Centromeric histone H3 is essential for vegetative cell division and for DNA elimination during conjugation in Tetrahymena thermophila

The Tetrahymena thermophila CNA1 gene encodes the centromeric H3, Cna1p. Green fluorescent protein (GFP)-tagged Cna1p localizes in micronuclei in dots whose number and behavior during mitosis and conjugation are consistent with centromeres. During interphase, Cna1p-GFP localizes in peripheral dots, suggesting centromeres are associated with the nuclear envelope. Newly synthesized Cna1p-GFP enters micronuclei in mitosis and accumulates in the nucleoplasm. Its deposition at centromeres starts at early S phase and continues through most of S phase. CNA1 is required for vegetative cell growth. Knockdown of CNA1 genes in the somatic macronucleus results in micronuclear DNA loss and delayed chromosome segregation during mitosis. During conjugation, Cna1p-GFP disappears from the centromeres in the developing macronucleus, consistent with centromeric sequences being internal eliminated sequences. Surprisingly, zygotic CNA1 is required for efficient elimination of germ line-specific sequences during development of the new macronuclei but not for the RNA interference pathway, through which sequences are targeted for elimination. Zygotically expressed Cna1p localizes in the spherical structures in which the later stages of DNA elimination occur, and these structures cannot be formed in the absence of zygotic CNA1, suggesting that, in addition to functioning in centromeres, Cna1p may also play a role in organizing the formation of the DNA elimination structures.     

Tetrahymena macronuclear genome mapping: colinearity Of macronuclear coassortment groups and the micronuclear map on chromosome 1l

The genetics of the ciliate Tetrahymena thermophila are richer than for most other eukaryotic cells, because Tetrahymena possesses two genomes: a germline (micronuclear) genome that follows a Mendelian model of genetic transmission and a somatic (macronuclear) genome, derived from the micronuclear genome by fragmentation, which follows a different genetic transmission model called phenotypic assortment. While genetic markers in the micronucleus fall into classical linkage groups under meiotic recombination and segregation, the same markers in the macronucleus fall into coassortment groups (CAGs) under phenotypic assortment by the random distribution of MAC chromosome pieces. We set out to determine whether genomic mapping in the macronucleus by genetic means is feasible. To investigate the relationship between the micronuclear map and coassortment groups, we systematically placed into CAGs all of the markers lying on chromosome 1L that are also found in the macronucleus. Sixteen CAGs were identified, 7 of which contain at least two loci. We have concluded that CAGs represent a fundamental genetic feature of the MAC. The MIC and MAC maps on 1L are colinear; that is, CAGs consist exclusively of markers that map to a continuous segment in a given region of the micronuclear map, with no intervening markers from other CAGs. These findings provide a solid foundation for exploiting the MAC chromosome pieces to build a physical map of the Tetrahymena genome.     

Stochastic developmental variation in the ratio of allelic rDNAs among newly differentiated, heterozygous macronuclei of Tetrahymena thermophila.

Ciliates possess nuclear dimorphism, i.e., they carry two structurally and functionally differentiated types of nuclei. The micronucleus and macronucleus serve as the germline and somatic nuclei, respectively, of the cell. The macronucleus differentiates from a mitotic sister of the micronucleus once per life cycle. Macronuclear differentiation is accompanied by a developmentally programmed set of DNA rearrangements, including chromosome fragmentation, telomere addition, and amplification. Given the diploidy of the MAC anlage, are both homologous copies of a chromosome processed and amplified equally and simultaneously in an individual differentiating MAC? We have approached this question for the case of the rDNA, exploiting previously identified DNA polymorphisms and the sensitivity of PCR. We determined allelic ratios in individual caryonide cells, i.e., the cells carrying the primary products of MAC differentiation, prior to the first division of the newly differentiated MAC. We observed stochastic variability in allelic ratios among caryonides that start with genetically identical heterozygous MACs. Either rDNA type can be in the majority. Appropriate controls make it unlikely that the ratios observed were significantly affected by variation in the assay itself. The variability may well result from the statistical variation associated with the relative timing of individual biochemical events initiating the processing and/or amplification of a few rDNA precursor molecules, presumably 4-8 at the most, in a MAC anlage. In addition to this stochastic variability, we observed a small but distinct bias in favor of the C3 rDNA. Thus the replication advantage of C3 relative to B rDNA in heterozygous MACs, previously detected during vegetative multiplication, may begin to be expressed during developmental amplification.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nowa1p and Nowa2p: novel putative RNA binding proteins involved in trans-nuclear crosstalk in Paramecium tetraurelia

