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H2A.Y is an essential, divergent Tetrahymena thermophila histone variant. It has a long nonhistone N terminus that contains leucine-rich repeats (LRR) and an LRR cap domain with similarity to Sds22p, a regulator of yeast protein phosphatase 1 (PP1) activity in the nucleus. In growing cells, H2A.Y is incorporated into micronuclei only during S phase, which occurs immediately after micronuclear mitosis. Depletion of H2A.Y causes prolonged retention of mitosis-associated histone H3-S10 phosphorylation and mitotic abnormalities that mimic S10E mutation. In cells where H2A.Y is depleted, an inducible chimeric gene, in which the H2A.Y N terminus is attached to H2A.X, is shown to regulate micronuclear H3-S10 phosphorylation. H2A.Y can also be specifically coimmunoprecipitated with a Tetrahymena PP1 ortholog (Ppo1p). Taken together, these results argue that the N terminus of H2A.Y functions to regulate H3-S10 dephosphorylation. This striking in vivo case of “cross-talk” between a H2A variant and a specific post-translational modification of another histone demonstrates a novel function for a histone variant.

Genetically programmed DNA rearrangements can regulate mRNA expression at an individual locus or, for some organisms, on a genome-wide scale. Ciliates rely on a remarkable process of whole-genome remodeling by DNA elimination to differentiate an expressed macronucleus (MAC) from a copy of the germline micronucleus (MIC) in each cycle of sexual reproduction. Here we describe results from the first high-throughput sequencing effort to investigate ciliate genome restructuring, comparing Sanger long-read sequences from a Tetrahymena thermophila MIC genome library to the MAC genome assembly. With almost 25% coverage of the unique-sequence MAC genome by MIC genome sequence reads, we created a resource for positional analysis of MIC-specific DNA removal that pinpoints MAC genome sites of DNA elimination at nucleotide resolution. The widespread distribution of internal eliminated sequences (IES) in promoter regions and introns suggests that MAC genome restructuring is essential not only for what it removes (for example, active transposons) but also for what it creates (for example, splicing-competent introns). Consistent with the heterogeneous boundaries and epigenetically modulated efficiency of individual IES deletions studied to date, we find that IES sites are dramatically under-represented in the ∼25% of the MAC genome encoding exons. As an exception to this general rule, we discovered a previously unknown class of small (

The Cd 2+ -inducible metallothionein ( MTT1 ) gene was cloned from Tetrahymena thermophila . Northern blot analysis showed that MTT1 mRNA is not detectable in the absence of Cd 2+ , is induced within 10 min of its addition, is expressed in proportion to its concentration, and rapidly disappears upon its withdrawal. Similarly, when the neo1 gene coding region flanked by the MTT1 gene noncoding sequences was used to disrupt the MTT1 locus, no transformants were observed in the absence of Cd 2+ , and the number of transformants was proportional to increased Cd 2+ concentration. The neo3 cassette, in which the MTT1 promoter replaced the histone gene HHF1 promoter of the previously used neo2 cassette , transformed cells at much higher frequencies than neo2 and produced germ-line knockouts where neo2 had failed. Rescuing the progeny of a mating of γ-tubulin gene, GTU1, knockout heterokaryons with a GTU1 gene inserted into the MTT1 locus yielded >75 times more transformants than rescuing with the wild-type GTU1 gene itself. When cells rescued with the MTT1-GTU1 chimeric gene were transferred to medium lacking Cd 2+ , they stopped growing and had phenotypic changes indistinguishable from cells containing only disrupted GTU1 genes. Thus, it is now possible to create conditional lethal mutants and study the terminal phenotypes of null mutations for essential genes by replacing the endogenous gene with one under the control of the MTT1 promoter. The MTT1 promoter also resulted in ≈30 times more overexpression of the IAG48[G1] surface antigen gene of the ciliate fish parasite Ichthyophthirius multifiliis than the highly expressed BTU1 promoter, accounting for ≈1% of the total cell protein. Thus, the MTT1 promoter should enable routine over-expression of endogenous and foreign genes in Tetrahymena .

Phosphorylation of histone H3 at serine 10 occurs during mitosis in diverse eukaryotes and correlates closely with mitotic and meiotic chromosome condensation. To better understand the function of H3 phosphorylation in vivo, we created strains of Tetrahymena in which a mutant H3 gene (S10A) was the only gene encoding the major H3 protein. Although both micronuclei and macronuclei contain H3 in typical nucleosomal structures, defects in nuclear divisions were restricted to mitotically dividing micronuclei; macronuclei, which are amitotic, showed no defects. Strains lacking phosphorylated H3 showed abnormal chromosome segregation, resulting in extensive chromosome loss during mitosis. During meiosis, micronuclei underwent abnormal chromosome condensation and failed to faithfully transmit chromosomes. These results demonstrate that H3 serine 10 phosphorylation is causally linked to chromosome condensation and segregation in vivo and is required for proper chromosome dynamics.

