Observations on the Life-cycle of a New Race of Blepharisma undulans from India

SYNOPSIS. A full account of the nuclear changes during binary fission and conjugation in a local race of Blepharisma is presented in this paper. The macronucleus consists of 2 nodes connected by a strand. Number of micronuclei varies from 6 to 18. During binary fission, condensation of macronucleus is followed by elongation and thinning of the middle region which finally breaks. Daughter nuclei later attain the typical vegetative form. Notably, during binary fission some micronuclei appear to complete their mitoses by the time the macronucleus attains the condensed form, while others lag behind and exhibit practically every stage of mitosis.
During conjugation, from 6 to 10 micronuclei undergo the first pregamic division, the same number through the second division, and two products of the second division take part in the third division. The rest degenerate. Division products of the nuclei in the paraoral region take part in synkaryon formation. The synkaryon undergoes either 2 or 3 divisions. In the former case, of the 4 products, 2 become the macronuclear anlagen, one the micronucleus and the fourth degenerates. In the latter case, of the 8 products, 3 to 4 become the macronuclear anlagen and the rest become micronuclei. Chromatin elimination has been observed during the division of the macronuclear anlage, followed by an extra metagamic fission of the cell.
Comparison with two other races from India and an American race indicates considerable diversity in the structure and behaviour of the nuclear apparatus in different races of Blepharisma undulans.     

Macronuclear Variation During the Life Cycle of Blepharisma dawsoni n.sp. and B. wardsi

SYNOPSIS. From characteristics of binary fission, conjugation, size and number of micronuclei, body size and incidence of giantism, a Blepharisma isolate hitherto called B. undulans is classified as B. dawsoni sp. nov. Binary fission in B. wardsi differs from fission in B. dawsoni in that the strand connecting the macronuclear nodes is severed; in B. dawsoni the strand persists.     

Electron Microscopy of the Nuclear Events During Binary Fission in Paramecium multimicronucleatum

SYNOPSIS. From the interphase to the early stage of binary fission in Paramecium multimicronucleatum , when the micro-nuclei are situated close to the macronucleus, the microtubules in the cytoplasm seem to connect the nuclear pores of macro- and micronuclei. During the 1st half of macronuclear division, the microtubules are formed outside the macronucleus, while during the latter half of division, numerous microtubules appear inside it. Chromatin bodies and nucleoli remain unchanged during macro-nuclear division, but the latter show temporory irregularity in shape. In late prophase of micronuclear division, spindle micro-tubules are formed, and a polar structure, composed of randomly dispersed twisting filaments, is formed at each pole of the micro-nucleus at anaphase. Spindle microtubules terminate on the surface of this structure. The nuclear envolope of the macro-and micronuclei remains intact thruout division. The envelope of the daughter micronuclei is derived from the pre-existing one.     

Repetitive Micronuclear Divisions in the Absence of the Macronucleus During Conjugation of Paramecium caudatum

ABSTRACT. The germinal micronucleus divides six times during conjugation of Paramecium caudatum : this includes two meiotic divisions and one mitosis of haploid nuclei during mating, and three mitoses of a fertilization nucleus (synkaryon). Microsurgical removal of the macronucleus showed that micronuclei were able to divide repeatedly in the absence of the macronucleus, after metaphase of meiosis I of the micronucleus and also after synkaryon formation. When the macronucleus was removed after the first division of synkaryon, in an extreme case the synkaryon divided five times and produced 32 nuclei, compared to three divisions and eight nuclei produced in the presence of the macronucleus. Treatment with actinomycin D (100 μ /ml) inhibited the morphological changes of the macronucleus during conjugation and induced a multimicronucleate state in exconjugants. However, in other cells, it induced production of a few giant micronuclei. We conclude that the micronucleus is able to undergo repeated divisions at any stage of conjugation in the absence of the macronucleus once the factor(s) for induction of the micronuclear division has been produced by the macronucleus. The macronucleus may also produce a regulatory factor required to stop micronucler division.     

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).     

