Replication of the extrachromosomal ribosomal RNA genes of Tetrahymena thermophilia.

Cultures of Tetrahymena thermophila were deprived of nutrients and later refed with enriched medium to obtain partial synchrony of DNA replication. Preferential replication of the extrachromosomal, macronuclear ribosomal RNA genes (rDNA) was found to occur at 40-80 min after refeeding. The rDNA accounted for one half of the label incorporated into cellular DNA during this period. Electron microscopy of the purified rDNA showed 1% replicative intermediates. Their structure was that expected for bidirectional replication of the linear rDNA from an origin or origins located in the central nontranscribed region of the palindromic molecule. Similar forms had previously been observed for the rDNA of a related species, Tetrahymena pyriformis. The electron microscopic data was consistent with an origin of replication located approximatley 600 base pairs from the center of the rDNA of T. thermophila, in contrast to a more central location in the rDNA of T. pyriformis. One implication of an off-center origin of replication is that there are two such sequences per palindromic molecule.     

Sequence and structure of the extrachromosomal palindrome encoding the ribosomal RNA genes in Dictyostelium

Ribosomal RNAs (rRNAs) are encoded by multicopy families of identical genes. In Dictyostelium and other protists, the rDNA is carried on extrachromosomal palindromic elements that comprise up to 20% of the nuclear DNA. We present the sequence of the 88 kb Dictyostelium rDNA element, noting that the rRNA genes are likely to be the only transcribed regions. By interrogating a library of ordered YAC clones, we provide evidence for a chromosomal copy of the rDNA on chromosome 4. This locus may provide master copies for the stable transmission of the extrachromosomal elements. The extrachromosomal elements were also found to form chromosome-sized clusters of DNA within nuclei of nocodazole-treated cells arrested in mitosis. These clusters resemble true chromosomes and may allow the efficient segregation of the rDNA during mitosis. These rDNA clusters may also explain the cytological observations of a seventh chromosome in this organism.     

Methylation of ribosomal RNA genes in the macronucleus of Tetrahymena thermophila.

We have investigated the occurrence of methylated adenine residues in the macronuclear ribosomal RNA genes of Tetrahymena thermophila. It has been shown previously that macronuclear DNA, including the palindromic ribosomal RNA genes (rDNA), of Tetrahymena thermophila contains the modified base N-6-methyladenine, but no 5-methylcytosine. Purified rDNA was digested with restriction enzymes Sau 3AI, MboI and DpnI to map the positions and levels of N-6-methyladenine in the sequence 5' GATC 3'. A specific pattern of doubly methylated GATC sequences was found; hemimethylated sites were not detected. The patterns and levels of methylation of these sites did not change significantly in different physiological states. A molecular form of the rDNA found in the newly developing macronucleus and for several generations following the sexual process, conjugation, contained no detectably methylated GATC sites. However, both the bulk macronuclear DNA and palindromic rDNA from the same macronuclei were methylated. Possible roles for N-6-methyladenine in macronuclear DNA are discussed in light of these findings.     

Low molecular weight DNA in the heteromeric macronuclei of two cyrtophorid ciliates

Summary— The size range of the native DNA molecules in the heteromeric macronuclei of two cyrtophorid ciliates (Trithigmostoma cucullulus, Chilodonella uncinata) was mainly investigated by using agarose gel electrophoresis. Numerous bands superimposed on a continuous spectrum of molecular sizes between about 0.35 kb and 30 kb were resolved by conventional electrophoresis. Species-specific banding patterns indicate a variation between species in the copy number of individual DNA fragments. A slight intra-specific variability of banding patterns can exist. Electrophoretic distributions for two strains of T cucullulus were indeed found to differ by at least one more intense band (‘overamplified’ sequences?). Fractionation by contour-clamped homogeneous electric field (CHEF) gel electrophoresis revealed that the size continum of macronuclear DNA molecules does not extend beyond 60–70 kb. The average size was estimated to be around 4 kb. Unresolved DNA fraction (> 1000 kb) accounted for less than 10% of the mass of cellular DNA entering CHEF gels. Macronuclear ribosomal DNA of each cyrtophorid species was identified by Southern hybridization with a Tetrahymena rDNA probe. The hybridization signal was observed on a single band of low molecular weight DNA. The corresponding size was close to 14.5 kb in Trithigmostoma and 15.5 kb in Chilodonella, which is about twice the size of monomeric rDNA in hypotrichous ciliates. We showed that S1 nuclease resistant duplexes wit half the length of the native rDNA can be formed by rapid renaturation of heat-denatured molecules and hybridized with native rDNA. This strongly suggests that the nucleotide sequence of this rDNA is a large palindrome. Unlike the hypotrichs, macronuclear rDNA in cyrtophorids should be organized into palindromic dimers as in Tetrahymena species.     