BACKGROUND: The germline genome of ciliates is extensively rearranged during development of a new somatic macronucleus from the germline micronucleus, a process that follows sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) and multicopy transposons are eliminated, whereas cellular genes are amplified to approximately 800 n. For a subset of IESs, introduction of the IES sequence into the maternal (prezygotic) macronucleus specifically inhibits excision of the homologous IES in the developing zygotic macronucleus. This and other homology-dependent maternal effects have suggested that rearrangement patterns are epigenetically determined by an RNA-mediated, trans-nuclear comparison, involving the RNA interference pathway, of germline and somatic genomes. RESULTS: We report the identification of novel developmentally regulated RNA binding proteins, Nowa1p and Nowa2p, which are required for the survival of sexual progeny. Green fluorescent protein (GFP) fusions show that Nowa1p accumulates into the maternal macronucleus shortly before meiosis of germline micronuclei and is later transported to developing macronuclei. Nowa1p/2p depletion impairs the elimination of transposons and of those IESs that are controlled by maternal effects, confirming the existence of distinct IES classes. CONCLUSIONS: The results indicate that Nowa proteins are essential components of the trans-nuclear-crosstalk mechanism that is responsible for epigenetic programming of genome rearrangements. We discuss implications for the current models of genome scanning in ciliates, a process related to the formation of heterochromatin by RNA interference in other eukaryotes.     

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.     

Differential role of CENP-A in the segregation of holocentric C. elegans chromosomes during meiosis and mitosis

Two distinct chromosome architectures are prevalent among eukaryotes: monocentric, in which localized centromeres restrict kinetochore assembly to a single chromosomal site, and holocentric, in which diffuse kinetochores form along the entire chromosome length. During mitosis, both chromosome types use specialized chromatin, containing the histone H3 variant CENP-A, to direct kinetochore assembly. For the segregation of recombined homologous chromosomes during meiosis, monocentricity is thought to be crucial for limiting spindle-based forces to one side of a crossover and to prevent recombined chromatids from being simultaneously pulled towards both spindle poles. The mechanisms that allow holocentric chromosomes to avert this fate remain uncharacterized. Here, we show that markedly different mechanisms segregate holocentric chromosomes during meiosis and mitosis in the nematode Caenorhabditis elegans. Immediately prior to oocyte meiotic segregation, outer-kinetochore proteins were recruited to cup-like structures on the chromosome surface via a mechanism that is independent of CENP-A. In striking contrast to mitosis, both oocyte meiotic divisions proceeded normally following depletion of either CENP-A or the closely associated centromeric protein CENP-C. These findings highlight a pronounced difference between the segregation of holocentric chromosomes during meiosis and mitosis and demonstrate the potential to uncouple assembly of outer-kinetochore proteins from CENP-A chromatin.     

Biological effects of a bifunctional DNA cross-linker. II. Generation of micronuclei and attached micronuclear-like structures.

Madin-Darby bovine kidney (MDBK) cells were treated with the bifunctional DNA cross-linker, L-7, to examine the generation of micronuclei and other nuclear abnormalities. The preceding paper demonstrates that L-7 treatment induces the formation of triradial and quadriradial chromosomes in MDBK cells. These chromosomes are believed to result from interduplex DNA cross-links formed between G-C rich centromeric satellite DNA regions on non-sister chromatids. Treatment produces a majority of centromere-positive micronuclei. In addition, many daughter cells remain attached by chromatin bridges which are sometimes beaded with micronuclei. Up to 15% of cell nuclei become lobular and fused with numerous micronuclear-like structures attached to their membranes. These attached structures are classified as attached micronuclear-like structures (AMNLS). Fluorescence in situ hybridization (FISH) using a centromeric satellite sequence was performed on treated cells. Hybridization reveals that intercellular bridges are composed of centromeric sequences and initiate at centromeric foci in daughter cells. Furthermore, the majority of junctions between AMNLS and nuclei contain an enhancement of centromeric signal. The frequency of AMNLS appears dependent on the concentration of L-7 and the duration of treatment. Similar results were found for the generation of cross-linked chromosome products in the previous paper. We suggest that AMNLS result from the abnormal mitotic segregation of cross-linked chromosome products.     

Human artificial chromosomes with alpha satellite-based de novo centromeres show increased frequency of nondisjunction and anaphase lag

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.     

