progeny. Taken together, these results show that HmgB3p is a germline micronuclear-specific marker protein. It may bind to micronucleus-specific DNA sequences or structures and is likely to have some function specific to micronuclei of T. thermophila.     

Cytogamy: An Inducible, Alternate Pathway of Conjugation in TETRAHYMENA THERMOPHILA

We report the occurrence of cytogamy in Tetrahymena thermophila. By analogy to Paramecium, cytogamy generates exconjugant clones that derive their entire genetic information from a single meiotic product of their cytoplasmic parent. Thus, "instant" whole-genome homozygotes are created. Cytogamy has been induced in every strain of T. thermophila tested, and most of the excytogamous progeny have exhibited high fertility. The high frequency with which cytogamy can be induced by hyperosmotic shock, coupled with the foregoing genetic properties, make this process a practical (and already proven) method for the isolation of recessive mutants in T. thermophila. We also report that the cytogamy-inducing treatment induces other rare abnormalities of genetic transmission, which have not yet been characterized.     

Role of the Cytosolic Heat Shock Protein 70 Ssa5 in the Ciliate Protozoan Tetrahymena thermophila

Heat shock protein 70 (Hsp70) is a member of a family of conserved chaperone proteins whose function is well investigated in many model organisms. Here we focus on an Hsp70 called Ssa5 in the ciliate protozoan Tetrahymena thermophila, and reveal that its translation is heat inducible as for general Hsps. Moreover, the protein is abundantly expressed in the cytoplasm during sexual reproduction (conjugation) as well as in response to heat‐stress. Knocking out of SSA5 (ΔSSA5) does not affect the survival of the cell under heat‐stress, likely due to other Hsp70 paralogs compensating for the defect. During conjugation, ΔSSA5 leads to a fertilization defect in which the two pronuclei are in close proximity but never fuse. The unfertilized pronuclei differentiate, resulting in a heterokaryon with developed haploid germline and somatic nuclei. In addition, degeneration of the parental somatic nucleus is not affected. These results suggest a specific involvement of Ssa5 in pronuclear fusion and fertilization.     

Involvement of 14-nm Filament-Forming Protein and Tubulin in Gametic Pronuclear Behavior during Conjugation in Tetrahymena

We have studied in detail the immunofluorescence localizations of Tetrahymena 14-nm filament-forming protein (49-kDa protein) in relation to tubulin in conjugating wild-type Tetrahymena thermophila (B strain) pairs and in pairs between B strain and star strains with defective micronuclei. The results suggest that germ nuclear behavior during conjugation may involve the following cytoskeletal structures: (1) during meiosis, microtubule structures are involved in micronuclear elongation and meiotic division; (2) at the postmeiotic stage, 49-kDa protein network structures that are formed independently of the existence of pronuclei are involved in the selection and the survival of one of four meiotic products; (3) during the third prezygotic division, gametic pronuclear transfer, and zygote formation, a cytoskeletal structure in which the 49-kDa protein colocalizes with microtubules and which is dependent on the existence of a normal gametic pronucleus is involved in gametic pronuclear behavior, and (4) during the postzygotic divisions, the microtubules are involved in nuclear behavior.     

"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.     

Nuclear Roles in the Post-Conjugant Development of the Ciliate Euplotes aediculatus. III. Roles of Old Macronuclear Fragments in Nuclear and Cortical Development1

Following conjugation of the hypotrichous ciliate Euplotes aediculatus, the posterior fragments of the old (prezygotic) macronucleus persist until after the first vegetative division. These fragments remain viable during exconjugant development as shown by their ability to regenerate should the cell's new macronucleus be damaged. It thus seemed possible that these parental nuclear fragments might participate in the development of the new macronucleus and/or the crucial post-conjugant cortical reorganization that restores the exconjugant cell's ability to feed. This idea was tested by damaging the posterior fragments with various doses of microbeam ultraviolet (UV) light and assessing the results of such treatment on subsequent cortical and nuclear development. When the posterior fragments of the macronucleus were irradiated at the beginning of cortical morphogenesis, the new macronucleus in 1/3 to 1/2 of the cells assumed a “folded” appearance but did not mature. These cells did not undergo cortical reorganization. Cells irradiated at earlier stages did not detectably develop an oral apparatus; their new macronucleus remained arrested at the spherical anlage stage. The results show that the posterior fragments of the parental macronucleus are necessary for normal nuclear and cortical development. These old nuclear fragments appear to influence the growing macronuclear anlage directly and probably the cortex as well. There also appears to be an information flow from the non-irradiated partner of a persistently joined exconjugant doublet to its irradiated counterpart, enabling normal anlage and cortex development in the irradiated cell.     

