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 micronuclear sequences that function as telomeres in yeast.

We explored the ability of S. cerevisiae to utilize heterologous DNA sequences as telomeres by cloning germline (micronuclear) DNA from Tetrahymena thermophila on a linear yeast plasmid that selects for telomere function. The only Tetrahymena sequences that functioned in this assay were (C4A2)n repeats. Moreover, these repeats did not have to be derived from Tetrahymena telomeres, although we show that micronuclear telomeres (like macronuclear telomeres) of Tetrahymena terminate in (C4A2)n repeats. Chromosome-internal restriction fragments carrying (C4A2)n repeats also stabilized linear plasmids and were elongated by yeast telomeric repeats. In one case, the C4A2 repeat tract was approximately 1.5 kb from the end of the genomic Tetrahymena DNA fragment that was cloned, but this 1.5 kb of DNA was missing from the linear plasmid. Thus, yeast can utilize internally located tracts of telomere-like sequences, after the distal DNA is removed. The data provide an example of broken chromo-some healing, and underscore the importance of the telomeric repeat structure for recognition of functional telomeric DNA in vivo.     

The cohering telomeres of Oxytricha.

We have studied the process by which purified Oxytricha macronuclear DNA associates with itself to form large aggregates. The various macronuclear DNA molecules all have the same terminal or telomeric DNA sequences that are shown below. 5' C4A4C4A4C4--mean length----G4T4G4T4G4T4G4T4G4 G4T4G4T4G4T4G4T4G4-----2.4 kb------C4A4C4A4C4. When incubated at high concentrations, these telomeric sequences cohere with one another to form an unusual structure--one that is quite different from any DNA structure so far described. The evidence for this is the following: 1) These sequences cohere albeit slowly, in the presence of relatively high concentrations of Na+, and no other cation tested. This contrasts with the rapid coherence of complementary single-chain terminals of normal DNA (sticky ends) which occurs in the presence of any cation tested. 2) If the cohered form is transferred into buffers containing a special cation, K+, it becomes much more resistant to dissociation by heating. We estimate that K+ increases the thermal stability by 25 degrees or more. The only precedent known (to us) for a cation-specific stabilization is that seen in the quadruplex structure formed by poly I. The thermal stability of double helical macronuclear DNA depends on the cation concentration, but not the cation type. Limited treatment with specific nucleases show that the 3' and 5'-ended strands are essential for the formation of the cohering structure. Once in the cohered form, the telomeric sequences are protected from the action of nucleases. Coherence is inhibited by specific, but not by non-specific, synthetic oligomers, and by short telomeric fragments with or without their terminal single chains. We conclude that the coherence occurs by the formation of a novel condensed structure that involves the terminal nucleotides in three or four chains.     

Internal sequences are eliminated from genes during macronuclear development in the ciliated protozoan Oxytricha nova

During the life cycle of the hypotrichous ciliate Oxytricha nova, a macronucleus containing short, gene-sized DNA molecules is produced from a copy of the chromosomal micronuclear genome. In order to characterize the process of macronuclear development, we have isolated and determined the DNA sequence of a particular macronuclear gene and its micronuclear precursor. The results of this analysis indicate that macronuclear telomeric sequences (5'C4A4(3') repeats) are not present at the ends of the gene in its micronuclear chromosomal location and must be added during development. In addition, the micronuclear copy of the gene contains three short blocks of sequence that must be removed during development, implying the involvement of a nucleic acid-splicing process in generating mature macronuclear genes.     

Telomere structure in Euplotes crassus: characterization of DNA-protein interactions and isolation of a telomere-binding protein.

The nucleoprotein structure of telomeres from Euplotes crassus was studied by using nuclease and chemical footprinting. The macronuclear telomeres were found to exist as DNA-protein complexes that are resistant to micrococcal nuclease digestion. Each complex encompassed 85 to 130 base pairs of macronuclear DNA and appeared to consist of two structural domains that are characterized by dissimilar DNA-protein interactions. Dimethyl sulfate footprinting demonstrated that very sequence-specific and salt-stable interactions occur in the most terminal region of each complex. DNase I footprinting indicated that DNA in the region 30 to 120 base-pairs from the 5' end lies on a protein surface; the interactions in this region of the complex are unlikely to be sequence specific. A 50-kilodalton telomere-binding protein was isolated. Binding of this protein protected telomeric DNA from BAL 31 digestion and gave rise to many of the sequence-specific DNA-protein interactions that were observed in vivo. The telomeric complexes from E. crassus were very similar in overall structure to the complexes found at Oxytricha telomeres. However, telomeric complexes from the two ciliates showed significant differences in internal organization. The telomeric DNA, the telomere-binding proteins, and the resultant DNA-protein interactions were all somewhat different. The telomere-binding proteins from the two ciliates were found to be less closely conserved than might have been expected. It appears that the proteins are tailored to match their cognate telomeric DNA.     

