Molecular Biology of the Genes for Immobilization Antigens in Paramecium1

Several genes for surface antigens of the Paramecium aurelia complex of species have been isolated. In addition lo known deletions of the 51A gene, we have obtained deletions involving the 51B gene and have developed a procedure for obtaining deletions of additional genes. Both Mendelian and non-Mendelian deletions of both the A and B genes have been found. In the non-Mendelian deletions the genes are present in the micronuclei and absent in the macronuclei. Processing of micronuclear DNA into new macronuclear DNA at conjugation and autogamy is under the control of the old macronucleus, and newly forming macronuclei become exactly like the old. Thus in the non-Mendelian mutants, macronuclei have a specific antigen gene deleted and also are impaired in their ability to direct normal DNA processing at the next conjugation or autogamy. These cases, along with others, show that this system of macronuclear control is a fundamental feature of ciliate genetics. The sequence of the 51A and 51C genes is described and compared with the 156G and 51H genes obtained by others. The 51A and 156G genes are remarkably similar while 51Cand 51H are rather different. No introns or pseudogenes have been observed. Some, possibly all, of the genes are on the ends of chromosomes. Characteristic upstream and downstream sequences adjacent to the coding portions of the genes are given. The sequences UAA and UAG are preferred over CAA and CAG for glutamine while UGA is the true stop codon.     

Molecular biology of the genes for immobilization antigens in Paramecium

Several genes for surface antigens of the Paramecium aurelia complex of species have been isolated. In addition to known deletions of the 51A gene, we have obtained deletions involving the 51B gene and have developed a procedure for obtaining deletions of additional genes. Both Mendelian and non-Mendelian deletions of both the A and B genes have been found. In the non-Mendelian deletions the genes are present in the micronuclei and absent in the macronuclei. Processing of micronuclear DNA into new macronuclear DNA at conjugation and autogamy is under the control of the old macronucleus, and newly forming macronuclei become exactly like the old. Thus in the non-Mendelian mutants, macronuclei have a specific antigen gene deleted and also are impaired in their ability to direct normal DNA processing at the next conjugation or autogamy. These cases, along with others, show that this system of macronuclear control is a fundamental feature of ciliate genetics. The sequence of the 51A and 51C genes is described and compared with the 156G and 51H genes obtained by others. The 51A and 156G genes are remarkably similar while 51C and 51H are rather different. No introns or pseudogenes have been observed. Some, possibly all, of the genes are on the ends of chromosomes. Characteristic upstream and downstream sequences adjacent to the coding portions of the genes are given. The sequences UAA and UAG are preferred over CAA and CAG for glutamine while UGA is the true stop codon.     

Genomic exclusion in Tetrahymena thermophila: A cytogenetic and cytofluorimetric study

Genomic exclusion is an aberrant form of conjugation of Tetrahymena thermophila in which the genome of a defective conjugant is excluded from the genotype of the exconjugant progeny. This paper is concerned with the cytogenetic and nucleocytoplasmic events of genomic exclusion in senescent clones A*III and C*. In crosses between A*III or C* and strain B, functional, haploid gametic nuclei are formed only in the strain B cell. In some instances one of the gametic nuclei divides prior to transfer of the migratory gametic nucleus, and both products then undergo DNA synthesis. Two alternative cytogenetic pathways are followed after transfer of the migratory nucleus. In the first, the conjugants separate without further micronuclear divisions. This pathway was most common in A*III genomic exclusion. In exconjugants the former gametic nuclei undergo both DNA synthesis and (presumably) intranuclear separation of centromeres to restore micronuclear diploidy. The old macronucleus of each exconjugant is retained without autolysis. This class of exconjugant survives and contributes genes to future sexual progeny. In the second cytogenetic pathway the gametic nuclei divide and macronuclear anlagen are formed, as in normal conjugation. This pathway was more common in C* genomic exclusion. The initial DNA content of the anlagen ranges from haploid to diploid. Following two to three rounds of DNA synthesis, further macronuclear development ceases and the anlagen appear to undergo autolysis. The old macronucleus condenses and also undergoes autolysis, as in normal conjugation. Except for rare C* exconjugants, in which macronuclear development is completed, anlagen-bearing genomic exclusion exconjugants die. Death may be caused by aneuploidy, errors in the timing or receptivity to signals for autolysis, or the inability of anlagen-bearing exconjugants to feed. Anlagenbearing conjugants are frequently abnormal with respect to the number of anlagen and micronuclei. Most of the anomalies can be explained by postulating errors in the timing of both developmental signals and nuclear divisions. Rare conjugants in which gametic nuclei divide but do not give rise to macronuclear anlagen are also observed. In these instances, the old macronuclei condense and undergo autolysis. Destruction of the old macronucleus therefore is independent of the presence of macronuclear anlagen and requires cell pairing in order to be initiated.     

