Mutation affecting cell separation and macronuclear resorption during conjugation in Tetrahymena thermophila: early expression of the zygotic genotype.

A new recessive conjugation lethal mutation was found in Tetrahymena thermophila which was named mra for macronuclear resorption arrest. Other events affected by the mra mutations are separation of pairs, DNA replication in the macronuclear anlagen, and resorption of one of the two micronuclei. In wild-type crosses 50% of the pairs had separated by 12 hr after mixing two mating types and had completed resorption of the old macronucleus 1-2 hr later. In contrast most mra conjugants did not separate even by 24 hr after mixing and the old relic (condensed) macronucleus was seen in over 90% of them. After addition of 10 mM calcium to the conjugation medium, the mra conjugants did separate but they still failed to complete resorption of the old macronucleus and to replicate macronuclear anlagen DNA in the exconjugants. The calcium induced separation of the mra conjugants occurred later than the separation of control pairs. During normal conjugation cell separation occurs before the first expression of known macronuclear genes and prior to processing of the macronuclear DNA. Therefore, the mra phenotype infers that separation of conjugants requires a signal which is produced by the macronuclear anlagen at an unusually early time.     

Macronuclear persistence of sequences normally eliminated during development in Tetrahymena thermophila

During conjugation in the ciliated protozoan, Tetrahymena thermophila, a somatic MAC-ronucleus develops from the germinal MICronucleus. Ten to 20 percent of the MIC genome is eliminated during this process. Three repetitive families have been identified which have different levels of repetition in the MIC and are eliminated to different degrees in the MAC. Some members of two of these families persist in the MAC. In this study, we have looked at these persistent sequences in the MAC of cell lines from a variety of sources including several inbed strains, two sets of caryonides, caryonidal subclones, and vegetatively aged cell clones. The results suggest that the sequences that remain in the MAC have a genetic predisposition to persist. However, epigenetic variations occur as the MAC develops so that only some of the persistent sequences are actually observed in a particular MAC. Polymorphisms may be generated if alternative processing of a single MIC segment occurs. These polymorphisms can later be resolved by phenotypic assortment during vegetative growth. These facultatively persistent sequences appear to differ from sequences previously described in this organism.     

Locus-dependent profiles of the rescue of nonexcitable behavioral mutants during conjugation in Tetrahymena thermophila

The Tetrahymena nonreversal (TNR) mutants of Tetrahymena thermophila are behavioral mutants with nonexcitable membranes. When cells of the tnrB mutant were mated with wild type, a phenotypic change occurred about l h after pair formation. The pairs began to lose their heterotypic character in stimulation solution containing high potassium and, within 1 1/2h, they were not distinguishable from the wild-type homotypic pairs. On the contrary, although pairs of the tnrA and wild type also lost their heterotypic character about 1 1/2 h after pair formation, they never showed a full response as wild-type homotypic pairs. When tnrA was mated with tnrB more than 50% of pairs expressed a heterotypic pair character 2 h after pair formation, consistent with the tnrB defect having been rescued but not the tnrA defect. Thus, conjugation rescue of the mutant phenotype is locus dependent and probably reflects the nature of the gene products controlling voltage-dependent Ca²⁺ channels. © 1992 Wiley-Liss, Inc.     

Regulation of Macronuclear DNA Content in Tetrahymena thermophila*†

Synopsis.
Unequal macronuclear division in Tetrahymena thermophila introduces variance into G1 macronuclei; unless eliminated such variance would result in continuous variation in DNA content. Analysis of G1 and G2 macronuclear variances reveals that the added variance is eliminated by action on the extremes of macronuclear DNA content. In this model (Model II), macronuclei with small amounts of DNA have an additional complete S phase, while those with large amounts of DNA skip S. From available data, chromatin extrusion is shown not to contribute significantly, if at all, to the elimination of variance. Computer simulations utilizing haploid subunits indicate that model II predictions apply reasonably well to experimental data in terms of coefficients of variation, mean DNA content, and frequency of additional and skipped S phases. The simulations reveal also that within certain constraints, particularly the thresholds for additional and skipped S phases, macronuclear assortment is unaffected by Model II regulation. The relationships between Model II and other aspects of the cell cycle are briefly discussed.     

