Tetrahymena glochidiophila n. sp., a new species of Tetrahymena (Ciliophora) that causes mortality to glochidia larvae of freshwater mussels (Bivalvia).

A ciliate protozoan was discovered whose presence coincided with a rapid decrease in the viability (i.e. ability to close valves) of glochidia of the freshwater mussel Lampsilis siliquoidea. Microscopic examination showed it to be a histophagous tetrahymenine ciliate. Small subunit (SSU) rRNA and cytochrome c oxidase subunit 1 (cox1) barcode sequences from cultured cells showed that it belongs to the same new species isolated from water samples as a free-living ciliate. Phylogenetic analyses place this new ciliate in the same clade with the macrostome species Tetrahymena paravorax, and we propose the name T. glochidiophila n. sp. for this new species. The phylogeny provides further support for the hypothesis that histophagy was a life history trait of the ancestor of Tetrahymena.

Macronuclear Subunits of Tetrahymena thermophila Are Functionally Haploid.

In Tetrahymena thermophila the major argument for the existence of diploid subunits has been that some loci show a delay in the accumulation of stable subclones during macronuclear assortment. This delay is based on the assumption that throughout the life cycle there are 45 subunits. We find that for at least 50 fissions after conjugation there is sufficient DNA for 66 haploid subunits. These additional subunits early in the life cycle are sufficient to explain the observed accumulation of stable subclones in all instances. This removes the need to invoke diploidy to explain assortment, thus resolving the question of subunit ploidy in favor of haploidy.

Expression of a cell surface immobilization antigen during serotype transformation in Tetrahymena thermophila.

A temperature shift from 40 to 28 degrees C rapidly induced expression of a specific immobilization antigen at the cell surface in Tetrahymena thermophila. This transformation was inhibited by actinomycin D and cycloheximide but not by colchicine or cytochalasin B. The major surface antigen expressed at 28 degrees C in cells homozygous for the SerH3 allele was partially purified, and an antiserum against this preparation was raised in rabbits. Electrophoresis, immunoblot, and [35S]methionine incorporation studies are reported which support the conclusion that the H3 antigen is an acidic protein with an Mr of approximately 52,000 daltons. An induced synthesis of the H3 immobilization antigen was detected within 30 min after a shift from 40 to 28 degrees C. This protein appeared to be synthesized in the microsomal fraction and transferred without cleavage to the cell surface, where it was inserted first into nonciliated regions.

Regulatory Serotype Mutations in TETRAHYMENA PYRIFORMIS, Syngen 1.

A method utilizing allelic exclusion has been developed to isolate mutants of Tetrahymena pyriformis, syngen 1, in which the normal pattern of expression of mutally exclusive surface antigens is altered. Cells homozygous for the recessive mutant allele R-1(r) do not express the L, H and T serotypes when grown under conditions appropriate for their expression. Rather, a new immobilization antigen, r, is expressed. Cells homozygous for the recessive mutant allele R-3(r) also express the r antigen instead of H serotypes, but are normal in their expression of T antigens. Genetic analyses show that R-1 and R-3 are not closely linked, that R-1 is linked to T by 9.3 units, and that R-3 may be loosely linked to the mt locus. Different linkage values were obtained, however, when different inbred laboratory strains were used, suggesting the possible existence of crossover modifying genes. The rates of assortment of R-1(R)/R-1(r) and R-3(R)/R-3(r) heterozygotes into pure sublines expressing either H or r serotypes are close to the values observed for the differentiation of heterozygotes at other loci. The data confirm the previous observation that genetic coupling relationships are not maintained in macronuclear phenotypes and are consistent with the hypothesis that the macronucleus contains 45 assorting subunits. The assortment of the double heterozygote R-1(R)/R-1(r), R-3(R)/R-3(r) at R(f)=0.0112 suggests that the units of assortment are not individual genetic loci or chromosome fragments, but that the units may be complete genomes.

Nucleo-cytoplasmic interaction during macronuclear differentiation in ciliate protists: genetic basis for cytoplasmic control of SerH expression during macronuclear development in Tetrahymena thermophila.

A novel class of mutations affecting the developmental expression of SerH cell surface antigen genes of Tetrahymena thermophila is described. Unlike previous categories of mutation, the four independently isolated mutations of this class act through the cytoplasm to affect SerH genes during macronuclear development. That is, macronuclei which develop under the influence of mutant cytoplasm do not subsequently express H, most likely because the developmental processing of SerH genes is affected. The cytoplasmic effect is specific for the SerH locus and is independent of which SerH allele is present. In place of H, hitherto unknown antigens are expressed. Expression of SerH can be rescued during development either by wild-type cytoplasm exchanged between conjugants or by the homozygous wild-type genotype. The mutations segregate independently of the SerH genes and identify one, possibly two, bistable genes. Possible models to explain these results are discussed.