The transition from conjugal development to the first vegetative cell division is dependent on RAD51 expression in the ciliate Tetrahymena thermophila

Rad51p, the eukaryotic homolog of the prokaryotic recA protein, catalyzes strand exchange between single- and double-stranded DNA and is involved in both genetic recombination and double-strand break repair in the ciliate Tetrahymena thermophila. We have previously shown that disruption of the Tetrahymena RAD51 somatic macronuclear locus leads to defective germline micronuclear division and that conjugation of two somatic rad51 null strains results in an early meiotic arrest. We have constructed Tetrahymena strains that are capable of RAD51 expression from their parental macronuclei and are homozygous, rad51 nulls in their germline micronuclei. These rad51 null heterokaryons complete all of the early and middle stages of conjugation, including meiosis, haploid nuclear exchange, zygotic fusion, and the programmed chromosome fragmentations, sequence eliminations, and rDNA amplification that occur during macronuclear development. However, the rad51 null progeny fail to initiate the first vegetative cell division following conjugal development. Coincident with the developmental arrest is a disproportionate amplification of rDNA, despite the maintenance of normal total DNA content in the developing macronuclei. Fusion of arrested rad51 null exconjugants to wild-type cells is sufficient to overcome the arrest. Cells rescued by cytoplasmic fusion continue to divide, eventually recapitulating the micronuclear mitotic defects described previously for rad51 somatic nulls.     

STUDIES ON NUCLEAR STRUCTURE AND FUNCTION IN TETRAHYMENA PYRIFORMIS : II. Isolation of Macro- and Micronuclei

This report describes a rapid, efficient method for isolating macronuclei from Tetrahymena. The macronuclear fraction contains only small amounts of micronuclear material and little detectable whole cell or cytoplasmic contamination. A method is also described for preparing a "micronuclear fraction" which contains 20–40 micronuclei for every macronucleus present. Electron microscope observations indicate that the ultrastructure of the nuclei in the macronuclear fraction closely resembles that of nuclei in situ. The presence of ribosomes on the outer membrane of micronuclei and of pores in the micronuclear envelope is also described.     

A Transmissible developmental block in Tetrahymena thermophila

When the amicronucleate mutant BI3840 of Tetrahymena thermophila is mated with normal micronucleate cells, it receives a pronucleus from its partner but there is no further nuclear development and the conjugants separate, retaining their original macronuclei. Both of these sexually mature exconjugants and any cells with which they are mated show an unconditional block in macronuclear development. Although prezygotic nuclear divisions, nuclear transfer and post-zygotic nuclear divisions appeared normal upon cytological analysis of Giemsa-stained conjugants, macronuclear development was invariably aborted. Since the original macronucleus was resorbed, the cells were rendered amacronucleate and they died. When no macronuclear development was initiated, as in crosses with the aneuploid strain A* (III), the exconjugants were viable and retained their original macronuclei. This pattern was invariant with three different strains serving as the original micronuclear source, and in the case of one non-BI3840 exconjugant, persisted for over 200 cell generations. Exconjugants from a cross of one of the micronuclear donors with strain A* (III) did not show arrested development when crossed. It thus appears likely that there is conjugal transfer of non-nuclear information originating in BI3840 which is self-replicating and which causes an arrest in macronuclear development.     

Micronuclear DNA from Paramecium tetraurelia: serotype 51 A gene has internally eliminated sequences.