Conservation of sequences adjacent to the telomeric C4A2 repeats of ciliate macronuclear ribosomal RNA gene molecules.

We sequenced and compared the telomeric regions of linear rDNAs from vegetative macronuclei of several ciliates in the suborder Tetrahymenina. All telomeres consisted of tandemly repeated C4A2 sequences, including the 5' telomere of the 11 kb rDNA from developing macronuclei of Tetrahymena thermophila. Our sequence of the 11 kb 5' telomeric region shows that each one of a previously described pair of inverted repeats flanking the micronuclear rDNA (Yao et al., Mol. Cell. Biol. 5: 1260-1267, 1985) is 29 bp away from the positions to which telomeric C4A2 repeats are joined to the ends of excised 11 kb rDNA. In general we found that the macronuclear rDNA sequences adjacent to C4A2 repeats are not highly conserved. However, in the non-palindromic rDNA of Glaucoma, we identified a single copy of a conserved sequence, repeated in inverted orientation in Tetrahymena spp., which all form palindromic rDNAs. We propose that this sequence is required for a step in rDNA excision common to both Tetrahymena and Glaucoma.     

Synthesis of ribosomal DNA in conjugating Tetrahymena.

In the ciliate protozoan Tetrahymena thermophila, a single integrated gene coding for ribosomal RNA in the micronucleus is amplified during the sexual cycle to yield many copies of extrachromosomal palindromic rDNA in the macronucleus. Hybridization of newly synthesized DNA with rRNA has shown that extensive rDNA synthesis takes place early in the sexual cycle of Tetrahymena. The number of genes synthesized during this period is sufficient to account for gene amplification. A later period of rDNA synthesis occurs when new macronuclear anlagen are beginning to develop. This synthesis may represent preferential polyploidization of already amplified rDNA.     

Site-specific initiation of DNA replication within the non-transcribed spacer of Physarum rDNA.

Physarum polycephalum rRNA genes are found on extrachromosomal 60 kb linear palindromic DNA molecules. Previous work using electron microscope visualization suggested that these molecules are duplicated from one of four potential replication origins located in the 24 kb central non-transcribed spacer [Vogt and Braun (1977) Eur. J. Biochem., 80, 557-566]. Considering the controversy on the nature of the replication origins in eukaryotic cells, where both site-specific or delocalized initiations have been described, we study here Physarum rDNA replication by two dimensional agarose gel electrophoresis and compare the results to those obtained by electron microscopy. Without the need of cell treatment or enrichment in replication intermediates, we detect hybridization signals corresponding to replicating rDNA fragments throughout the cell cycle, confirming that the synthesis of rDNA molecules is not under the control of S-phase. The patterns of replication intermediates along rDNA minichromosomes are consistent with the existence of four site-specific replication origins, whose localization in the central non-transcribed spacer is in agreement with the electron microscope mapping. It is also shown that, on a few molecules, at least two origins are active simultaneously.     

Analysis of Paramecium Macronuclear DNA Using Pulsed Field Gel Electrophoresis

.We have analyzed the macronuclear DNA of Paramecium tetraurelia using orthogonal-field-altemation gel electrophoresis. The mean size of the linear macronuclear DNA molecules is approximately 450 kb. Less than 6% of the macronuclear DNA is larger than 800 kb. Using pulse times of 20, 40, 60 and 90 s we show that the macronuclear fragment containing the A type variable surface antigen gene migrates reproducibly as a 320-kb linear DNA. Over the same pulse times we describe the unusual migration of the ribosomal RNA gene (rDNA) of P. tetraurelia. At pulse times of 20 and 40 s the rDNA migrates at limit mobility (300 and 500 kb, respectively) whereas with 60- and 90-s pulse times, 2 components of rDNA are observed; 1 fraction independent of pulse time migrating at limit mobility, and a 2nd component migrating between 100-kb and 400-kb linear markers. Based upon previous electron micrographic studies of Paramecium rDNA as well as data presented here we conclude that the majority of Paramecium rDNA molecules are a circular DNA form.     