Genomic exclusion and other micronuclear anomalies are common in genetically defective clones of tetrahymena thermophila.

Genomic exclusion (GE) is an abnormal form of conjugation which has previously been described in detail for three hypodiploid strains of Tetrahymena thermophila. These strains cannot form gametic nuclei and by failing to participate in normal reciprocal fertilization their genes are excluded from exconjugants. To determine whether GE is a general property of infertile strains, we surveyed genetically and cytogenetically 19 additional strains of T. thermophila to determine why they failed to contribute genes to sexual progeny. Crosses to genetically marked tester strains showed that seventeen of these strains undergo GE. In each case GE appears to be due to the failure of the defective partner to form functional gametic nuclei. The normal conjugant, however, contributes to its defective partner a haploid nucleus identical to its own, and following diploidization of the unfertilized nuclei, the conjugants separate retaining the old macronuclei. Cytofluorimetric measurement of micronuclear DNA content in 18 strains suggests that aneuploidy is the proximate cause of GE; eleven strains were hypodiploid, five were diploid and three were hyperdiploid. Many irregular cytogenetic events were observed in conjugants presumably not undergoing GE, including, in some instances, abnormal meiosis in the normal partner. Since genomic exclusion was found in both wildtype and mutant clones, the results suggest that it should be possible by appropriate crosses to identify genomic exclusion strains of any genotype.

Recombination and assortment in the macronucleus of Tetrahymena thermophila: a theoretical study by computer simulation.

The compound nature of the macronucleus of Tetrahymena thermophila presents multiple opportunities for recombination between genes on the same macronuclear chromosome. Such recombinants should be detectable through their assortment at subsequent amitotic macronuclear divisions. Thus, a macronucleus that is initially AB/ab should produce recombinant assortees of the genotypes Ab/aB. Computer simulation shows that, when the recombination frequency is two or fewer times per cell cycle, recombinant assortees are produced at experimentally measurable frequencies of less than 40%. At higher recombination frequencies, linked genes appear to assort independently. The simulations also show that recombination during macronuclear development can be distinguished from recombination in subsequent cell cycles only if the first appearance of recombinant assortees is 100 or more fissions after conjugation. The use of macronuclear recombination and assortment as a means of mapping macronuclear genes is severely constrained by the large variances in assortment outcomes; with experimentally small sample sizes, such mapping is impossible.

Multiple effects of mutation on expression of alternative cell surface protein genes in Tetrahymena thermophila.

Genes at the SerH locus of the ciliated protist Tetrahymena thermophila specify the major (H) surface protein on cells grown at 20-36 degrees. Alternative proteins L, T, S and I are expressed under different conditions of temperature and culture media. Mutants unable to express SerH genes were examined for expression of these proteins, also called immobilization or i-antigens, at both H and non-H conditions. In all instances, one or more i-antigens were expressed in the absence of H, and, in most instances, expression of i-antigens under non-H conditions was also affected. Examples of the latter include both the continued expression of H-replacement antigens and the inability to express certain other i-antigens. Such multiple effects were observed in mutants with trans-acting (rseA, rseB, rseC, RseD) and cis-acting (H1-1 and H1-2) mutations, but not in mutants in which SerH is affected developmentally (B2092, B2101, B2103, B2107). These interactions suggest that the wild-type genes identified by mutation exert both positive and negative effects in the regulation of i-antigen gene expression.

High frequency intragenic recombination during macronuclear development in Tetrahymena thermophila restores the wild-type SerH1 gene.

Macronuclear development in ciliates is characterized by extensive rearrangement of genetic material, including sequence elimination, chromosome fragmentation and telomere addition. Intragenic recombination is a relatively rare, but evolutionarily important phenomenon occurring in mitosis and meiosis in a wide variety of organisms. Here, we show that high frequency intragenic recombination, on the order of 30%, occurs in the developing amitotic macronucleus of the ciliate Tetrahymena thermophila. Such recombination, occurring between two nonsense transition mutations separated by 726 nucleotides, reproducibly restores wild-type expression of the SerH1 surface protein gene, thus mimicking complementation in trans heterozygotes. Recombination must be considered a potentially important aspect of macronuclear development, producing gene combinations not present in the germinal micronucleus.

A temperature-sensitive mutation of the temperature-regulated SerH3 i-antigen gene of Tetrahymena thermophila: implications for regulation of mutual exclusion.