Mating Types, Breeding System, Conjuàtion and Nuclear Phenomena in the Marine Ciliate Euplotes cristatus Kahl from the Gulf of Naples

SYNOPSIS. The marine ciliate Euplotes cristatus Kahl (Ciliophora, Hypotrichida), collected off Capri, Gulf of Naples, is described in detail. From populations, 6 different mating types, representing 1 variety or syngen, have been isolated. The breeding relations revealed a multiple mating type system characteristic of other members of the Hypotrichida that have been investigated. Presumably a 7th mating type was found which does not mate with any of the others. Although this may belong to another syngen, it could represent a mating type which has not yet reached sexual maturity or 1 which may be in a period of decline. Animals of different mating type do not mate immediately after being mixed but usually 3 or more hours later. An agglutination reaction involving many specimens is absent. Instead, 2 ciliates engage in a “pairing play” before joining firmly in conjugation. Well-fed or actively feeding and dividing ciliates do not mate; mating occurs only after the food becomes gradually depleted or when the food supply is sharply cut off. All mating types appear to be extremely stable. Neither selfing pairs (intraclonal conjugation) nor autogamy have been observed within any clonal culture during the several years under investigation. Cell-free filtrates from 1 mating type do not elicit mating or induce conjugation with specimens of a different mating type. The general pattern of nuclear events in conjugation and exconjugant reorganization is as follows: 1 preliminary division, 3 pregamic (prezygotic) divisions, fertilization, and generally 1 or occasionally 2 postzygotic divisions. The fate of micronuclear products may be determined by their size and location. Those which are larger and close to the cell membranes of the joined conjugants persist and/or divide. Those which are smaller are carried by cyclosis toward the center of each ciliate and degenerate. The degenerating macronucleus of each conjugant becomes segmented in a more or less uniform manner resulting in 4 subspherical masses. Two become localized in the anterior end of a conjugant and 2 in the posterior end. Those in the posterior end are always the first to degenerate completely and disappear. In nuclear reorganization of the exconjugant, fusion of the macronuclear anlage with parts of the old macronucleus does not occur.     

Membrane Dynamics at the Nuclear Exchange Junction during Early Mating (One to Four Hours) in the Ciliate Tetrahymena thermophila

Using serial-section transmission electron microscopy and three-dimensional (3D) electron tomography, we characterized membrane dynamics that accompany the construction of a nuclear exchange junction between mating cells in the ciliate Tetrahymena thermophila. Our methods revealed a number of previously unknown features. (i) Membrane fusion is initiated by the extension of hundreds of 50-nm-diameter protrusions from the plasma membrane. These protrusions extend from both mating cells across the intercellular space to fuse with membrane of the mating partner. (ii) During this process, small membrane-bound vesicles or tubules are shed from the plasma membrane and into the extracellular space within the junction. The resultant vesicle-filled pockets within the extracellular space are referred to as junction lumens. (iii) As junction lumens fill with extracellular microvesicles and swell, the plasma membrane limiting these swellings undergoes another deformation, pinching off vesicle-filled vacuoles into the cytoplasm (reclamation). (iv) These structures (resembling multivesicular bodies) seem to associate with autophagosomes abundant near the exchange junction. We propose a model characterizing the membrane-remodeling events that establish cytoplasmic continuity between mating Tetrahymena cells. We also discuss the possible role of nonvesicular lipid transport in conditioning the exchange junction lipid environment. Finally, we raise the possibility of an intercellular signaling mechanism involving microvesicle shedding and uptake.     