Extrachromosomal rDNA of Tetrahymena thermophila is not a perfect palindrome

We have determined the restriction-endonuclease-cleavage map and the nucleotide sequence of the central 1.4 kb fragment of the macronuclear extrachromosomal rDNA of Tetrahymena thermophila. These data demonstrate that this molecule is not a perfect palindrome, having a 29 bp AT-rich non-palindromic sequence at its center. This observation is important in determining the mechanism by which a single chromosomally integrated rRNA gene in the micronucleus is rearranged and amplified during sexual development to yield multiple copies of extrachromosomal rDNA in the macronucleus.     

Tandemly repeated hexanucleotide at Tetrahymena rDNA free end is generated from a single copy during development

The linear extrachromosomal ribosomal DNA of Tetrahymena is generated from a single integrated copy during macronuclear development. The free ends of this extrachromosomal gene contain 20-70 tandem repeats of the hexanucleotide CCCCAA. We have determined the nucleotide sequence at the same (3') end of the single, integrated micronuclear gene. In contrast to the extrachromosomal gene, only a single CCCCAA sequence was found at this position. The same result was obtained from two independently isolated DNA clones, and was therefore not likely an artifact of cloning. Comparisons of the genomic DNA with the cloned fragment by Southern hybridization also supported this argument. Thus the tandemly repetitive hexanucleotide at the free ends of the extrachromosomal rDNA is not an inherited feature, and must be generated during the development of the macronucleus.     

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.     

Gene amplification in Tetrahymena thermophila: formation of extrachromosomal palindromic genes coding for rRNA.

Tetrahymena thermophila contains in the macronucleus multiple copies of extrachromosomal palindromic genes coding for rRNA (rDNA) which are generated from a single chromosomal copy during development. In this study we isolated the chromosomal copy of rDNA and determined the structure and developmental fate of the sequence surrounding its 5' junction. The result indicates that specific chromosomal breakage occurs at or near the 5' junction of rDNA during development. The breakage event is associated with DNA elimination and telomeric sequence addition. Similar results were also found previously for the 3' junction of this gene. These results could explain how the extrachromosomal rDNA is first generated. Near both junctions of the chromosomal rDNA, a pair of 20-nucleotide repeats was found. These sequences might serve as signals for site-specific breakage. In addition, we found a pair of perfect inverted repeats at the 5' junction of this gene. The repeats are 42 nucleotides long and are separated by 28 nucleotides. The existence of this structure provides a simple explanation for the formation of the palindromic rDNA.     

Homologies between the contiguous and fragmented rRNAs of the two Plasmodium falciparum extrachromosomal DNAs are limited to core sequences.

Plasmodium falciparum contains two extrachromosomal DNAs, a 6 kb linear element and a 35 kb circular DNA; both encode rDNA sequences. The 6 kb element rDNAs comprise fragments of both large and small subunit rRNAs. Comparison of these with corresponding rDNA sequences from the 35 kb DNA and E. coli show that sequences conserved between the three are largely confined to highly conserved core regions; in fact, most of the 6 kb rDNA sequences correspond to core regions. Both the 6 kb element and 35 kb rDNAs show less conservation to each other than to E. coli sequences, suggesting that the two extrachromosomal DNAs of P. falciparum are not closely related. The characteristics of the fragmented rRNAs from the 6 kb element suggest they are functional, possibly in mitochondrial ribosomes.     

Structure of the central spacer region of extrachromosomal ribosomal DNA in Physarum polycephalum

We have analyzed the sequence organization of the central spacer region of the extrachromosomal ribosomal DNA from two strains of the acellular slime mold Physarum polycephalum. It had been inferred previously from electron microscopy that this region, which comprises about one third of the 60 kb³ palindromic rDNA, contains a complex series of inverted repetitious sequences. By partial digestion of end-labeled fragments isolated from purified rDNA and from rDNA fragments cloned in Escherichia coli, we have constructed a detailed restriction map of this region. The 11 kb of spacer DNA of each half molecule of rDNA contains the following elements: (a) two separate regions, one of 1.1 kb and one of 2.1 kb, composed of many direct repeats of the same 30 base-pair unit; (b) a region of 4.4 kb composed of a complex series of inverted repeats of a 310 base-pair unit; (c) another region of 1.6 kb composed of inverted repeats of the same 310 base-pair unit located directly adjacent to the center of the rDNA; (d) two copies of a unique sequence of 0.85 kb, which probably contains a replication origin. Some of the CpG sequences in the spacer resist cleavage by certain restriction endonucleases and thus appear to be methylated. The lack of perfect symmetry about the central axis and the arrangement of inverted repeated sequences explain the complex pattern of branches and forks of the fold-back molecules previously observed by electron microscopy. Comparison of the rDNA restriction maps from the two strains of Physarum suggests that the repeat units in the spacer are undergoing concerted evolution. We propose a model to explain the evolutionary origin of the several palindromic axes in the Physarum rDNA spacer.     

