Numbers of 5S and tRNA genes in macro- and micronuclei of Tetrahymena pyriformis

Macronuclei of Tetrahymena pyriformis contain approximately 200 copies of the genes for 25S and 17S ribosomal RNA (rRNA) per haploid genome. Micronuclei, however, contain only a few copies of the rRNA genes per haploid complement. Since macronuclei develop from, products of meiosis, fertilization and division of micronuclei, we suggested that the multiple copies of the rRNA genes in macronuclei are generated by amplification of the small number of genes in micronuclei (Yao et al., 1974). This process provides a simple mechanism for maintaining the homogeneity of the repeated rRNA genes. To test if amplification is a general mechanism operating on all repeated genes in Tetrahymena, we have examined the numbers of 5S RNA and tRNA genes in macro- and micronuclei. 5S RNA was purified by polyacrylamide gel electrophoresis and hybridized to saturation against macro- and micronuclear DNA. Approximately 0.013–0.014% of macronuclear DNA and about 0.009% of micronuclear DNA is complementary to 5S RNA. After correcting for the differences in the DNA sequence complexities between the two nuclei, we calculate that there are 300–350 5S genes per haploid macro- or micronuclear genome. From these data we conclude that there is little or no detectable amplification of the 5S genes in macronuclei relative to micronuclei. Similar studies using tRNA indicate that these genes are also highly repeated in both nuclei; about 800 genes are present per haploid genome. Thus, amplification from a small number of genes can be excluded as the mechanism for generating the repeated copies of the 5S and tRNA genes in Tetrahymena and it is likely that another, as yet unidentified, mechanism operates to maintain the homogeneity of these genes.     

Characterization of the macronuclear DNA of different species ofTetrahymena

Summary The macronuclear DNAs from 20 different species ofTetrahymena were characterized using alternating Orthogonal Field (AOF) gel electrophoresis. Each species has approximately 300 different macronuclear DNA molecules that range in size from about 100–2000 kb pairs. Although the individual macronuclear DNA molecules are not well resolved on an AOF gel, most species have a unique profile of macronuclear DNA. The sequences that hybridize with histone H4 (Tetrahymena) and ubiquitin (yeast) genes were identified on the separated macronuclear DNA molecules of the different species. All species have 2 histone H4 genes located on macronuclear DNA molecules of different sizes. This is consistent with the duplication of the histone H4 gene prior to the speciation events leading to the various species ofTetrahymena. The number and sizes of the macronuclear DNA molecules that hybridize with the ubiquitin probe vary from species to species. A grouping of the different species ofTetrahymena based on this hybridization pattern paralels groupings of the species based on ribosomal RNA sequences and isoenzymes. Some intraspecific variation among different strains ofTetrahymena thermophila was detected using ubiquitin and 5S ribosomal RNA as probes.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

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.     

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.     

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.     

Genome structure of Tetrahymena pyriformiis

Reassociation kinetics of DNA from the macronucleus of the ciliate, Tetrahymena pyriformis GL, has been studied. The genome size determined by the kinetic complexity of DNA was found to be 2.0×10⁸ base pairs (or 1.2×10¹¹ daltons). About 90% of the macronuclear DNA fragments 200–300 nucleotides in length reassociate at a rate corresponding to single-copy nucleotide sequences, and 7–9% at a rate corresponding to moderate repetitive sequences; 3–4% of such DNA fragments reassociate at C₀t practically equal to zero. To investigate the linear distribution of repetitive sequences, DNA fragments of high molecular weight were reassociated and reassociation products were treated with Sl-nuclease. DNA double-stranded fragments were then fractionated by size. It has been established that in the Tetrahymena genome long regions containing more than 2000 nucleotides make up about half of the DNA repetitive sequences. Another half of the DNA repetitive sequences (short DNA regions about 200–300 nucleotides long) intersperse with single-copy sequences about 1,000 nucleotides long. Thus, no more than 15% of the Tetrahymena genome is patterned on the principle of interspersing single-copy and short repetitive sequences. Most of the so called zero time binding or foldback DNA seem to be represented by inverted self-complementary (palindromic) nucleotide sequences. The conclusion has been drawn from the analysis of this fraction isolated preparatively by chromatography. About 75% of the foldback DNA is resistant to Sl-nuclease treatment. The Sl-nuclease resistance is independent of the original DNA concentration. Heat denaturation and renaturation are reversible and show both hyper and hypochromic effects. The majority of the inverted sequences are unique and about 20% are repeated tens of times. According to the equilibrium distribution in CsCl density gradients the average nucleotide content of the palindromic fraction does not differ significantly from that of total macronuclear DNA. It was shown that the largest part of this fraction of the Tetrahymena genome are not fragments of ribosomal genes.     