Tetrahymena thermophila is a model eukaryotic organism. Functional genomic analyses in Tetrahymena present rich opportunities to address fundamental questions of cell and molecular biology. The Tetrahymena Gene Expression Database (TGED; available at http://tged.ihb.ac.cn) is the first expression database of a ciliated protozoan. It covers three major physiological and developmental states: growth, starvation, and conjugation, and can be accessed through a user-friendly web interface. The gene expression profiles and candidate co-expressed genes for each gene can be retrieved using Gene ID or Gene description searches. Descriptions of standardized methods of sample preparation and the opportunity to add new Tetrahymena microarray data will be of great interest to the Tetrahymena research community. TGED is intended to be a resource for all members of the scientific research community who are interested in Tetrahymena and other ciliates.

A previously identified Tetrahymena thermophila actin gene (C. G. Cupples and R. E. Pearlman, Proc. Natl. Acad. Sci. USA 83: 5160-5164, 1986), here called ACT1 , was disrupted by insertion of a neo3 cassette. Cells in which all expressed copies of this gene were disrupted exhibited intermittent and extremely slow motility and severely curtailed phagocytic uptake. Transformation of these cells with inducible genetic constructs that contained a normal ACT1 gene restored motility. Use of an epitope-tagged construct permitted visualization of Act1p in the isolated axonemes of these rescued cells. In ACT1Δ mutant cells, ultrastructural abnormalities of outer doublet microtubules were present in some of the axonemes. Nonetheless, these cells were still able to assemble cilia after deciliation. The nearly paralyzed ACT1Δ cells completed cleavage furrowing normally, but the presumptive daughter cells often failed to separate from one another and later became reintegrated. Clonal analysis revealed that the cell cycle length of the ACT1Δ cells was approximately double that of wild-type controls. Clones could nonetheless be maintained for up to 15 successive fissions, suggesting that the ACT1 gene is not essential for cell viability or growth. Examination of the cell cortex with monoclonal antibodies revealed that whereas elongation of ciliary rows and formation of oral structures were normal, the ciliary rows of reintegrated daughter cells became laterally displaced and sometimes rejoined indiscriminately across the former division furrow. We conclude that Act1p is required in Tetrahymena thermophila primarily for normal ciliary motility and for phagocytosis and secondarily for the final separation of daughter cells.

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.

In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta-tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of alpha-tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.

All three genes encoding histone H3 proteins were cloned and sequenced from Tetrahymena thermophila. Two of these genes encode a major H3 protein identical to that of T. pyriformis and 87% identical to the major H3 of vertebrates. The third gene encodes hv2, a quantitatively minor replication independent (replacement) variant. The sequence of hv2 is only 85% identical to the animal replacement variant H3.3 and is the most divergent H3 replacement variant described. Phylogenetic analysis of 73 H3 protein sequences suggests that hv2, H3.3, and the plant replacement variant H3.III evolved independently, and that H3.3 is not the ancestral H3 gene, as was previously suggested (Wells, D., Bains, W., and Kedes, L. 1986, J. Mol. Evol., 23: 224-241). These results suggest it is the replication independence and not the particular protein sequence that is important in the function of H3 replacement variants.

Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Although structurally and functionally dissimilar, these nuclei are products of a single postzygotic division during conjugation, the sexual phase of the life cycle. Immunocytochemical analyses during growth, starvation, and conjugation were used to examine the nuclear deposition of hv1, a histone H2A variant that is found in macronuclei and thought to play a role in transcriptionally active chromatin. Polyclonal antisera were generated using whole hv1 protein and synthetic peptides from the amino and carboxyl domains of hv1. The transcriptionally active macronuclei stained at all stages of the life cycle. Micronuclei did not stain during growth or starvation but stained with two of the sera during early stages of conjugation, preceding the stage when micronuclei become transcriptionally active. Immunoblot analyses of fractionated macro- and micronuclei confirmed the micronuclear acquisition of hv1 early in conjugation. hv1 staining disappeared from developing micronuclei late in conjugation. Interestingly, the carboxy-peptide antiserum stained micronuclei only briefly, late in development. The detection of the previously sequestered carboxyl terminus of hv1 may be related to the elimination of hv1 during the dynamic restructing of micronuclear chromatin that occurs as the micronucleus enters a transcriptionally incompetent state that is maintained during vegetative growth. These studies demonstrate that the transcriptional differences between macro- and micronuclei are associated with the loss of a chromatin component from developing micronuclei rather than its de novo appearance in developing macronuclei and argue that hv1 functions in establishing a transcriptionally competent state of chromatin.