Telomere proteins: specific recognition and protection of the natural termini of Oxytricha macronuclear DNA

The macronuclear DNA in the ciliated protozoan O. nova consists of integral of 10(7) gene-sized DNA molecules, all of which terminate with 20 bp of C4A4 repeats followed by a 3' (G4T4)2 single-stranded tail. Two immunologically distinct proteins of 55 and 26 kd, which are tenaciously, but noncovalently associated with Oxytricha macronuclear DNA termini, have been purified. These proteins protect DNA termini from degradation by the exonuclease Bal31. They also facilitate retention of natural and synthetic telomeric DNAs onto nitrocellulose. The Oxytricha proteins are not simply C4A4-binding proteins. Rather, their efficient binding requires both the 3' single-stranded (G4T4)2 tail and the adjacent duplex region. Thus, these proteins require both the sequence and the structure of natural DNA termini for efficient binding. As such they represent the first described example of telomeric-specific proteins.     

Organization of gene and non-gene sequences in micronuclear DNA of Oxytricha nova.

In order to study the derivation of the macronuclear genome from the micronuclear genome in Oxytricha nova micronuclear DNA was partially digested with EcoRI, size fractionated, and then cloned in the lambda phage Charon 8. Clones were selected a) at random b) by hybridization with macronuclear DNA or c) by hybridization with clones of macronuclear DNA. One group of these clones contains only unique sequence DNA, and all of these had sequences that were homologous to macronuclear sequences. The number of macronuclear genes with sequences homologous to these micronuclear clones indicates that macronuclear sequences are clustered in the micronuclear genome. Many micronuclear clones contain repetitive DNA sequences and hybridize to numerous EcoRI fragments of total micronuclear DNA, yielding similar but non-identical patterns. Some micronuclear clones containing these repetitive sequences also contained unique sequence DNA that hybridized to a macronuclear sequence. These clones define a major interspersed repetitive sequence family in the micronuclear genome that is eliminated during formation of the macronuclear genome.     

DNA recognition and binding by the Euplotes telomere protein.

The 51-kDa telomere protein from Euplotes crassus binds to the extreme terminus of macronuclear telomeres, generating a very salt-stable telomeric DNA-protein complex. The protein recognizes both the sequence and the structure of the telomeric DNA. To explore how the telomere protein recognizes and binds telomeric DNA, we have examined the DNA-binding specificity of the purified protein using oligonucleotides that mimic natural and mutant versions of Euplotes telomeres. The protein binds very specifically to the 3' terminus of single-stranded oligonucleotides with the sequence (T4G4) > or = 3 T4G2; even slight modifications to this sequence reduce binding dramatically. The protein does not bind oligonucleotides corresponding to the complementary C4A4 strand of the telomere or to double-stranded C4A4.T4G4-containing sequences. Digestion of the telomere protein with trypsin generates an N-terminal protease-resistant fragment of approximately 35 kDa. This 35-kDa peptide appears to comprise the DNA-binding domain of the telomere protein as it retains most of the DNA-binding characteristics of the native 51-kDa protein. For example, the 35-kDa peptide remains bound to telomeric DNA in 2 M KCl. Additionally, the peptide binds well to single-stranded oligonucleotides that have the same sequence as the T4G4 strand of native telomeres but binds very poorly to mutant telomeric DNA sequences and double-stranded telomeric DNA. Removal of the C-terminal 15 kDa from the telomere protein does diminish the ability of the protein to bind only to the terminus of a telomeric DNA molecule.     

DNA primase and the replication of the telomeres in Oxytricha nova.

An enzymatic activity in crude extracts of macronuclei from the hypotrichous ciliate Oxytricha nova catalyzes the synthesis of RNA consisting of (C4A4)n using an oligodeoxynucleotide template of the telomeric sequence (dG4T4)n. Single-stranded (dG4T4)n is an effective template if it has a random sequence at its 5' end. The enzyme will not use a (dG4T4)n template of any length (up to 64 bases) if it lacks a random sequence at the 5' end. With a random, single-stranded sequence at the 5' end, the (dG4T4)n oligodeoxynucleotide must be at least 36 bases long to work as a template. A 16-base, single-stranded region of (dG4T4)2 is an effective template when joined to a 20-base double-stranded region of (dG4T4)n/(dA4dC4)n, a structural arrangement that is the same as the native telomere of Oxytricha macronuclear DNA. The RNA-synthesizing activity is unaffected by 1.0 mg/ml of alpha-amanitin. Macronuclear extracts have an alpha-amanitin-insensitive, RNA-polymerizing activity that can use a random 55mer oligodeoxynucleotide as a template. This enzyme activity may be the same one that uses (dG4T4)n templates to make (C4A4)n RNA. The (C4A4)n RNA made in the reaction can prime DNA synthesis by the E. coli DNA polymerase I Klenow fragment. Therefore, the RNA polymerase activity fulfills the requirements of the telomere DNA primase that we postulated for replication of telomeres in hypotrichs (Zahler and Prescott, 1988, Nucleic Acids Research 16, 6953-6972).     