Rearrangement of the 5S ribosomal RNA gene clusters during the development and replication of the macronucleus in Tetrahymena thermophila

The organization of the 5S rRNA genes in the MACronuclear genome of Tetrahymena thermophila was examined during MAC development and replication. The 5S genes are arranged in several tandem arrays of alternating transcribed and spacer sequences in both MICronucleus and MAC. The number of EcoRI fragments bearing 5S gene clusters is similar in MIC and MAC. Most fragments occur in both the MIC and newly formed MAC genomes, a few being MIC-limited and a few MAC-limited. The same rearrangements are seen in the MACs of all four caryonides of a mating pair, and most rearrangements are seen in the newly formed MACs of different inbred strains. During replication of the MAC about half the fragments bearing 5S gene clusters disappear in different cell lines, and new fragments containing 5S genes appear. These fragments differ in size from those present in the MIC or newly formed MAC. These alterations occur in the MACs of all strains except strain B, which is more resistant to vegetative rearrangement. The losses and gains of fragments occur during clonal propagation of cell lines. The process begins by 35 fissions following conjugation, but once an alteration occurs, it is stably propagated. Clonal variation occurs with respect to which losses and gains occur, although a nonrandom distribution is seen among cell clones. We conclude that the alterations in MAC fragment size occur at two stages in the life cycle of Tetrahymena. The first stage occurs during conjugation, when the MAC develops from the MIC. The second stage becomes manifest during vegetative growth, when DNA replication occurs in the MAC and daughter molecules are distributed “amitotically” to daughter nuclei. The two-stage character to MAC alterations for the 5S genes is interpreted in terms of the two steps previously described for MAC differentiation: determination and phenotypic assortment. Possible molecular mechanisms are also discussed.     

嗜热四膜虫——具有发展潜力的功能基因组学研究模型

在真核生物的分子生物学和遗传学研究方面,纤毛类原生动物嗜热四膜虫(Tetrahymenathermophila)已经被证明是一种有价值的生物学模型。通过对它的研究,人们发现并且掌握了核酶的分子机制、RNA的自我拼接、端粒的结构和功能、DNA序列重组等机理。这种原生动物的基因组功能分别由两个细胞核执行,即二倍体的小核与生殖过程有关,而多倍体的大核决定细胞的基因转录,并为转化基因的表达提供了强有力的手段。     

How the mirror-image pattern specified by a janus mutation of Tetrahymena thermophila comes to expression

The initial changes of cell-surface organization that occurred as the recessive janAl (janus) mutation of Tetrahymena thermophila first became expressed were elucidated in a special mating scheme in which old macronuclei homozygous for janA⁺ were synchronously replaced by new macronuclei homozygous for janAl. During this period of onset of expression, the number, regularity, and asymmetry of the ciliary rows remained unchanged. New normal (primary) oral apparatuses (OAs) continued to be formed posterior to old OAs, as in normal cells. At about four fissions after conjugation, abnormal (secondary) OAs with a partial reversal of asymmetry began to appear nearly opposite to the primary OAs, close to but not at the eventual circumferential position of janAl secondary OAs. The array of contractile vacuole pores (CVPs), normally located adjacent to two ciliary rows centered near 22% of the cell circumference to the righ of the primary oral meridian, underwent a two-step transformation: first, the number of adjacent ciliary rows bearing CVPs increased to 3, 4, and sometimes 5, then “skipped” rows appeared within this broadened CVP-arc to split the single set of CVPs into two separated subsets. The CVP transformations occurred gradually and progressively. They began prior to the expression of secondary OAs but accelerated as secondary OAs appeared. As the CVP are became broader, its midpoint shifted somewhat to the right, away from the primary oral meridian, but ended up close to halfway between the primary and secondary oral meridians. The data provide a better fit to an intercalation model than to an alternative double gradient model, suggesting that the janAl mutation alters the large-scale organization of positional values by preventing the expression of a subset of these values and thus provoking reverse-intercalation of the remainder.     

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.     

镉和铜对嗜热四膜虫金属硫蛋白基因的诱导表达

本文在荧光定量PCR优化的基础上,利用该技术考察了不同浓度的重金属镉和铜对嗜热四膜虫金属硫蛋白基因(MTT1)诱导表达的变化规律。结果表明MTT1基因的表达对镉离子的诱导更灵敏,且在一定阈值浓度(≤35.2μmol/L)范围内,镉离子浓度升高会增加MTT1基因表达量,超过该阈值后表达量迅速下降;镉与铜同时诱导时MTT1基因的表达情况与镉单独诱导的类似,但阈值浓度减小为22μmol/L,表明二者的联合毒性为协同作用。镉离子浓度低于22μmol/L时,与铜离子的共同作用会大大增加MTT1基因的表达量,从而增强了四膜虫的解毒能力。     

Cdk3, a conjugation-specific cyclin-dependent kinase,is essential for the initiation of meiosis in Tetrahymena thermophila