[6N]METHYL ADENINE IN THE NUCLEAR DNA OF A EUCARYOTE, TETRAHYMENA PYRIFORMIS

DNA isolated from macronuclei of the ciliate, Tetrahymena pyriformis, has been found to contain [⁶N]methyl adenine (MeAde); this represents the first clear demonstration of significant amounts of MeAde in the DNA of a eucaryote. The amounts of macronuclear MeAde differed slightly between different strains of Tetrahymena, with approximately 0.65–0.80% of the adenine bases being methylated. The MeAde content of macronuclear DNA did not seem to vary in different physiological states. The level of MeAde in DNA isolated from micronuclei, on the other hand, was quite low (at least tenfold lower than in macronuclear DNA).     

Tetrahymena Telomerase RNA levels increase during macronuclear development

Telomeres, the G-rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)_n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady-state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000–40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2-to 5-fold at 9–15 h, and 1.5- to 3.5-fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells. © 1992 Wiley-Liss, Inc.     

Nucleotide sequence of the split tRNAUAALeu gene from Vicia faba chloroplasts: evidence for structural homologies of the chloroplast tRNALeu intron with the intron from the autosplicable Tetrahymena ribosomal RNA precursor

Summary The gene encoding the tRNA _UAA ^Leu from broad bean chloroplasts has been located on a 5.1 kbp long BamHI fragment by analysis of the DNA sequence of an XbaI subfragment. This gene is 536 bp long and is split in the anticodon region. The 451 bp long intron shows high sequence homology over about 100 bp from each end with the corresponding regions of the maize chloroplast tRNA _UAA ^Leu intron. These conserved sequences are probably involved in the splicing reaction, for they can be folded into a secondary structure which is very similar to the postulated structure of the intron from the autosplicable ribosomal RNA precursor of Tetrahymena. Very little sequence conservation is found in the 5-and 3-flanking regions of the broad bean and maize chloroplast tRNA _UAA ^Leu genes.     

Fine structure physical mapping of 4S RNA genes on mitochondrial DNA of Saccharomyces cerevisiae.

Summary We have localized the genes for mitochondrial 4S RNA on the physical map of themtDNA of severalSaccharomyces cerevisiae strains by hybridization of iodinated 4S RNA to the restriction fragments obtained with endonucleasesHindII+III,EcoRI andHapII. The data indicate that 5–8 of the 4S RNA genes are dispersed over a large area of the genome whereas the rest (about 18 genes) is located within an area of about 9000 bp in length (about 12% of the genome) between the markers for chloramphenicol and paromomycin resistance (RIB 1 and PAR 1 loci). Within this region a cluster is present of 5 genes on a DNA fragment of 460 bp.Abbreviations Used mtDNA mitochondrial DNA - mtRNA mitochondrial RNA - rRNA ribosomal RNA - tRNA transfer RNA - C, E, P and O cytoplasmically-inherited resistance markers for chloramphenicol, erythromycin, paromomycin and oligomycin, respectively - SSC 150 mM sodium chloride, 15 mM sodium citrate (pH 7.0) - SDS sodium dodecylsulphate - EDTA (sodium)ethylenediaminetetraacetate; TEMED - N,N,N N-tetramethylethylenediamine; (k)bp, (kilo)base pairs - EthBr ethidium bromide     

Nuclear differentiation and nucleic acid synthesis in well-fed exconjugants of Paramecium aurelia

