Organization of the 5S RNA genes in macro- and micronuclei of Tetrahymena pyriformis

The organization of the 5S genes in macro- and micronuclei of Tetrahymena pyriformis was studied using restriction endonucleases. After complete digestion of macronuclear DNA with BamH-I or Hpa I, 5S RNA hybridized to a DNA fragment of approximately 280 base pairs (bp). When macronuclear DNA was only partially digested with these enzymes, hybridization with ³²P-5S RNA demonstrated an oligomeric series with a spacing of 280 bp. These results indicate that the 5S genes are tandemly repeated in macronuclei and that the repeating unit is 280 bp (or 180,000 daltons). Since 5S RNA is 120 nucleotides, we conclude that the 5S repeat units contain a 120 bp transcribed region and a 160 bp spacer region. When macronuclear DNA was digested with Eco RI, Bgl I, or Eco RI + Bgl I, 5S RNA hybridized to DNA of molecular weight 3–4×10⁶, suggesting that these enzymes do not cleave within a 5S repeat. These 3–4×10⁶ dalton fragments define the maximum size of an average cluster of 5S repeated units. Assuming the size of the 5S repeat to be 0.18×10⁶ daltons, there are about 15–20 5S repeats per average tandem cluster, and since there are 350 5S-genes per haploid genome, there must be approximately 15–20 tandem arrays. Results obtained using micronuclear DNA suggest that organization of the 5S-genes is very similar in macro- and micronuclei. Macronuclear rRNA genes are extracnromosomal palindromic dimers. In contrast, 5S genes in Tetrahymena were found to be integrated within the genomes of both macro- and micronuclei and not linked to the rRNA genes. Moreover, it is unlikely that they are palindromes; rather they appear to be tandemly repeated in head-to-tail linkages. Thus, the organization of the 5S genes in Tetrahymena is similar to that of higher eukaryotes.     

Comparison of the sequences of macro- and micronuclear DNA of Tetrahymena pyriformis

Macro- and micronuclei were isolated from Tetrahymena pyriformis (Syngen 1, strain WH-6) and their DNAs compared by isopycnic centrifugation in neutral and alkaline CsCl, by analysis of thermal denaturation properties and by molecular hybridization. Unlike the situation observed in Stylonychia the buoyant densities and thermal denaturation patterns of Tetrahymena macro- and micronuclear DNAs were virtually identical—the only observable differences bordering on the limits of resolution of these techniques. DNA was isolated from the two nuclei which had been labelled with different radioactive isotopes (i.e. ¹⁴C-thymidine and ³H-thymidine), and the renaturation kinetics of mixtures of macro- and micronuclear DNA were examined using a single-strand specific deoxyribonuclease (S₁). Renaturation kinetics obtained using varying ratios of macro- and micronuclear DNA suggested that 80–90% of the sequences present in micronuclei were present in similar amounts in macronuclei. However, careful analyses of the renaturation kinetics indicate that approximately 10–20% of the sequences found in micronuclei are probably absent in macronuclei, and that most of these sequences are probably moderately repetitive (100 copies per genome or less). These findings place severe constraint on possible models concerning the structure of the Tetrahymena macronucleus, and are very different from the situation observed in Stylonychia where it has been suggested that only a small percentage of the sequences in micronuclei are present in significant amounts in macronuclei. Nonetheless, these results along with those in Stylonychia can be taken as an indication that the loss or under-replication of some DNA sequences accompanies macronuclear differentiation in ciliates.     

STUDIES ON NUCLEAR STRUCTURE AND FUNCTION IN TETRAHYMENA PYRIFORMIS : I. RNA Synthesis in Macro- and Micronuclei

Tetrahymena in the log phase of growth were pulse labeled with uridine-³H, fixed in acetic-alcohol, extracted with DNase, and embedded in Epon. 0.5-µ sections were cut, coated with Kodak NTB-2 emulsion, and developed after suitable exposures. Grains were counted above macronuclei, above 1000 micronuclei, and above 1000 micronucleus-sized "blanks" which were situated next to micronuclei in the visual field by means of a camera lucida. An analysis of grain counts showed that micronuclei were less than ½₀₀₀ as active as macronuclei on the basis of grains per nucleus. Since micronuclei contained, on the average, about ½₀ as much DNA as macronuclei, micronuclear DNA had less than 1% of the specific activity of macronuclear DNA in RNA synthesis. However, even this small amount of apparent incorporation was not significantly different from zero. Comparisons of the frequency distributions of labeled micronuclei with those of micronuclear "blanks" showed no evidence of a small population of labeled nuclei such as might be expected if micronuclei synthesized RNA for only a brief portion of the cell cycle. We conclude from these studies that there is no detectable RNA synthesis in Tetrahymena micronuclei during vegetative growth and reproduction.     