Meiosis is an important process in sexual reproduction. Meiosis initiation has been found to be highly diverse among species. In yeast, it has been established that cyclin-dependent kinases (Cdks) and cyclins are essential components in the meiosis initiation pathway. In this study, we identified 4 Cdks in the model ciliate, Tetrahymena thermophila, and we found one of them, Cdk3, which is specifically expressed during early conjugation, to be essential for meiosis initiation. Cdk3 deletion led to arrest at the pair formation stage of conjugation. We then confirmed that Cdk3 acts upstream of double-strand break (DSB) formation. Moreover, we detected that Cdk3 is necessary for the expression of many genes involved in early meiotic events. Through proteomic quantification of phosphorylation, co-expression analysis and RNA-Seq analyses, we identified a conjugation-specific cyclin, Cyc2, which most likely partners with Cdk3 to initiate meiosis.     

bcd: A mutation affecting the width of organelle domains in the cortex of Tetrahymena thermophila

Summary A single-gene recessive mutation, bcd (broadened cortical domains), of Tetrahymena thermophila is characterized by a variable broadening of the spatial domains within which cortical organelles, including both the contractile vacuole pores (CVP) and oral apparatus (OA), are formed. The phenotype is not temperature-sensitive. During the development of the organelles of the mutant prior to cell division, extra CVPs and extra oral primordia (OP) appear near ciliary rows adjacent to the rows at which these structures normally form. In the later stages of development, some, but not all, of these extra structures are resorbed, or in the case of the oral domain, multiple adjacent OPs may be completely or partially integrated into a single enlarged OA. When multiple OAs persist, one or more of these may display a reversed orientation reminiscent of those encountered in janus mutants. However, unlike janus, bcd cells do not express any sign of a mirror-image global organization.Our results can best be accounted for by postulating that the bcd mutation affects some common determinant of the widths of both CVP and OA domains. Studies are in progress which explore the relationship between this width-determining mechanism(s) and the mechanism(s) determining the location of cortical organelles around the cell circumference.     

Alternative gene expression in type I and type II cells may enable further nuclear changes during conjugation of Blepharisma japonicum

In contrast to most ciliates, meiosis and successive nuclear changes during conjugation occur only in heterotypic pairs in Blepharisma. It has been suggested that homotypic pairs are ready for conjugation, but lack a trigger to initiate the nuclear changes, and the conjugation process is arrested before the onset of meiosis. To explore the possible nature of the trigger, we previously identified the genes BjCdk1 (homologous to cdk1/cdc2), Bj4HPPD (4-hydroxy-phenylpyruvate dioxygenase) and BjCks (cyclin dependent kinase regulatory subunit) whose expression is up-regulated in gamone1-treated type II cells. In this study, we investigated the molecular structures of these three genes, and compared their expression patterns in homotypic and heterotypic pairs, finding remarkable differences. BjCdk1, Bj4HPPD and BjCks were expressed specifically in gamone1-treated type II cells, but not in gamone2-treated type I cells. In heterotypic pairs, the expression of these genes stayed at the same level or gradually decreased throughout the entire process of conjugation, but it rapidly decreased and ceased after 10hours in homotypic pairs. These results indicate that some genes are expressed in a mating-type specific manner. Alternative gene expression in mating type I and type II cells and merging of individual factors in a heterotypic pair may induce nuclear changes including meiosis.     

DNA elimination and its relation to quantities in the macronucleus of Tetrahymena

The macronucleus of Tetrahymena contains a large number of DNA molecules of subchromosomal size. They belong to about 270 species each one occurring at an average number of 45 copies Macronuclei divide unequally and nothing is known of segregation control. This and the elimination and degradation of DNA during macronuclear amitosis make the clonal stability of macronuclei a problem of qualitative and quantitative control on a subchromosomal level. We studied the contribution of DNA elimination to the quantitative composition of the macronucleus cytophotometrically in single cells of different strains. This was done under standard conditions and under conditions known to influence the amount of macronuclear DNA. The following results were found: Elimination of DNA occurs at almost every division. The size of the elimination body is highly variable but still positively correlated with the macronuclear DNA content. In T. thermophila the amount of eliminated DNA is 2.5% of the G2 content and is not dependent on the growth state. It varies with species, amounting to as much as 8% in T pigmentosa. During conditions which increase the macronuclear DNA content, very little DNA is eliminate. On the other hand, large amounts are eliminated under other conditions causing the macronuclear DNA content to decrease. DNA to be eliminated at division is synthesized at the same time as bulk DNA. We developed a computer program which helps us study the effects of DNA elimination and unequal divisions upon the copy numbers of subchromosomal DNA classes. The result indicates that in a given cell line at least one of the DNA molecules becoms extinct after 60 generations which we expect would cause the cell's extinction and restrict a clone's life to 60 generations. As this does not happen in nature, there must be some control of the copy numbers preventing their extinction during vegetative multiplication. Whether elimination increases or decreases the imbalance of genes remains to be investigated. © 1992 Wiley-Liss, Inc.     