Nuclear Behavior of Euplotes woodruffi During Conjugation

SYNOPSIS. During conjugation of E. woodruffi , the micro-nucleus divides repeatedly four times prior to synkaryon formation and twice thereafter. The first division resembles an ordinary somatic mitosis, resulting in the formation of two daughter nuclei in each conjugant. Both products of this division enter the second division which corresponds to the heterotypic division of other ciliates, characterized by a parachute stage. Following this stage sixteen bivalents appear and separate into dyads and pass to the poles. During the following divisions individualized chromosomes do not appear but only certain chromatin elements comparable to those seen in the somatic and preliminary divisions. These divide and pass to the poles. All daughter nuclei of the second division enter and complete the third division. Only two of the products of the third division enter the final pregamic division while the rest degenerate. Exchange of pronuclei and their fusion leads to synkaryon formation. The conjugants then separate and in each exconjugant the synkaryon divides twice in rapid succession. Of the four products one condenses to become the functional micronucleus, another enlarges rapidly to become the macronuclear anlage while the remaining two degenerate and disintegrate. The old macronucleus breaks into irregular and polymorphic bodies. As the macronuclear anlage enlarges the remnants of the old macronucleus reorganize and fuse with the macronuclear anlage to form a characteristic vegetative macronucleus.     

The Cytology of a New Species of Spirostomum

SUMMARY. A study has been made of the cytology of an undescribed species of Spirostomum. The species is much smaller than S. ambiguum. The peristome extends to about half the length of the animal, whereas in S. ambiguum it is about two-thirds the body length. The nuclear apparatus reveals some striking differences. The macronucleus is cylindrical, and not chain-like, in the vegetative animal. The micronuclei are far fewer than in S. ambiguum and number 6–15, but are larger in size. During binary fission, the macronucleus becomes condensed into an oval or polymorphic mass and is drawn out again into a cylinder before it is cut into two lengths. The micro-nuclei divide by mitosis and, whatever their number in the vegetative animal, only 7–8 take part in the division. The others presumably degenerate.     

Autogamy in Frontonia leucas (Ehrbg.)

Autogamy in Frontonia leucas is described for the first time. The process appears to occur at irregular intervals. From 7 to 10% of the individuals are affected. The beginning of autogamy is marked by a swelling of all the micronuclei which take part in the first two maturation divisions. The third division however affects only one of the second division products. Occasionally two or three may divide. A paroral cone is not prominent. But a small area close to the peristome is distinguishable as the region where the pronuclei fuse. The syn-karyon divides four times. Some of the division products disintegrate, after which 8 to 9 bodies are left which become differentiated into 4 to 5 macronuclear anlagen and 4 micro-nuclei. Mitotic division of the micronuclei results in their increase in number in the daughter individuals after metagamic divisions. Changes in the macronucleus during autogamy consist in its fragmentation and later absorption in the cytoplasm. There is some indirect evidence of a relationship between the dissolution of the old macronucleus and the development of the new.     

Nuclear Activity During Regeneration in Blepharisma intermedium Bhandary*

SYNOPSIS. Transection and regeneration of Blepharisma intermedium initiate complex macronuclear activity and micronuclear division. Reversible condensation of the macronucleus is achieved by contraction which involves primary and secondary coiling. Primary coiling, evident in vegetative cells, is enhanced by pronounced constriction of the macronuclear extremities during regeneration. Secondary coiling is restricted to the period of contraction and is initiated at the ends of the mauonucleus. Condensation terminates the coiling processes and gives rise to an amorphous structure. Re-elongation of the macronucleus is not followed immediately by coiling. Micronuclear division begins early in regeneration, but the peak of visible activity is not reached until the 3rd through 5th hours.     

Selection of the germinal micronucleus in Paramecium caudatum: nuclear division and nuclear death

Each cell of Paramecium caudatum has a germinal micronucleus. When a bi-micronucleate state was created artificially by micronuclear transplantation, both micronuclei divided for at least 2 cell cycles after nuclear transplantation. However, this bi-micronucleate state was unstable and reduced to a uni-micronucleate state after several fissions. Although the number of micronuclei was usually 1 during the vegetative phase, 4 presumptive micronuclei differentiated after conjugation. At the first post-conjugational fission, only 1 of the 4 micronuclei divided, indicating that there is tight regulation of micronuclear number in exconjugants. Micronuclei that did not divide at the first post-conjugational fission may persist through the first and second post-conjugational cell cycles. The decision to divide appears to be separate from the decision to degenerate, as evidenced by division of a remaining micronucleus upon removal of the dividing micronucleus at the first division. Degeneration of micronuclei in exconjugants differs from that of haploid nuclei after meiosis. Nutritional state affected micronuclear degeneration. Under well-fed conditions, the micronuclei destined to degenerate lost the ability to divide earlier than after starvation treatment, suggesting that micronuclear degeneration is an "apoptotic" phenomenon, probably under the control of the new macronuclei (macronuclear anlagen).     