The onset of DNA synthesis and its relation to morphogenetic events of the pronuclei in activated eggs of the sea urchin, Arbacia punctulata

Experiments were carried out in order to investigate the onset of DNA synthesis and its possible relation to pronuclear morphogenesis prior to, during, and following pronuclear fusion in the sea urchin, Arbacia punctulata. Analysis of the acid-soluble radioactivity of zygotes continuously incubated in tritiated thymidine (³H-TdR) at 22°C demonstrated the production of thymidine triphosphate prior to and during pronuclear migration (5–10 min post-insemination) which continued to increase until prophase (about 45 min post-insemination). DNA synthesis was initiated subsequent to pronuclear fusion, i.e., 15–20 min post-insemination. Little or no cytoplasmic label was detected during fertilization. Silver grains were first detected over the zygote nucleus in sections prepared for light and electron microscopic radioautography from zygotes fixed at 18 min post-insemination. Incubation of Arbacia zygotes at 12°C dramatically slowed development but did not alter the temporal relation between pronuclear fusion and DNA synthesis. Radioautographs of polyspermic zygotes demonstrated that unfused male pronuclei were able to synthesize DNA and substantiate the claim that pronuclear fusion is not required for the initiation of DNA synthesis. Analysis of autoradiographs of artificially activated eggs also lead to the same conclusion. Uptake of ³H-TdR or ³H-bromodeoxyuridine was not found in spermatozoa incorporated into polyspermic oocytes regardless of whether or not they had initiated or developed into male pronuclei. It is concluded that the relation between DNA synthesis and pronuclear fusion is not close or a direct one, i.e., pronuclear fusion does not appear to be a prerequisite for the onset of DNA synthesis in Arbacia.     

Nuclear functions in postconjugational development of Paramecium caudatum: Ability to form food vacuoles analyzed by nuclear elimination and implantation

Paramecium caudatum loses the ability to form food vacuoles at the crescent stage of the micronucleus from 5 to 6 hr after the initiation of conjugation and regains it immediately after the third division of the zygotic nucleus. To assess the micronuclear function in the development of the oral apparatus after coniugation, prezygotic micronuclei was removed from cells at various stages of conjugation, and their ability to form food vacuoles were examined. (1) When all of the prezygotic micronuclear derivatives were eliminated before the stage of formation of the zygotic nucleus, the exconjugant did not regain its ability. (2) When a zygotic nucleus or postzygotic nuclei were removed, in some cases the cell formed as many food vacuoles as did nonoperated cells after conjugation, while in other operated cells the number of food vacuoles was subnormal. (3) When a micronucleus from a cell at vegetative phase (G1) was transplanted into a cell of an amicronucleate mating pair at the stage between 8 and 9 hr after the initiation of conjugation, the implanted cell regained the ability to form food vacuoles. However, no cell regained the ability when the implantation was carried out within 1 hr after the separation of the mates. The results show that the micronucleus plays an indispensable role in the development of the oral apparatus at the stages of exchange of gametic nuclei and fertilization and that the micronucleus transplanted from asexual cells can fulfill this function. On the other hand, removal of the macronucleus from exconjugants showed that the maternal macronucleus also has an indispensable function in regaining the ability to form food Vacuoles. © 1992 Wiley-Liss, Inc.     

The zinc-finger protein ZFR is critical for Staufen 2 isoform specific nucleocytoplasmic shuttling in neurons

In mammalian neurons, transport and translation of mRNA to individual potentiated synapses is believed to occur via a heterogeneous population of RNA granules. To identify components of Staufen2-containing granules, we used the yeast two-hybrid system. A mouse fetal cDNA library was screened with the N-terminal fragment of Staufen2 as bait. ZFR, a three zinc finger protein, was identified as an interacting protein. Confocal microscopy showed that ZFR, although mainly nuclear, was also found in the somatodendritic compartment of primary hippocampal neurons where it localized as granule-like structures. Co-localization with Staufen2 was observed in several granules. Biochemical analyses (immunoprecipitation, cell fractionation) further confirmed the ZFR/Staufen2 association. ZFR was shown to interact with at least the Staufen2(62) isoform, but not with Staufen1. ZFR also co-fractionated with ribosomes and Staufen2(59) and Staufen2(52) in a sucrose gradient. Interestingly, knockdown expression of ZFR through RNA interference in neurons relocated specifically the Staufen2(62), but not the Staufen2(59), isoform to the nucleus. Our results demonstrate that ZFR is a native component of Staufen2-containing granules and likely plays its role during early steps of RNA transport and localization. They also suggest that one of these roles may be linked to Staufen2(62)-containing RNA granule formation in the nucleus and/or to their nucleo-cytoplasmic shuttling.     