A method for the isolation of micronuclear DNA from Paramecium tetraurelia has been developed. After cell lysis, a low speed centrifugation at 1,000 g is used to remove all of the unbroken cells and macronuclei and approximately two thirds of the macronuclear fragments. Next a higher speed centrifugation of 9,000 g sediments the micronuclei and frees them from small particulates and soluble constituents. Advantage is then taken of the fact that micronuclei have a lower density than do macronuclear fragments in 45%-60% Percoll. Micronuclei float to the top during centrifugation at 24,000 g, while macronuclear fragments sediment. After several cycles of centrifugation in Percoll, the micronuclei, although heavily contaminated with cytoplasmic components, are essentially free of macronuclei and macronuclear fragments. Micronuclear DNA can then be extracted from the suspension. The whole procedure is very rapid and in about an hour micronuclear and macronuclear DNA can be separated. About 2 micrograms of micronuclear DNA can be obtained from 6 x 10(7) paramecia. We find that there are internal sequences in the micronuclear A gene DNA in wild type cells which are eliminated when the micronuclei develop into macronuclei. They yield unique restriction fragments for micronuclei and macronuclei. Therefore the purity of the preparations is easily monitored by probing Southern blots of restriction enzyme-digested DNA with the cloned A gene. No differences have been found between the micronuclear A gene in wild type and the d48 mutant.     

Micronuclear DNA from Paramecium tetraurelia: Serotype 51 A Gene Has Internally Eliminated Sequences

A method for the isolation of micronuclear DNA from Paramecium tetraurelia has been developed. After cell lysis, a low speed centrifugation at 1,000 g is used to remove all of the unbroken cells and macronuclei and approximately two thirds of the macronuclear fragments. Next a higher speed centrifugation of 9,000 g sediments the micronuclei and frees them from small particulates and soluble constituents. Advantage is then taken of the fact that micronuclei have a lower density than do macronuclear fragments in 45%–60% Percoll. Micronuclei float to the top during centrifugation at 24,000 g , while macronuclear fragments sediment. After several cycles of centrifugation in Percoll, the micronuclei, although heavily contaminated with cytoplasmic components, are essentially free of macronuclei and macronuclear fragments. Micronuclear DNA can then be extracted from the suspension. The whole procedure is very rapid and in about an hour micronuclear and macronuclear DNA can be separated. About 2 μ g of micronuclear DNA can be obtained from 6 times 10⁷ paramecia. We find that there are internal sequences in the micronuclear A gene DNA in wild type cells which are eliminated when the micronuclei develop into macronuclei. They yield unique restriction fragments for micronuclei and macronuclei. Therefore the purity of the preparations is easily monitored by probing Southern blots of restriction enzyme-digested DNA with the cloned A gene. No differences have been found between the micronuclear A gene in wild type and the d48 mutant.     

Analysis of the micronuclear B type surface protein gene in Paramecium tetraurelia.

The micronuclear DNA of Paramecium contains sequences that are precisely excised during the formation of the macronuclear (somatic) genome. In this paper we show that four eliminated sequences ranging in size from 28 to 416 base pairs, are present in or near the micronuclear copy of the B surface protein gene. Each excised sequence is bounded by the dinucleotide 5'-TdA-3'. Comparison of the micronuclear B gene with the previously determined micronuclear sequence of the A surface protein gene shows that although the positions of at least three of the eliminated sequences are conserved in both genes, the sequences are highly divergent. Transformation of vegetative macronuclei with fragments of the micronuclear B gene results in replication and maintenance of the DNA, but the micronuclear specific sequences are not removed. Previous studies have shown that the correct incorporation of the B gene into the new macronucleus requires copies of the macronuclear B gene in the old macronucleus. Using macronuclear transformation, we show that the micronuclear B gene can substitute for the macronuclear B gene with regard to its role in DNA processing. This suggests that the macronuclear DNA is not acting as a guide for the excision of the micronuclear specific sequences.     

Micronuclear DNA of Oxytricha nova contains sequences with autonomously replicating activity in Saccharomyces cerevisiae.

Oxytricha nova is a hypotrichous ciliate with micronuclei and macronuclei. Micronuclei, which contain large, chromosomal-sized DNA, are genetically inert but undergo meiosis and exchange during cell mating. Macronuclei, which contain only small, gene-sized DNA molecules, provide all of the nuclear RNA needed to run the cell. After cell mating the macronucleus is derived from a micronucleus, a derivation that includes excision of the genes from chromosomes and elimination of the remaining DNA. The eliminated DNA includes all of the repetitious sequences and approximately 95% of the unique sequences. We cloned large restriction fragments from the micronucleus that confer replication ability on a replication-deficient plasmid in Saccharomyces cerevisiae. Sequences that confer replication ability are called autonomously replicating sequences. The frequency and effectiveness of autonomously replicating sequences in micronuclear DNA are similar to those reported for DNAs of other organisms introduced into yeast cells. Of the 12 micronuclear fragments with autonomously replicating sequence activity, 9 also showed homology to macronuclear DNA, indicating that they contain a macronuclear gene sequence. We conclude from this that autonomously replicating sequence activity is nonrandomly distributed throughout micronuclear DNA and is preferentially associated with those regions of micronuclear DNA that contain genes.     