On the evolution of the karyorelict ciliate life cycle: heterophasic ciliates and the origin of ciliate binary fission

Karyorelict ciliates have near diploid somatic nuclei (macronuclei) incapable of division. If selective pressure favors nuclear division, how could such macronuclei have evolved? I propose that they initially evolved in the context of a diplophase stage that consisted entirely of a non-dividing trophont that was terminated by the induction of meiosis. The diploid macronucleus then differentiated, functioned and was destroyed in the absence of cell division. Such a life cycle would necessarily be heterophasic, i.e. with alternating haploid and diploid generations. I call these ancestors heterophasic ciliates. I further propose that the ability of this diploid trophont to undergo binary fission arose de novo. Ciliate binary fission would then be a derived characteristic, which possibly evolved indepedently in more than one heterophasic ciliate lineage. A progression of steps, leading to the reduction of the haplophase and the generation of the karyorelict life cycle, is proposed. The shared possession of nuclear dimorphism with non-dividing macronuclei, conjugation, and a putative heterophasic ancestry invites further investigation of the phylogenetic relationship between heterokaryotic foraminifera and karyorelict ciliates.     

Macro- and Micronuclei of Tetrahymena pyriformis: A Model System for Studying the Structure and Function of Eukaryotic Nuclei*†

The macro- and micronucleus of Tetrahymena pyriformis are formed from a common diploid synkaryon during conjugation. Shortly after the 2nd postzygotic division, distinct morphologic and physiologic differences develop between the 2 nuclei. Micronuclei remain small, presumably diploid, and electronmicroscopic observations indicate that micronuclear DNA is contained in a dense, fibrous, chromosome-like coil. Macronuclei contain considerably more DNA than micronuclei, and the DNA of the macronucleus is found largely in the chromatin bodies typical of ciliate nuclei. The functional differences between macro- and micronuclei in vegetative cells also are striking. The template activity of DNA in the micronucleus is highly restricted compared to that in the macronucleus. Micronuclei synthesize and contain little RNA, and do not contain either nucleoli or ribonucleoprotein granules. Macronuclei, on the other hand, synthesize and contain large amounts of RNA and have many nucleoli and ribonucleoprotein granules. Macro- and micronuclei also have distinct differences in the timing of DNA synthesis during the cell cycle and in the timing and mechanism of nuclear division. Finally, during conjugation the macronucleus becomes pycnotic and disappears while the micronucleus undergoes meiosis and fertilization, ultimately giving rise to new macro- and new micronuclei. In short, the macro- and micronuclei of Tetrahymena provide an excellent system for studying the molecular mechanisms by which the same (or related) genetic information is maintained in different structural and functional states. Methods have been devised to isolate and purify macro- and micronuclei of Tetrahymena in the hope of correlating differences in the nucleoprotein composition of these nuclei with differences in their structure and function. The DNAs of macro- and micronuclei have been found to differ markedly in their content of a methylated base, N⁶-methyl adenine, and major differences in the histones of the 2 nuclei have been observed. Macronuclei contain histones similar to those found in vertebrate nuclei, while 2 major histone fractions seem to be missing in micronuclei. In addition, histone fraction F2A1 which is found in multiple, acetylated forms in macronuclei, is present only as a single, unacetylated form in micronuclei.     

Studies on Conjugation in Paramecium polycaryum

SYNOPSIS. The process of autogamy in unassociated individuals of Paramecium polycaryum was reported by the author in 1954. In May, 1955, conjugation was first seen in this species in cultures collected by me at Annamalainagar, South India, thus removing it from the list of non-conjugating species. This appears to be the first instance in which the process of autogamy was detected prior to observation of conjugation in the same species. Autogamy occurs in singles of the Indian race and appears to be similar, cytologically, to that of American races. The details of the micronuclear behavior in conjugation parallel those of autogamy in singles. In fact, the conjugation process seems to be one of double autogamy (cytogamy), rather than of reciprocal gametic interchange. Paroral cones, often of fair size, are formed but breakdown of the cones to permit micronuclear passage has not been observed. In conjugation there are the usual three pregamic divisions; the first shows four characteristic crescents. The resulting nuclei may all participate in the second division. Fertilization occurs in the paroral cone area. Frequently, separation of the conjugants takes place immediately after the first division of the synkaryon. The old macronucleus undergoes very little change prior to the last postzygotic micronuclear division in the ex-conjugant, when it goes into a skein condition. Four macronuclear and four micronuclear anlagen are formed in the ex-conjugants at the completion of reorganization. On occasion giant individuals of P. polycaryum were observed to have ingested numbers of Tetrahymena pyriformis. The presence of an unidentified rod-like organism in the cytoplasm of the paramecia (non-conjugating) was detected in one collection from Bangalore, India.     

