Tetrahymena Tubulins and In Vitro Translation of Tetrahymena RNA*

SYNOPSIS. Tetrahymena outer doublet tubulin was compared with neurotubulin and Chlamydomonas flagellar tubulin on SDS-polyacrylamide gels. Tetrahymenaα tubulin did not comigrate with either brain or flagellar α tubulins, although brain, flagellar, and ciliary β tubulins all comigrated. Axonemal tubulin from Tetrahymena strain ST was compared with this tubulin from strains W. S. HSM, and E, and all were found to have the same mobilities. Poly-A containing RNA was separated from whole cell Tetrahymena RNA by oligo-dT cellulose chromatography. Poly-A+ RNA from 24-h cultures (early exponential growth) stimulated greater incorporation of amino acids into polypeptides in the wheat germ cell-free translation system than did poly-A+ RNA from 36-h and 49-h cultures. When separated on SDS-polyacrylamide gels, the translation products of the 24-h poly-A + RNA had 2 prominent protein bands which comigrated with α and β tubulin isolated from Tetrahymena cilia. These bands were not found in the translation products of poly-A+ RNA isolated from 49-h cultures or in the translation products ofpoly-A- RNA.     

四膜虫接合过程中旧大核内基因的彻底降解

形态学和遗传学方法早巳证明四膜虫接合过程中旧大核退化消失,其基因型对接合后代不发生影响。本文以1种具有强大复制优势而且是抗药性的rDNA分子,rdna-A3,为指标,证明在接合过程中旧大核内上万个rDNA分子没有1个能进入新大核,从而在基因分子水平上证明旧大核的退化是极为彻底的。同时检测了接合过程中旧大核内DNA发生降解的时间。     

Death Harmony Played by Nucleus and Mitochondria: Nuclear Apoptosis During Conjugation of Tetrahymena

Tetrahymena programmed nuclear death or nuclear apoptosis is a unique process during conjugation in which only the parental macronucleus is eliminated from the progeny cytoplasm, and other nuclei such as new micro- and macronuclei are unaffected. The nuclear death process consists of three successive steps: chromatin cleavage into high-molecular mass DNA, oligonucleosomal laddering concomitant with nuclear condensation, and complete degradation of the nuclear DNA. Following the first step of the death process, the parental macronucleus is engulfed by a large autophagosome in which many mitochondria are incorporated. Those sequestered mitochondria simply break down and release endonuclease similar to mammalian endonuclease G that is responsible for the generation of the DNA ladder, leading to the conclusion that mitochondria play a crucial role in the execution of the death program. Thus, the parental macronucleus is subject to final death by autophagy in collaboration with caspase-like enzymes, resulting in the ultimate outcome of the nuclear resorption.     

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

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

"Fenestrin" and Conjugation in Tetrahymena thermophila

ABSTRACT Certain monoclonal antibodies interact with proteins of Tetrahymena thermophila found in the conjugation junction as well as around the gametic nuclei (pronuclei) of conjugating cells; they also react with the oral primordium and fission zone of vegetative cells and with the cytoproct and contractile vacuole pores of all cells. One of these (FXIX-3A7) was investigated in detail. Immunogold labelling suggests that the material labelled by the 3A7 monoclonal antibody, which we call “fenestrin,” is located beneath the epiplasm (membrane skeleton). Immunoblots reveal that the major and perhaps sole antigen is a 64 kDa polypeptide, found in two isoelectric variants. Developmental studies implicate fenestrin in two processes involved in conjugation. The first is “tip transformation.” During preliminary starvation (“initiation”), labelling of fenestrin first appeared as a spot at the anterior end of starved mature cells, then after mixing of different mating types (“costimulation”) it extended posteriorly along the anterior suture. After pairing, this region spread to form a widened plate. The second process is pronuclear transfer. Fenestrations representing channels between the conjugating cells began to appear 0.5 to 1 h after the conjugants united, and eventually merged to form a small number of temporary large holes during exchange of the transfer pronuclei. A fenestrin envelope also enclosed both the transfer and resident pronuclei; a strand of fenestrin connected the two. Shortly after pronuclear transfer, both transfer and resident pronuclei were released from fenestrin caps and fused to produce a zygotic nucleus (synkaryon) not associated with fenestrin. Fenestrin thus appears to be intimately involved in the process of pronuclear exchange.     