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.     

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.     

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.     

Distribution of 5S ribosomal RNA genes in somatic and germ cells of the house cricket,Acheta domesticus

In the house cricket,Acheta domesticus, the 110 genes per haploid genome encoding 18S and 28S rRNA are contained within rDNA repeats which are amplified during oogenesis. The 5S rRNA coding sequences of this cricket are found in two sizes of 5S DNA repeating units (measuring 2.1 and 3.0 kb). The 3.0 kb repeats account for more than 90% of the totalAcheta 5S DNA. We have determined the number of cricket 5S rRNA genes by RNA-DNA hybridization analysis: 310 5S DNA repeats/haploid genome clearly approximates the number of 18S and 28S rRNA genes. Because of the relatively low copy number of 5S rRNA genes the possibility of 5S DNA amplification in oocytes ofA. domesticus was also examined. Although amplification of rDNA is readily detectable, amplification of 5S DNA is not observed in oocytes ofA. domesticus. Unlike the genes coding for 18S and 28S rRNA which are localized at a single chromosomal site in the genome ofA. domesticus, the 5S rRNA genes occupy numerous sites distributed along the length of most chromosomes.     

DNA amounts in the nuclei ofParamecium aurelia andTetrahymena pyriformis

DNA amounts have been determined in the micronuclei and macronuclei of 8 strains ofParamecium aurelia and 6 strains ofTetrahymena pyriformis. In the case ofTetrahymena a distribution of values for the amount of DNA in the macronuclei of all the strains was observed but the lowest values were approximately the same, viz. 1.17×10⁻¹¹ g. There are two groups of strains in relation to micronuclear DNA values ofTetrahymena, one giving an average of 0.36×10⁻¹² g and the other 0.815×10⁻¹² g. The ratio of MIC/MAC DNA varies in the two groups.Paramecium again has a range of macronuclear values within each stock—lowest value 2.51×10⁻¹⁰ g—and the micronuclear values are similar in all stocks—approximately 0.613×10⁻¹² g. The ratio of MIC/MAC DNA is similar in each stock.—The haploid genome values calculated from these data show excellent agreement with the values obtained by DNA renaturation studies. Supported by a Research Grant B/SR/8276 from the Science Research Council. The Vickers densitometer was purchased with a grant from the Medical Research Council.     

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.     

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.     

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.     

DNA Damage and Repair in Stylonychia lemnae (Ciliata,Protozoa)1

ABSTRACT Irradiation with X rays, UV irradiation after incorporation of bromodeoxyuridine (BU) into the DNA, and cis-platinum (cis-Pt) treatment each cause the loss of micronuclei of Stylonychia lemnae while the macronuclei are not severely affected. The abilities of both nuclei to repair DNA were investigated. Unscheduled DNA synthesis could not be demonstrated after X-ray irradiation, but it was found after treatment with BU/UV and cis-Pt in macro- and micronuclei. The extent of the repair process in the micro- and macronuclei was alike, as indicated by grain counts of [6-³H]thymidine-treated cells. One reason for the different sensitivity of both nuclei to DNA-damaging treatment may be the different number of gene copies in the macro- and micronuclei.     

Physical and genetic mapping of cloned ribosomal DNA from Toxoplasma gondii: primary and secondary structure of the 5S gene.

