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.     

Characterization of two telomeric DNA processing reactions in Saccharomyces cerevisiae.

We have investigated two reactions that occur on telomeric sequences introduced into Saccharomyces cerevisiae cells by transformation. The elongation reaction added repeats of the yeast telomeric sequence C1-3A to telomeric sequences at the end of linear DNA molecules. The reaction worked on the Tetrahymena telomeric sequence C4A2 and also on the simple repeat CA. The reaction was orientation specific: it occurred only when the GT-rich strand ran 5' to 3' towards the end of the molecule. Telomere elongation occurred by non-template-directed DNA synthesis rather than any type of recombination with chromosomal telomeres, because C1-3A repeats could be added to unrelated DNA sequences between the CA-rich repeats and the terminus of the transforming DNA. The elongation reaction was very efficient, and we believe that it was responsible for maintaining an average telomere length despite incomplete replication by template-directed DNA polymerase. The resolution reaction processed a head-to-head inverted repeat of telomeric sequences into two new telomeres at a frequency of 10(-2) per cell division.     

Isolation of telomeric DNA from the filamentous fungus Podospora anserina and construction of a self-replicating linear plasmid showing high transformation frequency.

It has been previously shown that linear plasmids bearing Tetrahymena telomeric sequences are able to replicate autonomously in the filamentous fungus Podospora anserina (1). However, autonomous replication occurs in only 50-70% of the transformants, suggesting a defect in the recognition of the Tetrahymena telomeric template by the putative P. anserina telomerase so that only a fraction of entering DNA is stabilized into linear extrachromosomal molecules. We have cloned DNA sequences added to the Tetrahymena (T2G4)n ends of the linear plasmid. Nucleotide sequencing showed that these sequences are exclusively composed of T2AG3 repeat units. Hybridization experiments of Bal31 treated DNA showed that T2AG3 repeats are confined within 200 bp in chromosomal P. anserina telomeres. A new plasmid has been constructed so that after linearization, the terminal sequences contain T2AG3 repeats. This linear molecule transforms P. anserina with a high frequency (up to 1.75 x 10(4) transformants/micrograms), autonomous replication occurs in 100% of the transformants and the plasmid copy number is about 2-3 per nucleus. These results underscore the importance of the telomeric repeat nucleotide sequence for efficient recognition as functional telomeric DNA in vivo and provide the first step toward the development of an artificial chromosome cloning system for filamentous fungi.     

Bal31 sensitivity of micronuclear sequences homologous to C4A4/G4T4 repeats in Oxytricha nova

The sequence similarity and functional equivalence of telomeres from macronuclear linear DNA molecules in Oxytricha and telomeric sequences of true mitotic/meiotic chromosomes suggest that the (C4A4)n/(G4T4)n sequences found at macronuclear telomeres may also function as micronuclear telomeres in Oxytricha. In this study, radioactively labeled (C4A4)n have been hybridized to micronuclear DNA samples that have been treated with the enzyme Bal31, which has double-stranded exonuclease activity. A time course of digestion shows that approximately 50% of the micronuclear sequences that hybridize to a C4A4 probe disappear with mild digestion by Bal31, suggesting that these sequences are telomeric. The remainder of the hybridizing sequences are not degraded any more rapidly than the total genomic DNA. All of the C4A4/G4T4 sequences that can be detected by hybridization of C4A4 probes to Southern-blotted restriction enzyme digests of micronuclear DNA occur in regions of the genome that are highly resistant to restriction enzyme digestion and show a clustering of sites reminiscent of telomeres in other organisms. We propose that the micronuclear C4A4 hybridizable sequences that are Bal31 resistant may be located near the telomere and within telomere-associated repetitive sequences that are immediately internal to telomeric (Bal31 sensitive) C4A4 hybridizeable sequences.     