Colpidium-Produced RNA as a Growth Stimulant for Tetrahymena*

SYNOPSIS. During logarithmic growth, Colpidium campylum produced a complex, heat-stable material which elevated the reproductive ate of Tetrahymena subsequently inoculated into the culture. This material stimulated only early stages in the growth of Tetrahymena—effect accompanied by an increased number and size of food vacuoles. The Colpidium product was found to be RNA, precipitable from the medium by acetone. Lipids and proteins or peptides were also present in the complex; these appeared to protect the RNA from the action of chemical and physical agents.     

Conjugation Involving Dividing Cells of Tetrahymena thermophila

Feulgen-stained preparations of mixtures of starved Tetrahymena thermophila cells of complementary mating types have revealed an atypical form of conjugation involving cells which have completed the nuclear events of cell division, but have not undergone cytokinesis. Both micronuclei in the dividing cells are induced to undergo meiosis, but in 21 of 23 cases, the anterior micronucleus was activated 1st, suggesting that the meiotic inducer is synthesized near the mating junction and diffuses posteriad. Despite the induction of two micronuclei, “triad” conjugants appear to regulate nuclear events so as to produce a normal outcome.     

Conjugation involving dividing cells of Tetrahymena thermophila

Feulgen-stained preparations of mixtures of starved Tetrahymena thermophila cells of complementary mating types have revealed an atypical form of conjugation involving cells which have completed the nuclear events of cell division, but have not undergone cytokinesis. Both micronuclei in the dividing cells are induced to undergo meiosis, but in 21 of 23 cases, the anterior micronucleus was activated 1st, suggesting that the meiotic inducer is synthesized near the mating junction and diffuses posteriad. Despite the induction of two micronuclei, "triad" conjugants appear to regulate nuclear events so as to produce a normal outcome.     

Induction of Blocks in Nuclear Divisions and Overcondensation of Meiotic Chromosomes with Cycloheximide During Conjugation of Tetrahymena thermophila

During conjugation, the micronucleus of Tetrahymena thermophila undergoes five consecutive nuclear divisions: meiosis, third prezygotic division (pregamic mitosis) and two postzygotic mitoses of the synkaryon. The four products of the synkaryon differentiate into macronuclear anlagen and new micronuclei and the old macronucleus is resorbed. The protein synthesis inhibitor cycloheximide, applied during conjugation, induced several developmental blocks. Pairs shifted to the drug during early meiotic prophase (stages I–III) were arrested at prophase. Cycloheximide applied to cells at pachytene (stages IV-VI) to metaphase arrested the conjugants at the stage of modified prometaphase/metaphase with overcondensed, swollen bivalents. In contrast to other systems, in the presence of cycloheximide, separation of chromatids, decondensation of chromosomes and exit from metaphase I were inhibited in both diploid and haploid cells. Pairs shifted to the drug after metaphase I were arrested at postmeiotic interphase after completing one nuclear cycle. The same rule applied to the subsequent cycle; then cells were arrested at the stage of pronuclei, and those pairs with functional pronuclei and synkarya were arrested at the stage of two products of the first postzygotic division (pronuclei were not arrested in nuclear transfer and karyogamy). Only pairs with two products of the first postzygotic division were arrested at the same stage after the cycloheximide treatment. Pairs shifted to cycloheximide during the second postzygotic division were arrested in development of macronuclear anlagen and resorption of old macronuclei. The postmeiotic conjugants pulse-treated with cycloheximide (2 h) yielded heterokaryons retaining parental macronuclei (i.e. they exhibited macronuclear retention).     

Alteration of the Tetrahymena Genome During Nuclear Differentiation*†

Synopsis.
The DNA of the macro- and the micronucleus of Tetrahymena thermophila has been compared by various biochemical methods. It became evident from their thermal denaturation temperatures and buoyant densities that the 2 DNAs were very similar in overall composition. Small differences were detected when the sequence complexities of these DNAs were compared by DNA renaturation studies. The studies suggested that ˜ 10% of the micronuclear genome was lost or underrepresented in the macronucleus. Comparison of individual gene levels revealed further differences. By using the technic of gene cloning a micronuclear sequence was isolated which hybridized only with micronuclear, but not with macronuclear DNA. These results indicated the occurrence of elimination or underreplication of this sequence in the macronucleus. Gene amplification was also shown to occur. In the micronucleus only a single copy of rDNA was found integrated into the chromosome. During macro-nuclear development, amplification was observed to occur, and the amount of rDNA to increase, until there were ˜ 200 copies per haploid genome in the mature macronucleus. all of them extrachromosomal and palindromic. The 3rd case of alteration involved a simple repeated sequence, (CCCCAA)n, present in the termini of rDNA and also in many other locations of the genome. Restriction endonuclease digestion studies revealed drastic differences in the organization of the repeats between macro-and micronucleus. These differences may be interpreted as the results of chromosome fragmentation which occurs at every cluster of the repeats during macronuclear development. The relationship between this event and gene amplification and elimination is discussed.     