Background: The model eukaryote, Tetrahymena thermophila, is the first ciliated protozoan whose genome has been sequenced, enabling genome-wide analysis of gene expression. Methodology/Principal Findings: A genome-wide microarray platform containing the predicted coding sequences (putative genes) for T. thermophila is described, validated and used to study gene expression during the three major stages of the organism's life cycle: growth, starvation and conjugation. Conclusions/Significance: Of the,27,000 predicted open reading frames, transcripts homologous to only,5900 are not detectable in any of these life cycle stages, indicating that this single-celled organism does indeed contain a large number of functional genes. Transcripts from over 5000 predicted genes are expressed at levels >5x corrected background and 95 genes are expressed at >250x corrected background in all stages. Transcripts homologous to 91 predicted genes are specifically expressed and 155 more are highly up-regulated in growing cells, while 90 are specifically expressed and 616 are up-regulated during starvation. Strikingly, transcripts homologous to 1068 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation. The patterns of gene expression during conjugation correlate well with the developmental stages of meiosis, nuclear differentiation and DNA elimination. The relationship between gene expression and chromosome fragmentation is analyzed. Genes encoding proteins known to interact or to function in complexes show similar expression patterns, indicating that co-ordinate expression with putative genes of known function can identify genes with related functions. New candidate genes associated with the RNAi-like process of DNA elimination and with meiosis are identified and the late stages of conjugation are shown to be characterized by specific expression of an unexpectedly large and diverse number of genes not involved in nuclear functions.

HMG (high-mobility-group protein) B and HMG C are abundant nonhistone chromosomal proteins isolated from Tetrahymena thermophila macronuclei with solubilities, molecular weights, and amino acid compositions like those of vertebrate HMG proteins. Genomic clones encoding each of these proteins have been sequenced. Both are single-copy genes that encode single polyadenylated messages whose amounts are 10 to 15 times greater in growing cells than in starved, nongrowing cells. The derived amino acid sequences of HMG B and HMG C contain a highly conserved sequence, the HMG 1 box, found in vertebrate HMGs 1 and 2, and we speculate that this sequence may represent a novel, previously unrecognized DNA-binding motif in this class of chromosomal proteins. Like HMGs 1 and 2, HMGs B and C contain a high percentage of aromatic amino acids. However, the Tetrahymena HMGs are small, are associated with nucleosome core particles, and can be specifically extracted from macronuclei by elutive intercalation, properties associated with vertebrate HMGs 14 and 17, not HMGs 1 and 2. Thus, it appears that these Tetrahymena proteins have features in common with both of the major subgroups of higher eucaryotic HMG proteins. Surprisingly, a linker histone found exclusively in transcriptionally inactive micronuclei also has several HMG-like characteristics, including the ability to be specifically extracted from nuclei by elutive intercalation and the presence of the HMG 1 box. This finding suggests that at least in T. thermophila, proteins with HMG-like properties are not restricted to regions of transcriptionally active chromatin.

Histone fraction F1 has been isolated and purified from macronuclei of the ciliated protozoan, Tetrahymena pyriformis. In many respects, Tetrahymena F1 is similar to that of other organisms. It is the only Tetrahymena histone soluble in 5% perchloric acid or 5% trichloroacetic acid, has a higher molecular weight than any other Tetrahymena histone, is the histone most easily dissociated from Tetrahymena chromatin, and is susceptible to specific proteolytic cleavage. However, unlike F1 in all other organisms, Tetrahymena F1 is not the slowest-migrating histone fraction when analyzed by polyacrylamide gel electrophoresis at low pH. Tetrahymena F1 also exhibits unusual behavior in sodium dodecyl sulfate-containing polyacrylamide gels, migrating faster than calf thymus F1 at pH 10, and slower than calf thymus F1 at pH 7.6. Tetrahymena F1 was found to be highly phosphorylated in rapidly growing cells, suggesting that the relationship between cell replication and F1 phosphorylation previously observed in mammalian cells may extend to all eukaryotes. The observation that extensive F1 phosphorylation occurs in macronuclei, which divide amitotically, argues against a unique role for F1 phosphorylation in the process of chromosome condensation at mitosis.