Internal micronuclear DNA regions which include sequences homologous to macronuclear telomeres are deleted during development in Tetrahymena

C4A2 repeats are present in multiple clusters in both the macronucleus and micronucleus of Tetrahymena. Although the macronucleus is generated from the micronucleus after sexual conjugation, the repeats are telomeric sequences in the macronucleus but are internally located in the micronucleus (1). This study investigates the fate of the sequences adjacent to the micronuclear C4A2 repeats. Southern blot analyses of 21 C4A2-containing micronuclear clones show that extensive elimination of the adjacent sequences occurs during the formation of the macronucleus. Comparison of one C4A2-containing micronuclear clone with its derived macronuclear segment indicates that approximately 4.5 kb of DNA, which includes the C4A2 repeats and adjacent sequences on both sides is deleted from the macronucleus. The two regions adjoining the deletion are joined together to form a contiguous segment in the macronucleus. This excision of C4A2 repeats and surrounding sequences and the rejoining of the retained segments is probably the mechanism by which all or most of the other C4A2 adjacent sequences are eliminated.     

Cloning yeast telomeres on linear plasmid vectors

We have constructed a linear yeast plasmid by joining fragments from the termini of Tetrahymena ribosomal DNA to a yeast vector. Structural features of the terminus region of the Tetrahymena rDNA plasmid maintained in the yeast linear plasmid include a set of specifically placed single-strand interruptions within the cluster of hexanucleotide (C4A2) repeat units. An artificially constructed hairpin terminus was unable to stabilize a linear plasmid in yeast. The fact that yeast can recognize and use DNA ends from the distantly related organism Tetrahymena suggests that the structural features required for telomere replication and resolution have been highly conserved in evolution. The linear plasmid was used as a vector to clone chromosomal telomeres from yeast. One Tetrahymena end was removed by restriction digestion, and yeast fragments that could function as an end on a linear plasmid were selected. Restriction mapping and hybridization analysis demonstrated that these fragments were yeast telomeres, and suggested that all yeast chromosomes might have a common telomere sequence. Yeast telomeres appear to be similar in structure to the rDNA of Tetrahymena, in which specific nicks or gaps are present within a simple repeated sequence near the terminus of the DNA.     

Native yeast telomeres are sufficient to stabilize linear DNA in Xenopus laevis oocytes.

We have constructed a linear plasmid in yeast containing the entire bovine papillomavirus genome and tested its physical stability following microinjection into stage VI oocytes of Xenopus laevis. Our results show that unmodified telomeres, in contrast to the yeast-passaged telomeres, drastically affect the stability of the injected linear plasmid. Plasmids carrying unmodified Tetrahymena thermophila telomeric sequences are rapidly degraded in oocytes. When these plasmids are passed through yeast, the telomere ends become modified by the addition of yeast telomeric sequences. These plasmids are stably maintained in X. laevis oocytes, demonstrating that yeast-modified telomeres are sufficient to prevent linear DNA degradation.     

Rare internal C4A4 repeats in the micronuclear genome of Oxytricha fallax.

Approximately 20,000 different short, linear, macronuclear DNA molecules are derived from micronuclear sequences of Oxytricha fallax after conjugation. These macronuclear DNAs are terminated at both ends by 20 base pairs of the sequence 5'-dC4A4-3'. Sequences homologous to this repeat (C4A4+) are also abundant in the micronuclear chromosomes, but most reside at their telomeres. Here we show that nontelomeric C4A4 clusters of 20 base pairs or longer exist in only a few hundred copies per micronuclear genome. This demonstrates that nearly none of the 20,000 sequence blocks of micronuclear DNA destined to be macronuclear DNA molecules can be flanked by full-length (20-base pair) C4A4 clusters, and therefore C4A4 repeats must be added to most, if not all, macronuclear telomeres during macronuclear development. Six internal micronuclear C4A4+ loci were cloned, and their structural relationships with macronuclear and micronuclear sequences were examined. The possible origins and functions of these rare, micronuclear internal C4A4 loci are discussed.     

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.     