Hydroxyproline in Tetrahymena*

SYNOPSIS. Hydroxyproline (HP) can be quantified by the sensitive colorimetric procedure of Kivirikko et al. (1967). Without preliminary hydrolysis, only the free imino acid (I) can be detected. After hydrolysis in 6 N HCl at 120 C for 15 hr, also the peptide-bound (II) and polypeptide-bound (III) is detected. Cells grown in 2% (w/v) proteose peptone supplemented with 0.4% yeast (w/v) extract (PPY) contained 0.01 mg HP/mg cellular proteins. Over 95% was in the I or II form (soluble in cold 20% trichloro acetic acid). Cells grown in a chemically defined medium (DM), contained less than 0.34 μg/mg cellular proteins. Whereas the DM does not contain any HP, the PPY medium is rich in HP (0.032 mg/mg proteose peptone). In conclusion, the HP found in the cells grown on PPY is a “contaminant'’from this medium. No endogenous production of HP was demonstrated.     

Protein acylation in Tetrahymena

Examination of exhaustively delipidated Tetrahymena mimbres cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of several protein bands containing covalently linked fatty acids. Palmitic (16:0) and stearic (18:0) acids together accounted for approximately 90% of the protein-linked acyl chains, with myristic acid (14:0) comprising most of the remainder. Each of these three fatty acids was present mainly in alkali-stable linkage, indicating that unlike most other systems examined, fatty acids are attached to proteins of Tetrahymena principally by amide bonds. Smaller proportions of the acyl chains were susceptible to release by hydroxylaminolysis or by alkaline hydrolysis as would be expected from an ester linkage. The protein-bound acyl chains accounted for 0.3% of the cells' total fatty acids. They closely resembled in composition the highly saturated free fatty acid pool but not the vast pool of glycerolipid-associated fatty acids, which were mainly unsaturated. Cells subjected to thermal stress by rapid chilling from 39 to 15 degrees C responded by sharply increasing the ratio of palmitate to stearate in covalent association with proteins.     

Self-splicing RNA and an RNA enzyme in Tetrahymena

The RNA molecules transcribed from many eukaryotic genes are interrupted by intervening sequences, which are removed by a process called RNA splicing. One structurally related group of intervening sequences, the group I intervening sequences, are found in a variety of microorganisms. Some of these, including the group I intervening sequence from the ribosomal RNA precursor of Tetrahymena thermophila, have been shown to mediate their own splicing in an RNA-catalyzed reaction. Following its excision from the ribosomal RNA precursor, the Tetrahymena intervening sequence acts as an enzyme, cutting and rejoining RNA substrates.     

Nuclear Behavior of Spirostomum ambiguum During Conjugation with Special Reference to Macronuclear Development*

SYNOPSIS. During conjugation in Spirostomum ambiguum, the micronuclei divide thrice before synkaryon formation and 20 times thereafter. During the first meiotic division 18-24 bivalents, each about 0.5 μ or less appear on the spindle. They separate and pass to the poles. The details of the 2nd and 3rd prezygotic divisions and synkaryon formation by reciprocal exchange of gametic nuclei resemble those described for other ciliates in the literature. The synkaryon divides twice resulting in 4 nuclei; 2 of them become micronuclei and the remaining 2 macronuclear anlagen. The micronuclei enter into division, but this division is arrested in metaphase. The chromosomes in the macronuclear anlagen resemble those appearing in the Ist meiotic division in shape and size. In their maximum stage of development the macronuclear chromosomes are at least 3-4 times larger than those appearing in the arrested micronuclear metaphases in the same cell. There is no banding pattern of the chromosomes and therefore the possible extent of polyteny is difficult to evaluate. The chromosomes duplicate 3-4 times resulting in about 200–250 before they become indistinct as separate entities. Spirostomum is the only nonhypotrichous ciliate in which these cytologic features are described.     