Histone rearrangements accompany nuclear differentiation and dedifferentiation in Tetrahymena

Histone synthesis and deposition into specific classes of nuclei has been investigated in starved and conjugating Tetrahymena. During starvation and early stages of conjugation (between 0 and 5 hr after opposite mating types are mixed), micronuclei selectively lose preexisting micronuclear-specific histones α, β, γ, and H3^F. Of these histones, only α appears to accumulate in micronuclear chromatin through active synthesis and deposition during the mating process. Curiously, α is not observed (by stain or label) in young macronuclear anlagen (4C, 10 hr of conjugation). Thus, young macronuclear anlagen are missing all of the histones which are known to be specific to micronuclei of vegetative cells. By 14–16 hr of conjugation, we observe active synthesis and deposition of macronuclear-specific histones, hv1, hv2, and H1, into new macronuclear anlagen (8C). Thus macronuclear differentiation seems well underway by this time of conjugation. It is also in this time period (14–16 hr) that we first detect significant amounts of micronuclear-specific H1-like polypeptides β and γ in micronuclear extracts. These polypeptides do not seem to be synthesized during this period, which suggests that β and γ are derived from a precursor molecule(s). Since these micronuclear-specific histones do not appear in micronuclear chromatin until after other micronuclei have been selected to differentiate as macronuclei, we suspect that micronuclear differentiation is also an important process which occurs in 10–16 hr mating cells. Our results also suggest that proteolytic processing of micronuclear H3^S into H3^F (which occurs in a cell cycle dependent fashion during vegetative growth) is not operative during most if not all of conjugation. Thus micronuclei of mating cells contain only H3^S which also seems consistent with the fact that some micronuclei differentiate into new macronuclei (micronuclear H3^S is indistinguishable from macronuclear H3). Interestingly, the only H3 synthesized and deposited into the former macronucleus of mating cells is the relatively minor macronuclear-specific H3-like variant, hv2. These results demonstrate that significant histone rearrangements occur during conjugation in Tetrahymena in a manner consistent with the fact that during conjugation some micronuclei eventually differentiate into new macronuclei. Our results suggest that selective synthesis and deposition of specific histones (and histone variants) plays an important role in the nuclear differentiation process in Tetrahymena. The disappearance of specific histones also raises the possibility that developmentally regulated proteolytic processing of specific histones plays an important (and previously unsuspected) role in this system.     

Differential Nuclear Coloration in Ciliates

SYNOPSIS. Application of the triple stain of Grosso (methyl green-pyronin-orange G) to the exconjugants of different species of ciliates produces a differential coloration of their nuclei. Both the old macronucleus or its remnants and the micronuclei bind methyl green whereas the newly developing macronucleus (macronuclear anlage) has a selective affinity for the orange G present in the mixture. This finding holds true not only for hypotrichous ciliates, whose macronuclear anlagen are involved in the formation of polytene chromosomes, but also for other ciliates not included in the order Hypotrichida. Such a reaction of the new macronuclei apparently results from the presence of non-histone (acidic) proteins associated with the nucleic acids.
The staining method is fully described to provide a more general application.     

Elimination of germ-line tandemly repeated sequences from the somatic genome of the ciliate Oxytricha fallax

The ciliated protozoa exhibit nuclear dimorphism. The genome of the somatic macronucleus arises from the germ-line genome of the micronucleus following conjugation. We have studied the fates of highly repetitious sequences in this process. Two cloned, tandemly repeated sequences from the micronucleus of Oxytricha fallax were used as probes in hybridizations to micronuclear and macronuclear DNA. The results of these experiments show: (1) the cloned repeats are members of two apparently unrelated repetitious sequence families, which each appear to comprise a few percent of the micronuclear genome, and (2) the amount of either family in the macronuclei from which our DNA was prepared is about 1/15 that found in an equal number of diploid micronuclei. Most, if not all, of the apparent macronuclear copies of these repeats can be accounted for by micronuclear contamination, which strongly suggests that these sequences are eliminated from the macronuclei and have no vegetiative function.     