Histone fraction F2A1 has been isolated and purified from macronuclei of the ciliate Tetrahymena pyriformis. It migrates as a single species on sodium dodecyl sulphate-acrylamide gel electrophoresis, with a molecular weight indistinguishable from that of calf thymus F2A1. The solubility properties of Tetrahymena F2A1 are also similar to those of calf thymus F2A1. Electrophoretic analyses on urea-acrylamide gels indicate that Tetrahymena F2A1 consists of four or five subspecies, the two fastest having electrophoretic mobilities identical with those of the two major electrophoretically separable forms of calf thymus F2A1. High resolution (long gel) electrophoresis coupled with incorporation of radioactive acetate both in vivo and in vitro suggest that, as in the case of calf thymus F2A1, differentical acetylation of a parent molecule can explain the observed electrophoretic heterogeneity of Tetrahymena F2A1. Electrophoretic analysis of histones isolated from the micronucleus, which is genetically less active than the macronucleus, indicates that it contains largely the relatively unacetylated (parent) form of histone F2A1.

Nineteen mutants that are conditional for both the ability to regain motility following deciliation and the ability to grow were isolated. The mutations causing slow growth were placed into five complementation groups. None of the mutations appears to affect energy production as all mutants remained motile at the restrictive temperature. In three complementation groups protein synthesis and the levels of mRNA encoding alpha-tubulin or actin were largely unaffected at the restrictive temperature, consistent with the hypothesis that mutations in these three groups directly affect the assembly of functional cilia and growth. Complementation group 1 was chosen for further characterization. Both phenotypes were shown to be linked, suggesting they are caused by a single mutation. Group 1 mutants regenerated cilia at the restrictive temperature, but the cilia were nonmotile. This mutation also caused a block in cytokinesis at the restrictive temperature but did not affect nuclear divisions or DNA synthesis. The block in cell division was transiently rescued by wild-type cytoplasm exchanged when mutants were paired with wild-type cells during conjugation (round 1 of genomic exclusion). Thus, at least one mutation has been isolated that affects assembly of some microtubule-based structures in Tetrahymena (cilia during regeneration) but not others (nuclei divide at 38 degrees), and the product of this gene is likely to play a role in both ciliary function and in cytokinesis.

Heterologous probes for yeast H4 and H3 histone genes have been used to study the corresponding histone mRNAs in growing and starved Tetrahymena. Histone mRNAs in both physiological states are polyadenylated. Two types of H4 protein and two types of H3 protein have previously identified in Tetrahymena. Two size classes of H4 messages and three classes of H3 messages have been detected by northern analyses. Southern blot analysis indicate that the number of different kinds of H3 and H4 genes is the same or slightly greater than the number of different messages, suggesting that each message is derived from a different gene. Growing cells have -30 times more histone mRNA than starved cells, even though their total mRNA content is only 4 times greater. The relative abundance of different H4 and H3 messages in growing and starved cells is different, demonstrating that the different messages for a particular type of histone are regulated non-coordinately. In starved cells the presence of a single size class of H3 messages correlates with the preferential synthesis of a previously described macronuclear-specific H3 variant. The fraction of histone messages loaded in growing and starved cells is the same as for bulk mRNAs, and the relative concentrations of the multiple messages for H4 and H3 are the same in polysomal and total RNAs of each cell type. These observations suggest that histone synthesis in Tetrahymena is controlled largely at the level of message abundance, and that very little, if any, control occurs at the translational level.

Macronuclei of the ciliated protozoan Tetrahymena pyriformis contain at least five classes of histones, including two with properties like those of histones F3 and F1 of higher eukaryotes. Micronuclei isolated under identical conditions contain little or no detectable F3 or F1. Digestion of both macronuclei and micronuclei with staphylococcal nuclease results in DNA fragments of discrete sizes. The electrophoretic mobilities of the larger fragments suggest that they are oligomers of the smallest ones. These results indicate that the periodic subunit structure observed in the chromatin of higher organisms also occurs in protozoans, and that this structure does not depend on the presence of either histone F1 or F3, even in an organism which has the genetic information for synthesizing these proteins.