Paramecium aurelia exconjugants contain new macronuclear anlagen and numerous fragments of the old pre-zygotic macronucleus. Macronuclear anlagen develop during the first two cell cycles after conjugation. During this time their volume increases from about 11 m³ to about 3700 m³ and more than 10 doublings of DNA content occur. The rate of DNA synthesis is between two and three times as great as in the vegetative macronucleus. — In macronuclear fragments, however, DNA synthesis is suppressed. The rate of DNA synthesis in macronuclear fragments during the extended first cell cycle after conjugation (11 1/2 hr. vs. 5 1/2 hr. for the vegetative cell cycle) is only about one-third of the rate in vegetative macronuclei and there is only a 65% increase in the mean DNA content of fragments. The rate of fragment DNA synthesis continues to decrease during each of the subsequent two cell cycles. — Unlike the rate of DNA synthesis, the rate of RNA synthesis per unit of DNA is similar in macronuclear anlagen, macronuclear fragments and fully developed macronuclei. Macronuclear fragments continue to synthesize RNA at the normal rate long after the new macronuclei are fully developed. Fragments contribute about 80% of all RNA synthesized during the first two cell cycles after conjugation. RNA synthesis begins very early in the development of macronuclear anlagen and nucleolar material appears during the first half-hour of anlage development. — Chromosome-like structures were never observed during anlage development and there was no evidence of two periods of DNA synthesis separated by a DNA poor stage as has been observed in several hypotrichous Ciliates.     

Free genes for rRNAs in the macronuclear genome of the ciliate Stylonychia mytilus

When separated on an agarose gel, macronuclear DNA of the hypotrichous ciliate Stylonychia mytilus gives rise to many well-defined bands ranging in molecular weight from 0.3×10⁶ to 14×10⁶ dalton. Hybridization of 25 S rRNA, 17 S rRNA or 5 S RNA to such a gel revealed sharp hybridization bands. This suggests that this banding pattern is not an artefact due to nonspecific degradation of macronuclear DNA but that the DNA in the macronucleus of Stylonychia occurs in discrete fragments, each coding for at least one gene. The size of the DNA fragment coding for rRNA was found to be 4.5×l0⁶ dalton, the fragment coding for 5 S RNA has a molecular weight of 150,000–250,000 dalton.     

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.     

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.     

DNA replication in macronuclei of Tetrahymena pyriformis GL

DNA replication in macronuclei of Tetrahymena pyriformis GL has been studied to discriminate between hypotheses developed for the interpretation of intraclonal differentiation in ciliated protozoa (the diploid subnuclear, and the ‘master’-‘slave’ hypotheses). Tetrahymena cells were grown in a heavy ¹⁵N-³H medium and then transferred to a light ¹⁴N-¹⁴C medium. DNA was isolated after various periods following this transfer and studied in equilibrium CsCl density gradient centrifugation. Time-related changes in the DNA buoyant density pattern were investigated. The data obtained are interpreted to mean that all DNA in macronuclei of asynchronously growing Tetrahymena at exponential phase replicates semiconservatively once in a cell cycle. These data are in good agreement with the findings of Andersen & Zeuthen obtained on synchronous Tetrahymena cultures in the presence of BUdR.These results are not consistent with the ‘master’-‘slave’ hypothesis. The diploid subnuclear hypothesis is not in accord with other experimental evidence. An alternative hypothesis has been proposed concerning the nature of the macronuclear units and the process of determination. The two main points of this hypothesis are: (a) macronuclear units are diploid genome fragments (‘nucleosomes’); (b) determination is a process of haploidization by ‘allelic splitting’ at a definite macronuclear fission. Consistency with experimental data is discussed and some predictions of the hypothesis are given.     

Intron length variation of the Adh gene in Brachyscome (Asteraceae)

Eighteen polymerase chain reaction (PCR) products of the partial sequence of the Adh (alcohol dehydrogenase) gene from 10 Brachyscome species were sequenced and compared. These products contained the 5 three fourths of exon 4 and whole sequences of intron 3. They varied extensively in length due to the differences in length of intron 3. A total of 10 long insertions were flanked by direct repeats of 5 to 12 bp sequences, indicating inserted elements. These inserted elements were classified into the following five categories based on nucleotide sequence characteristics and length; (1) a region homologous to that of 5S RNA genes (5S DNA), (2) A-rich structure at the 3 end-like short interspersed elements (SINEs) in animals, (3) a sequence of 280 bp with no characteristic features, (4) a sequence of 125 bp with no characteristic features, (5) termini of 11 bp inverted repeats flanked by 5 bp sequence of direct repeats characteristics of a transposon.     

Variable copy number of macronuclear DNA molecules in Tetrahymena

In Tetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000-kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species of Tetrahymena using histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins. © 1992 Wiley-Liss, Inc.     

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.     

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.