Sequence characterization of Tetrahymena macronuclear DNA ends.

Tetrahymena is a ciliated protozoan which has two nuclei: a micronucleus, which maintains the genetic continuity of the cell, and the macronucleus which is derived from the micronucleus after sexual conjugation. A macronuclear DNA library was constructed to contain DNA ends. A probe containing C4A2 repeats which are known to be present at macronuclear DNA ends (1) was used to screen the library. Three clones were characterized by sequencing, restriction enzyme mapping and Bal 31 digestion. The data indicate that these three clones represent macronuclear DNA ends which were generated by DNA fragmentation during macronuclear formation. The sequencing data at the C4A2 repeat junction show a conserved sequence of five nucleotides, TTATT. Sequences further away show no obvious homologies except that they are highly enriched in AT. This structure is quite different from the subtelomeric sequences of other organisms.     

Multiple sequence versions of the Oxytricha fallax 81-MAC alternate processing family

The 81-MAC family consists of three sizes of macronuclear chromosomes in Oxytricha fallax. Clones of these and of micronuclear homologs have been classified according to DNA sequence into three highly homologous (95.9-97.9%), but distinct versions. Version A is represented by a micronuclear clone and by clones of two different-sized macronuclear chromosomes, showing that alternate processing of micronuclear DNA is responsible for the variety of sizes of macronuclear chromosomes. Three Internal Eliminated Sequences (IES's) are demonstrated in Version A micronuclear DNA. Two have been sequenced and show short, flanking direct repeats but no inverted terminal repeats. Version C micronuclear DNA has interruptions in the macronuclear homology which correspond closely to the Version A IES's. Whether they are true IES's is unknown because no Version C macronuclear DNA has been demonstrated. Version C micronuclear DNA may be "macronuclear-homologous" but "micronucleus-limited" and not "macronucleus-destined." Version B is represented by macronuclear DNA clones, but no micronuclear clones. Vegetative micronuclear aneuploidy is suggested. The possible role of micronuclear defects in somatic karyonidal senescence is discussed in light of the precise macronuclear chromosome copy controls demonstrated within the 81-MAC family. These controls apparently operate throughout karyonidal life to maintain 1) a constant absolute amount of 81-MAC sequences in the macronucleus and 2) a constant stoichiometry within the family, both according to version and chromosome size.     

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.     

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.     

A unique pattern of intrastrand anomalies in base composition of the DNA in hypotrichs

The 50 non-coding bases immediately internal to the telomeric repeats in the two 5′ ends of macronuclear DNA molecules of a group of hypotrichous ciliates are anomalous in composition, consisting of 61% purines and 39% pyrimidines, A>T (ratio of 44:32), and G>C (ratio of 17:7). These ratio imbalances violate parity rule 2, according to which A should equal T and G should equal C within a DNA strand and therefore pyrimidines should equal purines. The purine-rich and base ratio imbalances are in marked contrast to the rest of the non-coding parts of the molecules, which have the theoretically expected purine content of 50%, with A = T and G = C. The ORFs contain an average of 52% purines as a result of bias in codon usage. The 50 bases that flank the 5′ ends of macronuclear sequences in micronuclear DNA (12 cases) consist of ~50% purines. Thus, the 50 bases in the 5′ ends of macronuclear sequences in micronuclear DNA are islands of purine richness in which A>T and G>C. These islands may serve as signals for the excision of macronuclear molecules during macronuclear development. We have found no published reports of coding or non-coding native DNA with such anomalous base composition.     

Cloning and expression of genes for the Oxytricha telomere-binding protein: specific subunit interactions in the telomeric complex.