Progressive alteration of telomeric sequences at one end of a yeast linear plasmid and its possible association with reduced plasmid stability

After selection for migration into the nucleus, a cytoplasmic yeast linear plasmid bearing an inverted terminal repeat (ITRs) at each end replicates in Saccharomyces cerevisiae in a linear form, called pTLU, which carries host telomeric repeats (TG(1-3))(n) of about 300-350 bp added to the ITR ends. We previously showed that the nucleotide composition of the added telomeric sequences varied among individual pTLU isolates, while those on the two ends of any given pTLU were always identical. The telomeric sequences of pTLU remained unchanged over numbers of cell generations when cells were selected for expression of the plasmid-borne nuclear marker. We report here that progressive alterations in telomeric sequences can be detected in cells which are grown under non-selective conditions. Surprisingly, in any given molecule, the telomeric alterations occur exclusively on one side, either the left or the right end, while the sequence at the opposite end remained identical to the original, suggesting a difference in the mode of DNA replication between the plasmid ends. These alterations occur over a broad area extending from the termini of telomeres to nucleotides near the junction between the telomeric sequences and the pTLU-ITR, implying that the plasmid ends undergo successive rounds of extension and contraction. Clonal analysis under non-selective conditions indicated that the alterations in telomeric sequences are generally associated with extreme instability of the pTLU plasmid.     

Recognition and elongation of telomeres by telomerase

Telomeres stabilize chromosomal ends and allow their complete replication in vivo. In diverse eukaryotes, the essential telomeric DNA sequence consists of variable numbers of tandem repeats of simple, G + C rich sequences, with a strong strand bias of G residues on the strand oriented 5' to 3' toward the chromosomal terminus. This strand forms a protruding 3' over-hang at the chromosomal terminus in three different eukaryotes analyzed. Analysis of yeast and protozoan telomeres showed that telomeres are dynamic structures in vivo, being acted on by shortening and lengthening activities. We previously identified and partially purified an enzymatic activity, telomere terminal transferase, or telomerase, from the ciliate Tetrahymena. Telomerase is a ribonucleoprotein enzyme with essential RNA and protein components. This activity adds repeats of the Tetrahymena telomeric sequence, TTGGGG, onto the 3' end of a single-stranded DNA primer consisting of a few repeats of the G-rich strand of known telomeric, and telomere-like, sequences. The shortest oligonucleotide active as a primer was the decamer G4T2G4. Structural analysis of synthetic DNA oligonucleotides that are active as primers showed that they all formed discrete intramolecular foldback structures at temperatures below 40 degrees C. Addition of TTGGGG repeats occurs one nucleotide at a time by de novo synthesis, which is not templated by the DNA primer. Up to 8000 nucleotides of G4T2 repeats were added to the primer in vitro. We discuss the implications of this finding for regulation of telomerase in vivo and a model for telomere elongation by telomerase.     

Extragenomic double-stranded DNA circles in yeast with linear mitochondrial genomes: potential involvement in telomere maintenance

Although the typical mitochondrial DNA (mtDNA) is portrayed as a circular molecule, a large number of organisms contain linear mitochondrial genomes classified by their telomere structure. The class of mitochondrial telomeres identified in three yeast species, Candida parapsilosis, Pichia philodendra and Candida salmanticensis, is characterized by inverted terminal repeats each consisting of several tandemly repeating units and a 5' single-stranded extension. The molecular mechanisms of the origin, replication and maintenance of this type of mitochondrial telomere remain unknown. While studying the replication of linear mtDNA of C.parapsilosis by 2-D gel electrophoresis distinct DNA fragments composed solely of mitochondrial telomeric sequences were detected and their properties were suggestive of a circular conformation. Electron microscopic analysis of these DNAs revealed the presence of highly supertwisted circular molecules which could be relaxed by DNase I. The minicircles fell into distinct categories based on length, corresponding to n x 0.75 kb (n = 1-7). Similar results were obtained with two other yeast species (P.philodendra and C. salmanticensis) which possess analogous telomeric structure.     

A linear shuttle vector for yeast and the hypotrichous ciliate Stylonychia

A linear plasmid was constructed in vitro using the telomeres of the rDNA of Tetrahymena pyriformis. These telomeres were added to a yeast circular vector containing an ARS sequence from Dictyostelium, the LEU2 gene of yeast and the neo gene from Escherichia coli Tn5 fused with a eukaryotic promoter. The resulting plasmid was used to transform yeast. During the replication of the linear plasmid in yeast it was spontaneously modified at the extremity by the addition of 300 bp of yeast telomeric sequence for each end. Total DNA prepared from yeast transformants was used to transform the hypotrichous ciliate Stylonychia lemnae. The same plasmid isolated from Stylonychia can again be replicated in yeast.     