Stochastic developmental variation in the ratio of allelic rDNAs among newly differentiated,heterozygous macronuclei of Tetrahymena thermophila

Ciliates possess nuclear dimorphism, i.e., they carry two structurally and functionally differentiated types of nuclei. The micronucleus and macronucleus serve as the germline and somatic nuclei, respectively, of the cell. The macronucleus differentiates from a mitotic sister of the micronucleus once per life cycle. Macronuclear differentiation is accompanied by a developmentally programmed set of DNA rearrangements, including chromosome fragmentation, telomere addition, and amplification. Given the diploidy of the MAC anlage, are both homologous copies of a chromosome processed and amplified equally and simultaneously in an individual differentiating MAC? We have approached this question for the case of the rDNA, exploiting previously identified DNA polymorphisms and the sensitivity of PCR. We determined allelic ratios in individual caryonide cells, i.e., the cells carrying the primary products of MAC differentiation, prior to the first division of the newly differentiated MAC. We observed stochastic variability in allelic ratios among caryonides that start with genetically identical heterozygous MACs. Either rDNA type can be in the majority. Appropriate controls make it unlikely that the ratios observed were significantly affected by variation in the assay itself. The variability may well result from the statistical variation associated with the relative timing of individual biochemical events initiating the processing and/or amplification of a few rDNA precursor molecules, presumably 4–8 at the most, in a MAC anlage. In addition to this stochastic variability, we observed a small but distinct bias in favor of the C3 rDNA. Thus the replication advantage of C3 relative to B rDNA in heterozygous MACs, previously detected during vegetative multiplication, may begin to be expressed during developmental amplification. We discuss the relevance of this stochastic developmental variability to classical genetic observations of Nanney and their collaborators on other T. thermophila loci. © 1992 Wiley-Liss, Inc.     

Fluorescent staining of L cell centromeres and chromocenters with 1-dimethylaminonaphthalene-5-sulfonyl chloride and G-bandings

Fluorescent staining patterns of L cell chromosomes with 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride) were studied. Ordinary air-dried L cell metaphase chromosomes exhibited relatively uniform and bright yellowish green fluorescence by dansyl-staining under the fluorescence microscope. However, after the chromosome preparations were treated with 10 mM NaCl for 24 h at 4 °C, which produced distinctive G-bands with Giemsa-staining, the centromeric regions and several interstitial regions of some particular chromosomes were clearly fluorescent but other regions showed only dull fluorescence. After the treatment of chromosome slides with cupric sulfite reagent, which converts sulfhydryls and disulfides to thiosulfates chromosomes showed clear G-bands which were indistinguishable from those after 10 mM NaCl treatment. By dansyl-staining, however, the cupric sulfite-treated chromosomes exhibited very faint fluorescence on their contour alone, and neither centromeric regions nor some interstitial regions of marker chromosomes had distinctly bright fluorescence.Although Giemsa-staining disclosed dark chromocenters in approx. 75% of interphase nuclei irrespective of pretreatments, dansyl-staining revealed bright chromocenters in approx. 60% of interphase nuclei in control slides, in about 40% of nuclei in 10 mM NaCl-treated slides, and in only about 30% of nuclei in cupric sulfite-treated preparations.These observations indicated that in the air-dried chromosome preparations, the distribution of protein over the metaphase chromosome is relatively uniform along its length, and that G-bands in the chromosome and Giemsa-staining of chromocenters in interphase nuclei are not significantly affected by apparent loss of protein from the preparations. It was also suggested that particular protein may be associated with the centromeric regions of L cell chromosomes. Some technical details of dansyl fluorochroming and the significance of the observations were discussed.     

Sgo1 is required for co-segregation of sister chromatids during achiasmate meiosis I

The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation, called meiosis I and meiosis II. While meiosis II is similar to mitosis in that sister kinetochores are bi-oriented and segregate to opposite poles, recombined homologous chromosomes segregate during the first meiotic division. Formation of chiasmata, mono-orientation of sister kinetochores and protection of centromeric cohesion are three major features of meiosis I chromosomes which ensure the reductional nature of chromosome segregation. Here we show that sister chromatids frequently segregate to opposite poles during meiosis I in fission yeast cells that lack both chiasmata and the protector of centromeric cohesion Sgo1. Our data are consistent with the notion that sister kinetochores are frequently bi-oriented in the absence of chiasmata and that Sgo1 prevents equational segregation of sister chromatids during achiasmate meiosis I.Key words: meiosis, chromosome segregation, recombination, kinetochore, Sgo1, fission yeast     

The use of the in vitro micronucleus assay to detect and assess the aneugenic activity of chemicals

The successful validation of the in vitro micronucleus assay by the SFTG now provides the opportunity for this highly cost effective assay to be used to screen chemicals for their ability to induce both structural (clastogenic) and numerical (aneugenic) chromosome changes using interphase cells. The use of interphase cells and a relatively simple experimental protocol provides the opportunity to greatly increase the statistical power of cytogenetic studies on chemical interactions. The application of molecular probes capable of detecting kinetochores and centromeres provides the opportunity to classify mechanisms of micronucleus induction into those which are primarily due to chromosome loss or breakage. When a predominant mechanism of micronucleus induction has been shown to be based upon chromosome loss then further investigation can involve the determination of the role of non-disjunction in the induction of aneuploidy. The binucleate cell modification of the in vitro micronucleus assay can be combined with the use of chromosome specific centromere probes to determine the segregation of individual chromosomes into daughter nuclei. The combination of these methods provides us with powerful tools for the investigation of mechanisms of genotoxicity particularly in the low dose regions.     