The Ser genes of Tetrahymena thermophila specify alternative forms of a major cell surface glycoprotein, the immobilization or i-antigen (i-ag). Regulation of i-ag expression assures that at least one i-ag gene is expressed at all times. To learn more about the regulatory system and the possible role of i-ag itself, we studied SerH3-ts1, a temperature-sensitive allele of the temperature-regulated SerH3 gene normally expressed from 20-36 degrees. In homozygotes grown at the nonpermissive temperature (> 32 degrees), H3 is not present on the cell surface, but the gene continues to be transcribed until its 36 degrees cutoff. H3 formed at the permissive temperature is stable at nonpermissive temperatures, indicating that SerH3-ts1 is temperature-sensitive for synthesis rather than function. At nonpermissive temperatures, the S i-ag is expressed in place of H3. This result suggests that normal H protein may play a role in regulating S expression. SerH3-ts1 was isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Sequencing of SerH3-ts1 revealed a single A --> G transition at nucleotide 473, resulting in the substitution of glycine for aspartate. The affected residue is conserved in the internal repeats comprising the H protein, and the charge difference correlates with changes in electrophoretic mobility of the H3 protein.

Transitory Heterosis in Numbers of Basal Bodies in TETRAHYMENA PYRIFORMIS.

The numbers of kinetosomes in ciliary rows on the ventral surface of cells of Tetrahymena pyriformis, syngen 1, are greater by about 10% in young heterozygotes established by crossing two inbred strains than in either homozygote. The difference, apparent at 20 fissions post-fertilization, is essentially gone by 100 fissions. This observation is consistent with a generalized "allelic exclusion" at all loci completed by perhaps 60 fissions after fertilization.

Isolation and genetic analysis of mutations at the SerH immobilization antigen locus of Tetrahymena thermophila.

Multiple alleles at the SerH locus specify the major cell surface protein (immobilization antigen) of the ciliate Tetrahymena thermophila. Following mutagenesis of SerH1 homozygotes, two mutations, H1-1 and H1-2, were recovered in heterozygous form. Mutant homozygotes do not express H1 antigen, nor is H1 expressed in F1 progeny of crosses to wild-type strains homozygous for SerH2 or SerH3. H1-1 and H1-2 segregate without recombination from these wild-type alleles in expected F2 and testcross Mendelian ratios. H1-1 and H1-2 do, however, complement each other to express H1 antigen. Experiments suggest this complementation is due neither to recombination during macronuclear development nor to interallelic complementation of defective SerH1 gene products. These results suggest that SerH1 is intact in one mutant, and possibly both, although no such allele has been segregated in testcross progeny (N = 205). The hypothesis is presented that complementation between H1-1 and H1-2 is due to interaction between allele-specific regulators closely linked to the SerH1 gene.

Mutations affecting cell division in Tetrahymena pyriformis. I. Selection and genetic analysis.

Fourteen nitrosoguanidine-induced mutations that bring about temperature-sensitive morphological abnormalities resulting from a specific effect on cell division have been isolated as heterozygous phenotypic assortants in Tetrahymena pyriformis syngen 1. Genetic analysis revealed all to be single-gene recessives. Detailed analysis of the kinetics of assortment for one of the mutated alleles revealed a rate (0.0104 pure lines per fission) consistent with that previously observed at other loci in this organism. The mutations fall into six complementation groups (mo1, mo2, mo3, mo6, mo8, and mo12). Homozygotes of mo2 are unconditionally expresed, while all alleles of mo1, mo6, mo8, and mo12 are heat sensitive for division arrest. At the mo3 locus two alleles are heat senstivie, one is primarily cold sensitive, while two are sensitive to both heat and cold. Two out of three combinations of different mo3 alleles show conventional Mendelian segregation of conditions of expression. Different alleles of mo1, mo3, mo8, and mo12 also manifest differences in penetrance at the restrictive temperature. Despite these differences involving expression, the abnormal phenotypes themselves are locus-specific and distinctive; in the one case (mo1a and mo1b) in which two alleles manifest somewhat different phenotypes, the F1 between them is intermediate. One additional recessive mutation (fat1) brings about a nonconditional lengthening of the cell cycle, with some arrest of cell division at the restrictive temperature. These findings demonstrate that selection of heterozygotes undergoing phenotypic assortment can be an effective method for obtaining substantial numbers of a desired class of temperature-sensitive mutations in T. pyriformis.

Sequence and expression of the SerJ immobilization antigen gene of Tetrahymena thermophila regulated by dominant epistasis.