Isolation and characterization of a human telomere.

A method is described that allows cloning of human telomeres in S. cerevisiae by joining human telomeric restriction fragments to yeast artificial chromosome halves. The resulting chimeric yeast-human chromosomes propagate as true linear chromosomes, demonstrating that the human telomere structure is capable of functioning in yeast and suggesting that telomere functions are evolutionarily conserved between yeast and human. One cloned human telomere, yHT1, contains 4 kb of human genomic DNA sequence next to the tandemly repeating TTAGGG hexanucleotide. Genomic hybridizations using both cloned DNA and TTAGGG repeats have revealed a common structural organization of human telomeres. This 4 kb of genomic DNA sequence is present in most, but not all, human telomeres, suggesting that the region is not involved in crucial chromosome-specific functions. However, the extent of common features among the human telomeres and possible similarities in organization with yeast telomeres suggest that this region may play a role in general chromosome behavior such as telomere-telomere interactions. Unlike the simple telomeric TTAGGG repeats, our cloned human genomic DNA sequence does not cross-hybridize with rodent DNA. Thus, this clone allows the identifications of the terminal restriction fragments of specific human chromosomes in human-rodent hybrid cells.     

Elimination of specific DNA sequences from the somatic nucleus of the ciliate Tetrahymena

Tetrahymena micronuclear DNA fragments have been cloned in the plasmid pBR322. One clone, pTt 2512, has been found to contain the C-C-C-C-A-A hexanucleotide repeat which is also present in the macronuclear rDNA. Further restriction enzyme digestion and hybridization studies suggest that the clone also contains sequences that are not present in the somatic macronucleus. The flanking sequences of the C4A2 repeats in this clone were separated into four restriction fragments, one from one side and three from the other. These fragments were used as probes for Southern hybridization to study the organizations of similar sequences in the macronucleus and micronucleus. All four fragments hybridized to many fragments of restriction enzyme digested micronuclear DNA. However, none of these hybridizations were detected in the macronucleus. Thus, these families of repetitive DNA are completely eliminated from the macronucleus. Further analysis suggested that the four different sequences may be linked at other locations of the genome. Using nullisomic strains of Tetrahymena, it is found that at least one of these sequences is present in more than one chromosome. Studies of various normal and star strains of Tetrahymena suggest that these sequences are stable in the normal micronucleus but are altered drastically in the defective micronuclei of the star strains. Eliminated DNA of similar nature has also been found in at least five other randomly selected clones of micronuclear DNA and may be present widely in the genome.     

A novel minisatellite at a cloned hamster telomere

The ends of eukaryotic chromosomes have special properties and roles in chromosome behavior. Selection for telomere function in yeast, using a Chinese hamster hybrid cell line as the source DNA, generated a stable yeast artificial chromosome clone containing 23 kb of DNA adjacent to (TTAGGG)n, the vertebrate telomeric repeat. The common repetitive element d(GT)n appeared to be responsible for most of the other stable clones. Circular derivatives of the TTAGGG-positive clone that could be propagated in E. coli were constructed. These derivatives identify a single pair of hamster telomeres by fluorescence in situ hybridization. The telomeric repeat tract consists of (TTAGGG)n repeats with minor variations, some of which can be cleaved with the restriction enzyme MnlI. Blot hybridization with genomic hamster DNA under stringent conditions confirms that the TTAGGG tracts are cleaved into small fragments due to the presence of this restriction enzyme site, in contrast to mouse telomeres. Additional blocks of (TTAGGG)n repeats are found 4–5 kb internally on the clone. The terminal region of the clone is dominated by a novel A-T rich 78 bp tandemly repeating sequence; the repeat monomer can be subdivided into halves distinguished by more or less adherence to the consensus sequence. The sequence in genomic DNA has the same tandem organization in probably a single primary locus of >20–30 kb and is thus termed a minisatellite.     