The ribosomal DNA (rDNA encoding rRNA) of the obligately intracellular protozoan parasite, Toxoplasma gondii, was identified, cloned, physically mapped, its copy number determined, and the 5S gene sequenced. Using total RNA as a probe, a collection of recombinant lambda phages containing copies of rDNA were isolated from a lambda 2001 tachyzoite genomic library. Northern gel hybridization confirmed specific homology of the 7.5-kb rDNA unit, subcloned into pTZ18R, to T. gondii rRNA. The mapped rDNA found in pTOX1 contained small ribosomal subunit (SS; 18S)- and large ribosomal subunit (LS; 26S)-encoding genes localized using intragenic heterologous probes from the conserved sequences of the SS (18S) and LS (28S) Xenopus laevis genes. the physical mapping data, together with partial digestion experiments and Southern gel hybridization, confirmed a 7.5-kb rDNA unit arranged in a simple head-to-tail fashion that is tandemly repeated. We estimated the rDNA repeat copy number in T. gondii to be 110 copies per haploid tachyzoite genome. Parts of the SS gene and the complete 5S gene were sequenced. The 5S gene was found to be within the rDNA locus, a rare occurrence found only in some fungi and protozoa. Secondary-structure analysis revealed an organization remarkably similar to the 5S RNA of eukaryotes.     

Interspersion of histone and 5S RNA genes in Artemia

Four recombinant lambda phage containing histone genes were selected from a library of Artemia genomic DNA fragments. The histone gene organization of Artemia resembles that of other invertebrates in that all five genes are clustered and repeated in tandem with approximate repeat lengths of 8.5 kb and 9.3 kb. Each recombinant lambda phage isolate hybridizes with five histone mRNAs and unexpectedly also with 5S ribosomal RNA. Hybridization kinetics have shown the number of histone genes to be about 95-100 copies per haploid genome. An identical number of copies was determined for a hybridization probe containing the 5S gene but no histone genes. We have not found any evidence for a separate set of repeated 5S genes outside this histone + 5S block.     

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.     

Sequence and organization of 5S ribosomal RNA-encoding genes of Arabidopsis thaliana.

We have isolated a genomic clone containing Arabidopsis thaliana 5S ribosomal RNA (rRNA)-encoding genes (rDNA) by screening an A. thaliana library with a 5S rDNA probe from flax. The clone isolated contains seven repeat units of 497 bp, plus 11 kb of flanking genomic sequence at one border. Sequencing of individual subcloned repeat units shows that the sequence of the 5S rRNA coding region is very similar to that reported for other flowering plants. Four A. thaliana ecotypes were found to contain approx. 1000 copies of 5S rDNA per haploid genome. Southern-blot analysis of genomic DNA indicates that 5S rDNA occurs in long tandem arrays, and shows the presence of numerous restriction-site polymorphisms among the six ecotypes studied.     

Chromatin elimination and the genetic organisation of the macronucleus in Tetrahymena thermophila

In exponentially growing Tretrahymena thermophila the DNA content of the following structures was determined by cytophotometry: macronuclei of sister cells immediately after division; micronuclei; extranuclear chromatin in dividing cells and postdividers. Further, the development of macro-nuclear DNA amount in successive cell generations was determined. It was found that chromatin elimination is a frequent process reducing DNA content by about 4% per fission. This chromatin disappears within 20 min after division. The quantity of DNA extruded is highly variable and is different from the micronuclear DNA amount or multiples of it. The frequency of generations with two replication rounds as well as those without replication is estimated to be in the range of 2% each. These findings together with the qualitative difference between micro- and macronuclear DNAs suggest that the macronucleus of Tetrahymena is not entirely composed of complete genomes and that parts of the genetic material must be treated specifically for different sequences either during extrusion or during replication.     

Non-coding RNAs mediate the rearrangements of genomic DNA in ciliates

Most eukaryotes employ a variety of mechanisms to defend the integrity of their genome by recognizing and silencing parasitic mobile nucleic acids. However, recent studies have shown that genomic DNA undergoes extensive rearrangements, including DNA elimination, fragmentation, and unscrambling, during the sexual reproduction of ciliated protozoa. Non-coding RNAs have been identified to program and regulate genome rearrangement events. In Paramecium and Tetrahymena, scan RNAs (scnRNAs) are produced from micronuclei and transported to vegetative macronuclei, in which scnRNA elicits the elimination of cognate genomic DNA. In contrast, Piwi-interacting RNAs (piRNAs) in Oxytricha enable the retention of genomic DNA that exhibits sequence complementarity in macronuclei. An RNA interference (RNAi)-like mechanism has been found to direct these genomic rearrangements. Furthermore, in Oxytricha, maternal RNA templates can guide the unscrambling process of genomic DNA. The non-coding RNA-directed genome rearrangements may have profound evolutionary implications, for example, eliciting the multigenerational inheritance of acquired adaptive traits.     

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.