Localization of genes for ribosomal RNA in the nuclei of Oxytricha fallax

The location of ribosomal RNA (rRNA) genes in the nuclei of the ciliated protozoan, Oxytricha fallax, was analysed by in situ hybridization. The micronuclear genome of O. fallax has typical chromosomal DNA organization. Macronuclei, although derived from micronuclei, lack chromosomes and instead contain short pieces of DNA ranging from 500 to 20 000 base pairs in length. In situ hybridization was carried out to determine if specific DNA sequences are limited to certain locations within the macronucleus, or if sequences are randomly arranged. Cells were fixed, squashed and then hybridized with ³H-labelled RNA synthesized in vitro using cloned O. fallax rDNA as a template. After autoradiography, silver grains were found to be distributed uniformly over the entire macronucleus without any detectable localization to specific regions. The uniformity of hybridization indicates that rDNA molecules are randomly dispersed throughout the macronucleus and suggests that the macronuclear genetic apparatus lacks any substantial multimolecular organization. S phase macronuclei also showed a uniform distribution of rDNA molecules, irrespective of the position of the replication band at which DNA synthesis takes place. The micronuclei, in contrast, did not show any hybridization, even in cells in which macronuclei were heavily labelled. Macronuclear anlagen, in which the micronuclear chromosomes are polytenized, also do not hybridize. This absence of hybridization indicates a much lower concentration of rDNA in the micronucleus than in the macronucleus. The change in rDNA concentration of rRNA genes presumably occurs during the complicated process of development of a macronucleus from a micronucleus.     

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).     

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.     

Scheduled and unscheduled DNA synthesis during development in conjugating Tetrahymena

Autoradiography has been used to confirm and to extend previous microspectrophotometric studies (Doerder and DeBault, 1975) on the timing of DNA synthesis during conjugation in Tetrahymena thermophila. The majority of DNA synthesis occurs at the expected periods preceding gamete formation and the two postzygotic divisions and during macronuclear development. DNA in new macronuclei is endoreplicated in an extremely discontinuous fashion. Under starvation conditions, the first endoreplication (2C to 4C) occurs immediately after the second postzygotic division when both new macronuclei and new micronuclei replicate. The second endoreplication (4C to 8C) does not occur until after separation of conjugants. If mating cells are kept under prolonged starvation conditions (20-24 hr), refeeding induces a partially synchronous division, after which an unexpectedly high percentage of cells incorporate tritiated thymidine into both macro- and micronuclei. Two previously undescribed periods of DNA synthesis were observed in the micronuclei of conjugating Tetrahymena. The first occurs during the early stages of meiotic prophase, before full crescent elongation. The second takes place in an extended period corresponding to macronuclear anlagen development, before conjugants have separated. CsCl gradient analyses indicate that, in micronuclear fractions, only main band DNA is being synthesized in both of these periods. However, in macronuclear fractions from both stages, a significant fraction (approximately 20%) of the DNA being synthesized has the buoyant density of ribosomal DNA. The finding that macro- and micronuclear DNA can be synthesized simultaneously in a single cell, both during conjugation and after refeeding starved exconjugants, raises interesting questions of how macro- or micronuclear-specific histones are targeted to the appropriate nuclei.     

Non-Mendelian inheritance of paralogs of 2 cytoskeletal genes in the ciliate Chilodonella uncinata

Recognition of the role of non-Mendelian inheritance is on the rise, particularly as epigenetic phenomena are shown to shape the transformation of genomes into phenotypes. Ciliates provide a model system in which to explore the role of epigenetics because ciliates have both a germ line (micronuclear) and somatic (macronuclear) genome within every cell. In the ciliate Chilodonella uncinata, the macronucleus is extensively fragmented such that many genes end up on their own chromosomes. Hence, it is possible to track the fate of unlinked genes within macronuclei of C. uncinata. Here we demonstrate that the pattern of inheritance in isolates of C. uncinata is complex and involves both Mendelian transmission between micronuclei and macronuclei and epigenetic phenomena. The macronuclei from 2 isolates of C. uncinata and their progeny share identical rDNA loci and 2 identical beta-tubulin paralogs, yet have different actin paralogs and some beta-tubulin paralogs that are not shared. We propose a model in which all the divergent paralogs are present in the ciliate micronuclei. Under this model, different paralogs are retained in developing macronuclei following conjugation. We further speculate that an epigenetic mechanism, such as RNA interference, is involved in selective retention of specific paralogs within lines. This system allows the exploration of epigenetic phenomena that shape somatic genomes and provides parallels to studies of the development of somatic nuclei within animals.