Cloning and sequence analysis of linear plasmid telomeres of the bacterium Borrelia burgdorferi

Borrelia burgdorferi, the Lyme disease agent, has double-stranded linear plasmids with covalently closed ends. DNA at the ends, or telomeres, of two linear plasmids of B. burgdorferi strain B31 was examined. Telomeric sequences from both ends of a 16 kb linear plasmid and from one end of a 49 kb linear plasmid were cloned and sequenced. An 18 bp AT-rich inverted repeat was found at each end of the 16 kb linear plasmid. The sequences of the two ends of this plasmid were different beyond these short inverted terminal repeats. The cloned end of the 49 kb linear plasmid had sequence identity with one end of the 16 kb linear plasmid. The end sequence common to both plasmids contained a series of phased, short direct repeats and a 52 bp palindrome adjacent to a highly AT-rich region. These findings indicate that Borrelia linear plasmid telomeres have structural features different from those of other known replicons.     

Dynamics of telomere length variation in Tetrahymena thermophila

We have analyzed the mechanism and dynamics of telomere length variation in the macronucleus of Tetrahymena thermophila. In a newly differentiated macronucleus, the average length of the telomeric repeated sequence, (C4A2 X T2G4)n, is closely regulated. In contrast, in vegetatively dividing cells in log phase, all macronuclear telomeric sequences lengthen coordinately by 3-10 bp per generation until up to 1000 bp are added. In both elongated and short telomeres, characteristic single-stranded breaks on both strands are distally located. Reduction of elongated telomeres to their original length involves either the appearance of a novel type of variant cell, incapable of net telomere elongation, or, under stationary phase conditions, a reversible removal of telomeric sequences. The demonstration that telomeres are dynamic structures provides evidence for a model of telomere length regulation by activities that add and remove telomeric repeats.     

Organization of DNA sequences and replication origins at yeast telomeres

We have shown that the DNA sequences adjacent to the telomeres of Saccharomyces cerevisiae chromosomes are highly conserved and contain a high density of replication origins. The salient features of these telomeres can be summarized as follows. There are three moderately repetitive elements present at the telomeres: the 131 sequence (1 to 1.5 kb), the highly conserved Y sequence (5.2 kb), and the less conserved X sequence (0.3 to 3.75 kb). There is a high density of replication origins spaced about 6.7 kb apart at the telomeres. These replication origins are part of the X or the Y sequences. Some of the 131-Y repetitive units are tandemly arranged. The terminal sequence T (about 0.33 to 0.6 kb) is different from the 131, X, or Y sequences and is heterogeneous in length. The order of these sequences from the telomeric end towards the centromere is T-(Y-131)n-X-, where n ranges from 1 to no more than 4. Although these telomeric sequences are conserved among S. cerevisiae strains, they show striking divergence in certain closely related yeast species.     

Stability of telomeric G-quadruplexes

In most eukaryotes, telomeric DNA consists of repeats of a short motif that includes consecutive guanines and may hence fold into G-quadruplexes. Budding yeasts have telomeres composed of longer repeats and show variation in the degree of repeat homogeneity. Although telomeric sequences from several organisms have been shown to fold into G-quadruplexes in vitro, surprisingly, no study has been dedicated to the comparison of G-quadruplex folding and stability of known telomeric sequences. Furthermore, to our knowledge, folding of yeast telomeric sequences into intramolecular G-quadruplexes has never been investigated. Using biophysical and biochemical methods, we studied sequences mimicking about four repetitions of telomeric motifs from a variety of organisms, including yeasts, with the aim of comparing the G-quadruplex folding potential of telomeric sequences among eukaryotes. G-quadruplex folding did not appear to be a conserved feature among yeast telomeric sequences. By contrast, all known telomeric sequences from eukaryotes other than yeasts folded into G-quadruplexes. Nevertheless, while G(3)T(1-4)A repeats (found in a variety of organisms) and G(4)T(2,4) repeats (found in ciliates) folded into stable G-quadruplexes, G-quadruplexes formed by repetitions of G(2)T(2)A and G(2)CT(2)A motifs (found in many insects and in nematodes, respectively) appeared to be in equilibrium with non-G-quadruplex structures (likely hairpin-duplexes).