The complete DNA sequence is presented of H4-II, the second of the pair of histone H4 genes of the ciliated protozoan, Tetrahymena thermophila. Both H4 genes code for the same protein. Codon usage in these and other Tetrahymena genes is severely restricted and is similar to that in yeast. Flanking regions are AT-rich (greater than or equal to 75%), relative to coding sequences (approximately 45% GC). Except for small, similarly positioned homologies, flanking sequences of the two genes are different. Canonical sequences in higher eukaryotic promoters are not obvious in these genes. Instead, short, localized, base composition eccentricities characterize the 5' flanking sequences of all Tetrahymena genes analyzed. The consensus, P yP u(A)3-4 ATGG initiates translation in these and all other known Tetrahymena genes. Nuclear transcripts and messages of both growing and starved cells begin at multiple sites, mainly at the first or second A residue following a pyrimidine. The palindrome typical of histone message 3' termini in higher organisms is not present. Downstream of both genes are sequences similar to the processing/polyadenylation signal of higher eukaryotes, although the unique 3' ends are not those predicted by the location of the signals.

Micronuclei of Tetrahymena thermophila contain two electrophoretically distinct forms of histone H3. The slower migrating micronuclear species, H3 S , is indistinguishable from macronuclear H3 by electrophoretic analyses in three gel systems and by partial proteolytic peptide mapping. The faster species, H3 F , is unique to micronuclei. Pulse-chase experiments with radioactive amino acids show that H3 S is a precursor to H3 F . We present evidence that the in vivo processing of H3 S into H3 F requires cell growth and/or division and may occur regularly each generation at a specific point in the cell cycle. The processing event must occur after H3 F is deposited on micronuclear chromatin, since both H3 S and H3 F can be isolated from sucrose gradient-purified mononucleosomes (Allis, Glover and Gorovsky, 1979). Partial proteolytic peptide mapping coupled with 3 H-N-ethylmaleimide labeling suggest that the processing event involves a proteolytic cleavage from the amino terminal end of H3 F . Automated sequence analyses of 14 C-lysine-labeled macronuclear H3 together with either 3 H-lysine-labeled H3 S or H3 F demonstrated that H3 F is derived from H3 S by a proteolytic cleavage which removes six residues from the amino terminus. These observations represent the first demonstration of a physiologically regulated proteolytic processing event in histone metabolism.

Publisher Summary This chapter discusses the isolation of micro- and macronuclei of Tetrahymena Pyriformis. With the exception of a few amicronucleate or partially amicronucleate strains of Tetrahymenaa each cell contains two types of nuclei, a smaller micronucleus and a larger macronucleus. Although these two nuclei are formed from daughter nuclei of a single mitotic division (the second postzygotic division) during conjugation, they differ in most of their structural and functional properties in vegetative cells. Micronuclei divide mitotically prior to cell division, undergo DNA synthesis early in the cell cycle, do not contain nucleoli or heterogeneous ribonucleoprotein particles, and are genetically inactive. Macronuclei divide amitotically when the cell divides, synthesize DNA in the middle third of the cell cycle, have large numbers of RNA-containing inclusions, and are responsible for the genetic activity which controls the phenotype of the vegetative cell. Micro- and macronuclei thus have a common origin during conjugation and reside in the same cytoplasm during vegetative growth, yet differ strikingly in structure and function. Therefore, it is suggested that these two nuclei may be used as a model system to explore the mechanisms whereby the same (or closely related) genetic information is maintained in different structural and functional states in eukaryotic cells.

Two-dimensional gel electrophoresis (triton-acid-urea followed by SDS) has been used to resolve two previously uncharacterized, quantitatively minor histone variants in acid extracts from macronuclei of Tetrahymena thermophila. Utilizing techniques which allow characterization of these variants without purifying them in significant quantities, we identify one protein as a subtype of H3. The other protein is a moderately lysine-rich histone whose tryptic peptide map differs from that of both H2A and H2B. However, its pattern of secondary modifications, its detergent-binding properties and its methionineless nature all suggest that it is more like H2A than any other histone. Both variants are associated with nucleosomes derived from macronuclei. Thus primary sequence variants of the inner histones, presumably indicative of nucleosome heterogeneity, exist in a lower eucaryote, in an amitotic nucleus, and within the nucleus of a clonally propagated organism. Evidence is presented that these newly described minor variants are absent in micronuclei, suggesting that they play an important role in the structural and functional differentiation of macronuclear chromatin.

Histones were extracted from macro- and micronuclear chromatin of the ciliated protozoan Tetrahymena pyriformis. Conditions that resulted in macronuclear chromatin containing large amounts of histone F1 yielded micronuclear chromatin in which this histone was absent. Evidence is presented indicating that the absence of F1 from micronuclei is not a preparative artifact and that histone F1 is replaced by other histone fractions. Since micronuclei divide mitotically, while macronuclei divide amitotically, these results suggest that histone F1 and its phosphorylation do not play an indispensable role in the process of mitotic chromosome condensation, in chromosome replication, or in the separation of newly synthesized chromatids.