Sequence-specific fragmentation of macronuclear DNA in a holotrichous ciliate

Macronuclear DNA from the protozoan G. chattoni, a holotrichous ciliate, was analyzed. Most, if not all, of the macronuclear DNA is subchromosomal, ranging in size from above 100 kb down to 2.1 kb, with molecules in the lower molecular weight range being resolvable by gel electrophoresis into reproducible, specific, discrete size classes. A prominent class of linear 9.3 kb molecules consists of single free rRNA genes. Upon denaturation and partial renaturation, a high percentage of total macronuclear DNA was found as single-stranded circles. Sequence analysis showed that a minimum of 38 tandem repeats of the sequence CCCCAA is present in inverted orientation at each end of most or all Glaucoma macronuclear DNA molecules, including the rDNA. This sequence must therefore be recognized during site-specific fragmentation of chromosomes in macronuclear development.     

Localization by high resolution in situ hybridization of the ribosomal minichromosomes during the nucleolar cycle of Physarum polycephalum.

We have used biotinylated rDNA probes to localize by in situ hybridization the extrachromosomal genes for ribosomal RNA in the slime mold Physarum polycephalum. We established conditions that allow for highly specific hybridization at the ultrastructural level and determined that the 60-kb palindromic rDNA molecules are confined to the nucleolus in interphase. Our study definitively locates these extrachromosomal genes in mitosis in the form of thin DNA fibers contained within nucleolar remnants. We further show that these rDNA minichromosomes do not condense and that they segregate as entities independent of the condensed chromosomal DNA. In telophase, these minichromosomes migrate from the poles toward the equatorial region of the nucleus in a direction opposite that of the chromosomes. Our results illustrate the discontinuous nature of the nucleolar organizing region in Physarum.     

Position effect takes precedence over target sequence in determination of adenine methylation patterns in the nuclear genome of a eukaryote, Tetrahymena thermophila.

Approximately 0.8% of the adenine residues in the macronuclear DNA of the ciliated protozoan Tetrahymena thermophila are modified to N 6-methyladenine. DNA methylation is site specific and the pattern of methylation is constant between clonal cell lines. In vivo, modification of adenine residues appears to occur exclusively in the sequence 5'-NAT-3', but no consensus sequence for modified sites has been found. In this study, DNA fragments containing a site that is uniformly methylated on the 50 copies of the macronuclear chromosome were cloned into the extrachromosomal rDNA. In the novel location on the rDNA minichromosome, the site was unmethylated. The result was the same whether the sequences were introduced in a methylated or unmethylated state and regardless of the orientation of the sequence with respect to the origin of DNA replication. The data show that sequence is insufficient to account for site-specific methylation in Tetrahymena and argue that other factors determine the pattern of DNA methylation.     

Phylogeny of the Large Extrachromosomal DNA of Organisms in the Phylum Apicomplexa

ABSTRACT. Organisms in the phylum Apicomplexa appear to have a large extrachromosomal DNA which is unrelated to the mitochondrial DNA. Based on the apparent gene content of the large (35 kb) extrachromosomal DNA of Plasmodium falciparum , it has been suggested that it is a plastid-like DNA, which may be related to the plastid DNA of rhodophytes. However, phylogenetic analyses have been inconclusive. It has been suggested that this is due to the unusually high A + T content of the Plasmodium falciparum large extrachromosomal DNA. To further investigate the evolution of the apicomplexan large extrachromosomal DNA, the DNA sequence of the organellar ribosomal RNA gene from Toxoplasma gondii , was determined. The Toxoplasma gondii rDNA sequence was most similar to the large extrachromosomal rDNA of Plasmodium falciparum , but was much less A + T rich. Phylogenetic analyses were carried out using the LogDet transformation to minimize the impact of nucleotide bias. These studies support the evolutionary relatedness of the Toxoplasma gondii rDNA with the large extrachromosomal rDNA of Plasmodium falciparum and with the organellar rDNA of another parasite in the phylum Apicomplexa, Babesia bovis. These analyses also suggest that the apicomplexan large extra-chromosomal DNA may be more closely related to the plastid DNA of euglenoids than to those of rhodophytes.