The Relation of Concanavalin A Receptor Distribution to the Conjugation Process in Tetrahymena thermophila

By using fluorescent isothiocyanate-conjugated concanavalin A (FITC-Con A), cell surface events were examined under a light microscope during the early period of the conjugation process in Tetrahymena thermophila. Until the two complementary mating types (D-III and IV) were mixed, Con A-binding activities were hardly detected on the cell surface of ciliates. After mixing, however, the FITC-Con A (25 μg/ml) bound especially to the anterior cell surface at the early stage of conjugation, followed by characteristic changes of the Con A-binding pattern and, subsequently, by formation of a bright fluorescent ring around the area of contact between conjugants. Such alterations of FITC-Con A-binding pattern were found to be interrupted or eliminated by cycloheximide (2 μg/ml). These findings are related to the onset and subsequent conjugation in T. thermophila.     

The Zinc Finger Protein Zfr1p Is Localized Specifically to Conjugation Junction and Required for Sexual Development in Tetrahymena thermophila

Conjugation in Tetrahymena thermophila involves a developmental program consisting of three prezygotic nuclear divisions, pronuclear exchange and fusion, and postzygotic and exconjugant stages. The conjugation junction structure appears during the initiation of conjugation development, and disappears during the exconjugant stage. Many structural and functional proteins are involved in the establishment and maintenance of the junction structure in T. thermophila. In the present study, a zinc finger protein-encoding gene ZFR1 was found to be expressed specifically during conjugation and to localize specifically to the conjugation junction region. Truncated Zfr1p localized at the plasma membrane in ordered arrays and decorated Golgi apparatus located adjacent to basal body. The N-terminal zinc finger and C-terminal hydrophobic domains of Zfr1p were found to be required for its specific conjugation junction localization. Conjugation development of ZFR1 somatic knockout cells was aborted at the pronuclear exchange and fusion conjugation stages. Furthermore, Zfr1p was found to be important for conjugation junction stability during the prezygotic nuclear division stage. Taken together, our data reveal that Zfr1p is required for the stability and integrity of the conjugation junction structure and essential for the sexual life cycle of the Tetrahymena cell.     

Conjugation in Tetrahymena thermophila: A temporal analysis of cytological stages

The time course and synchrony of the stages of conjugation in Tetrahymena thermophila as defined by cytologically observable changes in the morphology and position of the nuclei were established. The time required for 50% of the pairs to enter or pass a particular stage, as well as the duration of each stage were determined. The relative synchrony of the pairs as they went through conjugation was followed by correlating the maximum percentage of the population found in a stage with the duration of that stage. The degree of synchrony between the pairs was found to be high under the conditions of this study, with very little decrease in synchrony seen during the initial 9 h of conjugation. Although some variability in the degree of synchrony was seen between different matings, there was little change detected in the duration of each cytological stage. Prolonged starvation of the cells prior to their mating resulted in a gradual loss of synchrony.     

Incorporation of Tritium from Thymidine-methyl-H3 into DNA,RNA, Lipids and Protein in Logarithmic and Stationary Phase Tetrahymena pyriformis,Strain W*

SYNOPSIS. Numerous reports may be found in the literature on cytoplasmic and non-DNA utilization of tritium from H³-thymidine. Such reports underscore the need to clarify the metabolic fate of H³-thymidine. This investigation outlines the fate of thymidinemethyl-H³ (TMH³) in logarithmic phase and stationary phase Tetrahymena pyriformis, strain W. Isotope identification by liquid scintillation spectrometry in chemically derived fractions of log phase cultures grown thruout the initial 48 hours of population growth with TMH³ revealed the majority of the radioactivity (90% of intracellular recovery) to be in the DNA fraction. The remainder of the intracellular label was recovered in the acid soluble fraction, lipid fraction, and a small amount in the RNA and cell residue. On chromatographs, tritium appeared only in the thymine moiety of the nucleic acid derivatives. Hence in dividing cells, thymidine-methyl-H³ is “essentially” specific for DNA at the dosage used although some incorporation into other compounds was detected. Fractionation of the lipid extract from the above experiment on a florisil column localized most of the label to the triglyceride and phospholipid fractions with some recovery in the cholesterol-esters. Similar scintillation counting of the various fractions of early stationary phase cells incubated for the last 48 hours of culture with TMH³ revealed limited tritium distribution in all fractions.