Telomeres of Oxytricha nova macronuclear chromosomes consist of a repeated T4G4 sequence, single-stranded at the 3' terminus, bound by a heterodimeric protein. The cloning of genes for the two polypeptides and their separate expression in E. coli have enabled evaluation of their individual contributions to DNA binding. The 56 kd alpha subunit binds single-stranded DNA by itself, one polypeptide per T4G4 block; multiple subunits can coat a (T4G4)n multimer. The derived amino acid sequence of alpha does not reveal any known DNA-binding motif, so it appears to represent a novel type of DNA-binding protein. The previously cloned 41 kd beta subunit does not by itself protect DNA from methylation, but is required along with alpha to recreate the pattern of methylation protection indicative of telomeres in vivo. The unusual ability of the protein to engage in two different interactions with the same telomeric DNA sequence might provide the versatility necessary for diverse telomere functions.     

Multiple Sequence Versions of the Oxytricha fallax 81-MAC Alternate Processing Family1

The 81-MAC family consists of three sizes of macronuclear chromosomes in Oxytricha fallax. Clones of these and of micronuclcar homologs have been classified according to DNA sequence into three highly homologous (95.9–97.9%), but distinct versions. Version A is represented by a micronuclear clone and by clones of two different-sized macronuclear chromosomes, showing that alternate processing of micronuclear DNA is responsible for the variety of sizes of macronuclcar chromosomes. Three Internal Eliminated Sequences (IES's) are demonstrated in Version A micronuclcar DNA. Two have been sequenced and show short, flanking direct repeats but no inverted terminal repeats. Version C micronuclear DNA has interruptions in the macronuclear homology which correspond closely to the Version A IES's. Whether they are true IES's is unknown because no Version C macronuclear DNA has been demonstrated. Version C micronuclear DNA may be “macronuclear-homologous” but “micronucleus-limited” and not “macronucleusdestined.” Version B is represented by macronuclear DNA clones, but no micronuclear clones. Vegetative micronuclear aneuploidy is suggested. The possible role of micronuclear defects in somatic karyonidai senescence is discussed in light of the precise macronuclear chromosome copy controls demonstrated within the 81-MAC family. These controls apparently operate throughout karyonidai life to mairitain 1) a constant absolute amount of 81-MAC sequences in the macronuclcus and 2) a constant sioichiometry within the family, both according to version and chromosome size.     

Macrostructure of the tomato telomeres.

The macrostructure of the tomato telomeres has been investigated by in situ hybridization, genomic sequencing, and pulsed-field gel electrophoresis. In situ hybridizations with a cloned telomeric sequence from Arabidopsis thaliana indicated that the telomeric repeat of tomato cross-hybridizes with that of Arabidopsis and is located at all telomeres. Bal31 digestion kinetics confirmed that the tomato telomeric repeat represents the outermost DNA sequence of each tomato chromosome. Genomic sequencing of enriched tomato telomeric sequences, using primers derived from the Arabidopsis sequence, revealed that the consensus sequence of the tomato telomeric repeat is TT(T/A)AGGG compared with the Arabidopsis consensus sequence of TTTAGGG. Furthermore, as shown by pulsed-field gel electrophoresis, the telomeric repeat of tomato is separated by not more than a few hundred kilobases from a previously described 162-base pair satellite DNA repeat of tomato (TGR I) at 20 of the 24 telomeres. Together, these sequences are found in the heterochromatic terminal knob observed in pachytene chromosomes. Therefore, these two repeats determine the structure of 20 of the 24 tomato chromosome ends over approximately 2% of the total chromosome length.     

Conserved DNA sequences adjacent to chromosome fragmentation and telomere addition sites in Euplotes crassus.

During the formation of a new macronucleus in the ciliate Euplotes crassus, micronuclear chromosomes are reproducibly broken at approximately 10 000 sites. This chromosome fragmentation process is tightly coupled with de novo telomere synthesis by the telomerase ribonucleoprotein complex, generating short linear macronuclear DNA molecules. In this study, the sequences of 58 macronuclear DNA termini and eight regions of the micronuclear genome containing chromosome fragmentation/telomere addition sites were determined. Through a statistically based analysis of these data, along with previously published sequences, we have defined a 10 bp conserved sequence element (E-Cbs, 5'-HATTGAAaHH-3', H = A, C or T) near chromosome fragmentation sites. The E-Cbs typically resides within the DNA destined to form a macronuclear DNA molecule, but can also reside within flanking micronuclear DNA that is eliminated during macronuclear development. The location of the E-Cbs in macronuclear-destined versus flanking micronuclear DNA leads us to propose a model of chromosome fragmentation that involves a 6 bp staggered cut in the chromosome. The identification of adjacent macronuclear-destined sequences that overlap by 6 bp provides support for the model. Finally, our data provide evidence that telomerase is able to differentiate between newly generated ends that contain partial telomeric repeats and those that do not in vivo.     

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.