Mating type differentiation in tetrahymena thermophila: Characterization of the delayed refeeding effect and its implications concerning intranuclear coordination

Mating type determination in Tetrahymena thermophila involves developmentally programmed, heritable alterations of the macronucleus, localized to the mtd locus. This determination can be predictably controlled by the environmental conditions during macronuclear development, eg, temperature and time of refeeding. In this article we have further characterized the effects of delayed refeeding on mating type determination, as revealed by the frequency of mating types among the progeny of a cross. Our results show that 1) the magnitude of this starvation effect decreases with temperature of conjugation and becomes undetectable at 18°C; 2) starvation during the interval 14 to 22 hr (after conjugation is induced at 30°C) is a necessary and sufficient condition for the induction of starvation effects; 3) relative mating type frequencies vary monotonically with nutrient concentration present during this critical period; and 4) sister macronuclei, developing under starvation conditions in the same cytoplasm, differentiate majority mating types characteristic of early or late refeeding; sister macronuclei show no apparent correlation with each other. On the basis of our observations on early and late refed cells, we propose that the composition of the newly developed macronucleus is the outcome of two key events: 1) mating type determination at the mtd locus and 2) differential molecular cloning of generally one or two autonomously replicating fragments (ARFs) of the macronuclear DNA bearing the mtd locus.     

嗜热四膜虫金属硫蛋白基因MMT2和MTT4的表达规律比较与进化模式分析

多个金属硫蛋白基因异构型已在四膜虫中被鉴定,这些异构型可分为7a和7b两个亚家族。该文利用实时荧光定量PCR技术检测了嗜热四膜虫金属硫蛋白基因MTT2和MTT4在Hg、Cu、Cd、Zn、H2O2暴露下的表达水平,结果显示两者表达规律相似,均为:Cu暴露下上调最高(>200倍),Hg次之,Cd、Zn上调倍数不大,H2O2有下调趋势。此表达规律明显有别于7a亚家族,具有7b亚家族的表达特征。同种诱导物暴露下MTT4的上调表达幅度比MTT2高出数倍,结合生物信息学分析结果,推测可能与MTT2和MTT4上游调控元件(如AP-1、MRE等)的数量差异有关。基于MTT2和MTT4在结构和功能上的高度相似性,推测两者可能是经近期基因复制事件产生,并遵循基因剂量模型进化而来。     

The influence of fission line expression on the number and positioning of oral primordia in the cdaA1 mutant of Tetrahymena thermophila

During cytokinesis, furrowing creates new boundaries for daughter cells. Following a shift to a restrictive temperature, cells of the temperature-sensitive cell-division-arrest (cdaA1) mutant of Tetrahymena thermophila complete development of the oral apparatus for the prospective posterior daughter cell before becoming arrested in cytokinesis. When maintained under weak restrictive conditions (35°C), some of the chains were arrested prior to the start of fission line formation (D-shaped chains), whereas others manifested rudimentary unilateral furrowing on the ventral side (B-shaped chains). In their second cell cycle following the temperature shift, the D-shaped chains usually formed only one oral primordium, at a position highly correlated with the length of the entire chain. The B-shaped chains always produced two separate oral primordia, located at irregular positions anterior and posterior to the division furrow, often close to the posterior oral apparatus produced during the first cycle. These results suggest that the formation of the fission line sets a reference boundary to assess the number of oral primordia and influence their position, that appear during subsequent morphogenetic episodes. They also indicate that, during cell division cycles, pre-existing oral apparatuses do not strongly inhibit the formation of new oral apparatuses in their close vicinity. © 1992 Wiley-Liss, Inc.     

Control of tubulin and actin gene expression in Tetrahymena pyriformis during the cell cycle

Poly(A)-containing mRNA was isolated from division synchronized populations of the ciliated protozoan, Tetrahymena pyriformis. The level of tubulin and actin mRNA at specific cell cycle stages was analyzed by hybridization to tubulin and actin cDNA probes and by gel analysis of their in vitro translation products. The pattern of fluctuation of tubulin mRNA levels was similar to that observed for the in vivo tubulin synthesis previously reported [1]. This suggests that as the cells progress through the cell cycle, tubulin synthesis is controlled at the mRNA level. There was little fluctuation of actin synthesis or actin mRNA levels during the cell cycle, which may be indicative of a different regulatory mechanism for actin than for tubulin.