Nuclear Differentiation in Exconjugants of Paramecium caudatum: Role of Nuclear Division in Differetiation

It has been known that, immediately after the third division of fertilization nucleus (synkaryon), nuclei localized near the posterior region of exconjugant are to be macronuclear anlagen and those near the anterior region are to be presumptive micronuclei in Paramecium caudatum. One of such posterior nuclei was transplanted into amicronucleate cell at vegetative phase in this work. The implanted nuclei were able to divide at every fission. Their DNA content was nearly equal to or less than ordinary micronuclei during vegetative phase. When conjugation was induced between clones obtained and amicronucleates, macronuclear anlagen developed from the division products of implanted nuclei and thereafter derivative caryonides were true to the marker gene of implanted nuclei. The results indicate that there was no intrinsic difference between nuclei localized anteriorly and those situated posteriorly in exconjugant. Differentiation of nuclei into macronucleus may be irreversible at the stage of anteroposterior localization of the nuclei. The role of nuclear division in differentiation may be only to transport the daughter nuclei into the cytoplasm/cortex differentiated anteroposteriorly.     

Taxonomy of Genus Blepharisma with Special Reference to Blepharisma undulans

SYNOPSIS. Three new species of Blepharisma are presented: B. seshachari sp. nov., B. intermedium sp. nov., and B. tropicum sp. nov. Two sub-species of Suzuki are elevated to specific rank, B. undulans americanum to B. americanum and B. undulans japonicum Suzuki to B. japonicum Suzuki. Stein's taxa for the species Blepharisma undulans are considered to be diagnostic for that species.
Important features of morphology and life-cycle of the above mentioned species are given. Discussion and position of the species Blepharisma undulans are presented and a proposal for the reorganization of the species is made. The possibility of a new key derived from a hypothesis of the phylogeny on an evolutionary basis of the genus is presented. This hypothesis has been extended to include ciliates like Stentor and Spirostomum and its implications discussed.     

Effect of the antitubulin drug nocodazole on meiosis and postmeiotic development in Tetrahymena thermophila. Induction of achiasmatic meiosis

Nocodazole (ND), a potent antitubulin drug, can be used to dissect the steps of meiosis in Tetrahymena, presumably by interfering with the assembly of microtubules. Its effects depend upon the time during conjugation at which the drug is applied. When applied prior to the elongation of the micronucleus into the characteristic 'crescent' configuration, no crescent is formed and the chromosomes of prepachytene and pachytene condense into spherical nuclei. If ND is applied after micronuclear elongation has begun, but before it is fully elongated, the chromosomes fail to synapse and appear in metaphase I as unpaired monovalents. In contrast, the metaphase I chromosomes appear as bivalents when ND is applied later, during or after the crescent has reached its maximum elongation. Still later, application of ND inhibits chromosome movements during anaphase and telophase of either meiotic division, but does not prevent separation of kinetochores. In some of the blocked restitutive nuclei an additional round of chromosome replication occurs, corresponding to the third pregamic division in normal conjugation. The hyperploid micronuclei produced by such treatment may be useful in certain genetic manipulations and in studying the regulation of nuclear DNA content.     

嗜热四膜虫染色体浓缩调节因子(RCC1)的定位及功能分析

真核细胞中染色体浓缩调节因子（regulator of chromosome condensation 1, RCC1）是 RanGTPase 唯一的鸟嘌呤核苷酸交换因子. 染色质结合的RCC1和RanGTPase相互作用,催化细胞核内RanGDP向RanGTP的转化,进而调控了核质间的定向运送、有丝分裂期纺锤体的组装以及核膜的形成. 本实验从原生生物嗜热四膜虫大核基因组中鉴定了1个新的RCC1（TTHERM_00530380）基因. 该基因全长2 541 bp,包含2个内含子序列,开放阅读框为2 181 bp,编码726个氨基酸. 实时荧光定量PCR表明,RCC1在四膜虫营养生长、饥饿以及有性生殖时期都有表达,且在有性生殖转录水平达到最高. 免疫荧光定位分析表明, HA RCC1在营养生长和饥饿时期,定位于大核和小核中|在有性生殖时期,定位于亲本大核、减数分裂的小核、新生成的大核和凋亡的大核中. 过表达RCC1导致大核的无丝分裂异常, 细胞增殖变慢,最终产生无大核的后代细胞. 敲减RCC1导致了多小核的产生. 结果表明,RCC1参与调控了四膜虫细胞核的分裂, RCC1的正常表达对核分裂以及细胞增殖起到重要的调控作用.