Sgo1 is required for co-segregation of sister chromatids during achiasmate meiosis I

The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation, called meiosis I and meiosis II. While meiosis II is similar to mitosis in that sister kinetochores are bi-oriented and segregate to opposite poles, recombined homologous chromosomes segregate during the first meiotic division. Formation of chiasmata, mono-orientation of sister kinetochores and protection of centromeric cohesion are three major features of meiosis I chromosomes which ensure the reductional nature of chromosome segregation. Here we show that sister chromatids frequently segregate to opposite poles during meiosis I in fission yeast cells that lack both chiasmata and the protector of centromeric cohesion Sgo1. Our data are consistent with the notion that sister kinetochores are frequently bi-oriented in the absence of chiasmata and that Sgo1 prevents equational segregation of sister chromatids during achiasmate meiosis I.     

Maternal phosphatase inhibitor-2 is required for proper chromosome segregation and mitotic synchrony during Drosophila embryogenesis

Protein phosphatase-1 (PP1) is a major Ser/Thr phosphatase conserved among all eukaryotes, present as the essential GLC7 gene in yeast. Inhibitor-2 (I-2) is an ancient PP1 regulator, named GLC8 in yeast, but its in vivo function is unknown. Unlike mammals with multiple I-2 genes, in Drosophila there is a single I-2 gene, and here we describe its maternally derived expression and required function during embryogenesis. During oogenesis, germline expression of I-2 results in the accumulation of RNA and abundant protein in unfertilized eggs; in embryos, the endogenous I-2 protein concentrates around condensed chromosomes during mitosis and also surrounds interphase nuclei. An I-2 loss-of-function genotype is associated with a maternal-effect phenotype that results in drastically reduced progeny viability, as measured by reduced embryonic hatch rates and larval lethality. Embryos derived from I-2 mutant mothers show faulty chromosome segregation and loss of mitotic synchrony in cleavage-stage embryos, patchy loss of nuclei in syncytial blastoderms, and cuticular pattern defects in late-stage embryos. Transgenic expression of wild-type I-2 in mutant mothers gives dose-dependent rescue of the maternal effect on embryo hatch rate. We propose that I-2 is required for proper chromosome segregation during Drosophila embryogenesis through the coordinated regulation of PP1 and Aurora B.     

The yeast RSC chromatin-remodeling complex is required for kinetochore function in chromosome segregation

The accurate segregation of chromosomes requires the kinetochore, a complex protein machine that assembles onto centromeric DNA to mediate attachment of replicated sister chromatids to the mitotic spindle apparatus. This study reveals an important role for the yeast RSC ATP-dependent chromatin-remodeling complex at the kinetochore in chromosome transmission. Mutations in genes encoding two core subunits of RSC, the ATPase Sth1p and the Snf5p homolog Sfh1p, interact genetically with mutations in genes encoding kinetochore proteins and with a mutation in centromeric DNA. RSC also interacts genetically and physically with the histone and histone variant components of centromeric chromatin. Importantly, RSC is localized to centromeric and centromere-proximal chromosomal regions, and its association with these loci is dependent on Sth1p. Both sth1 and sfh1 mutants exhibit altered centromeric and centromere-proximal chromatin structure and increased missegregation of authentic chromosomes. Finally, RSC is not required for centromeric deposition of the histone H3 variant Cse4p, suggesting that RSC plays a role in reconfiguring centromeric and flanking nucleosomes following Cse4p recruitment for proper chromosome transmission.     

Defective Micronuclei and Genomic Exclusion in Selected C* Subclones of Tetrahymena1

SYNOPSIS. C* is a structurally heterogeneous clone containing normal and crinkled cells which are amicronucleate. Selection for structurally normal subclones did not result in the genesis of a diploid cell line. The stem cell of C* appears to be aneuploid with about 4 chromosomes instead of the usual 10, and from this line cells with fewer chromosomes or without micronuclei are generated. Only cells containing a portion of the micronucleus are viable. Amicronucleate cells die. They also mate very poorly. Consequently, little difference is observed between subclones having different proportions of amicronucleate cells in their ability to generate viable offspring by genomic exclusion. Our observations are discussed in terms of the separate functions of micronucleus and macronucleus and ways in which the nuclei seem to interact.