In ciliates, variable surface protein genes encoding the immobilization antigen (-ag) are expressed under different environmental conditions, including temperature and salt stress. These i-ags are GPI-linked and coat the entire external surface of the cell, including the cilia. In Tetrahymena thermophila-ag in natural isolates is the result of dominant epistasis masking the expression of the H i-ag ordinarily expressed at 20-36 degrees C. This report describes the expression and sequence of the Ser-ag. J is present on the cell surface up to 38 degrees C; above 38 degrees C SerSeranked by an A-rich 5' UTR and a 3' UTR containing putative mRNA destabilization motifs. The encoded J polypeptide consists of 438 amino acids and is rich in alanine, cysteine, serine and threonine. The N- and resemble signal peptide and GPI-anchor addition sites, respectively. The majority of the molecule consists of four imperfect repeats with 10 periodic cysteines per repeat in the pattern CX(6)CX(2)CX(21)CX(4)CX(13-15)CX(2)CX(18)CX(3)CX(11)CX(9-10). Although H i-ags encoded by paralogous SerH genes have 3.5 imperfect repeats with eight periodic cysteines per repeat, J nevertheless resembles H with respect to amino acid composition, codon usage, N- and C-termini, the arrangement of the cysteine periods, and regulation by mRNA stability. However, despite these similarities and epistasis, the evolutionary relationship between SerH and SerJ is unclear.

Sequence, codon usage and cysteine periodicity of the SerH1 gene and in the encoded surface protein of Tetrahymena thermophila.

The temperature-regulated SerH1 gene coding for an immunodominant surface glycoprotein (i-Ag H1) of Tetrahymena thermophila has been sequenced. The gene is reproducibly rearranged during macronuclear development and steady state mRNA levels are present at < 36 degrees C. The deduced i-Ag H1 amino acid (aa) sequence is rich in Ser, Thr and Cys, and contains three periods each consisting of 85 aa punctuated by eight Cys with the general formula, CX6CX17CX2CX18CX2CX11CX2CX19 (where X = any aa). Such Cys periodicity is common to ciliate i-Ag. Codon usage in Tt, Paramecium primaurelia and P. tetraurelia i-Ag encoding genes is similar, with approx. 80% A+T in the 3' position which is in marked contrast to the approx. 54% 3' A+T in other ciliate genes.

Age-associated changes in the micronuclear cycle of Tetrahymena thermophila A III heterokaryons. A brief note.

The micronuclear cycle of Tetrahymena thermophila A III heterokaryons is shown to change with increasing clonal age. Autoradiographic and cytofluorimetric studies suggest that alteration may be due to (1) loss of late replicating sequences, or (2) (more likely) changes in the timing of micronuclear division and S with respect to cytokinesis.

Age-dependent micronuclear deterioration in Tetrahymena pyriformis, syngen 1.

In the ciliate Tetrahymena pyriformis, syngen 1, senescence is marked both by mitotic and meiotic cytogenetic irregularities of the micronucleus (germinal nucleus) and by an increased frequency of cell death. Full vigor can be restored through the age-dependent process of genomic exclusion, which in prematurely senscent clones C, A III and A V results in the replacement of defective micronuclei but not in the development of new macronuclei (somatic nuclei). After a period of normal behavior, which lasts about 40 fissions in C and 100 fissions in A III and A V, the new micronucleus placed in the same cytoplasm as the old macronucleus again becomes defective and the rejevenated clone again shows signs of senescence. The origin of these defective micronuclei is traced to action of the old macronucleus. It is suggested that the time of course of aging is regulated through the accumulation of unrepaired damage to the micronuclear genetic material.

Dominant mutations regulating i-antigen expression in Tetrahymena thermophila.

Two dominant mutations at the RseD locus regulating the differential expression of alternative cell surface immobilization antigens of the ciliate Tetrahymena thermophila are described. RseD1 and RseD2 express I to the exclusion of H (28 degrees C) and are leaky for I when expressing either L (15 degrees C) or T (40 degrees C). Complementation was not observed in RseD1/RseD2 heterozygotes, and in 326 testcross progeny no wild-type (micronuclear) recombinants were observed. Macronuclear recombination also was not observed. RseD is located on chromosome 5, at least 50 map units from rseB, which also regulates antigen expression. This brings to four the number of loci known to regulate antigen expression.

Identification of a cDNA coding for the SerH3 surface protein of Tetrahymena thermophila.

We have identified a Tetrahymena thermophila cDNA-containing plasmid (pC6) which hybridizes to a 1.47-kB RNA whose changes in cellular concentration parallel the changes in synthetic rate of a major cell surface protein. From a molecular and genetic analysis of strains expressing the gene (SerH3) encoding this protein, and of strains expressing immunologically distinct alleles of this gene, we conclude that pC6 encodes a portion of the SerH3 allele.