The p53-induced mouse zinc finger protein wig-1 binds double-stranded RNA with high affinity

The p53-induced mouse wig-1 gene encodes a Cys₂His₂-type zinc finger protein of unknown function. The zinc fingers in wig-1 are connected by long (56–75) amino acid linkers. This distribution of zinc finger domains resembles that of the previously described double-stranded (ds)RNA-binding proteins dsRBP-ZFa and JAZ. Ectopically expressed FLAG-tagged mouse wig-1 protein localized to nuclei and in some cells to nucleoli, whereas GFP-tagged mouse wig-1 localized primarily to nucleoli. Electrophoretic mobility shift assay using a recombinant GST–wig-1 fusion protein showed that wig-1 preferentially binds dsRNA rather than single-stranded RNA or dsDNA. A set of deletion/truncation mutants of wig-1 was tested to determine the dsRNA-binding domain(s) or region(s) in wig-1 that is involved in the stabilization of wig-1–dsRNA complexes in vitro. This revealed that the first zinc finger in wig-1 is essential for binding to dsRNA, whereas zinc fingers 2 and 3 are dispensable. wig-1 protein expressed in mammalian cells also showed a high affinity for dsRNA. wig-1 represents the first confirmed p53-induced gene that encodes a dsRNA-binding protein. This suggests that dsRNA binding plays a role in the p53-dependent stress response.     

A Role for a Protease in Morphogenic Responses during Yeast Cell Fusion

Cell fusion during yeast mating provides a model for signaling-controlled changes at the cell surface. We identified the AXL1 gene in a screen for genes required for cell fusion in both mating types during mating. AXL1 is a pheromone-inducible gene required for axial bud site selection in haploid yeast and for proteolytic maturation of a-factor. Two other bud site selection genes, RSR1, encoding a small GTPase, and BUD3, were also required for efficient cell fusion. Based on double mutant analysis, AXL1 in a MATα strain acted genetically in the same pathway with FUS2, a fusion-dedicated gene. Electron microscopy of axl1, rsr1, and fus2 prezygotes revealed similar defects in nuclear migration, vesicle accumulation, cell wall degradation, and membrane fusion during cell fusion. The axl1 and rsr1 mutants exhibited defects in pheromone-induced morphogenesis. AXL1 protease function was required in MATα strains for fusion during mating. The ability of the Rsr1p GTPase to cycle was required for efficient cell fusion, as it is for bud site selection. During conjugation, vegetative functions may be redeployed under the control of pheromone signaling for mating purposes. Since Rsr1p has been reported to physically associate with Cdc24p and Bem1p components of the pheromone response pathway, we suggest that the bud site selection genes Rsr1p and Axl1p may act to mediate pheromone control of Fus2p-based fusion events during mating.     

RAD51 is required for propagation of the germinal nucleus in Tetrahymena thermophila

RAD51, the eukaryote homolog of the Escherichia coli recA recombinase, participates in homologous recombination during mitosis, meiosis, and in the repair of double-stranded DNA breaks. The Tetrahymena thermophila RAD51 gene was recently cloned, and the in vitro activities and induction of Rad51p following DNA damage were shown to be similar to that of RAD51 from other species. This study describes the pattern of Tetrahymena RAD51 expression during both the cell cycle and conjugation. Tetrahymena RAD51 mRNA abundance is elevated during macronuclear S phase during vegetative cell growth and with both meiotic prophase and new macronuclear development during conjugation. Gene disruption of the macronuclear RAD51 locus leads to severe abnormalities during both vegetative growth and conjugation. rad51 nulls divide slowly and incur rapid deterioration of their micronuclear chromosomes. Conjugation of two rad51 nulls leads to an arrest early during prezygotic development (meiosis I). We discuss the potential usefulness of the ciliates' characteristic nuclear duality for further analyses of the potentially unique roles of Tetrahymena RAD51.     

A Marine Euplotes (Ciliophora, Hypotrichida) with Reduced Number of Prezygotic Micronuclear Divisions

In contradistinction to the pattern of 3 prezygotic micronuclear divisions found in 10 species of Euplotes, a marine species resembling Euplotes crassus in structure, has only 2 divisions. This atypical division pattern was observed in all matings involving the 4 available mating types. The critical stages of the nuclear events are demonstrated by using special strains having the micronuclear DNA content and chromosome number only 1/2 the normal values. The employment of such strains facilitates differentiation between the stationary and migratory pronuclei in a given conjugant and determination of the times of conclusion of prezygotic divisions and of the pronuclear exchange.