Sequence features of Oxytricha trifallax (class Spirotrichea) macronuclear telomeric and subtelomeric sequences

We sequenced and analyzed the subtelomeric regions of 1356 macronuclear "nanochromosomes" of the spirotrichous ciliate Oxytricha trifallax. We show that the telomeres in this species have a length of 20 nt, with minor deviations; there is no correlation between telomere lengths at the two ends of the molecule. A search for open reading frames revealed that the 3' and 5' untranslated regions are short, with a median length of approximately 130 nt, and that surprisingly there are no detectable differences between sequences upstream and downstream of genes. Our results confirm a previously reported purine bias in the first approximately 80 nucleotides of the subtelomeric regions, but with this larger data set we curiously detected a 10 bp periodicity in the bias; we relate this finding to the possible regulatory and structural functions these regions must serve. Palindromic sequences in opposing subtelomeric regions, although present in most sequences, are not statistically significant.     

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.     

Processing of double-strand breaks is involved in the precise excision of paramecium internal eliminated sequences

In ciliates, the development of the somatic macronucleus involves the programmed excision of thousands of internal eliminated sequences (IES) scattered throughout the germ line genome. Previous work with Tetrahymena thermophila has suggested that excision is initiated by a staggered double-strand break (DSB) at one IES end. Nucleophilic attack of the other end by the 3'OH group carried by the firstly broken chromosome end leads to macronuclear junction closure. In this study, we mapped the 3'OH and 5'PO(4) groups that are developmentally released at Paramecium IES boundaries, which are marked by two conserved TA dinucleotides, one of which remains in the macronuclear genome after excision. We show that initiating DSBs at both ends generate 4-base 5' overhangs centered on the TA. Based on the observed processing of the 5'-terminal residue of each overhang, we present a new model for the precise closure of macronuclear chromosomes in Paramecium tetraurelia, different from that previously proposed for tetrahymena. In our model, macronucleus-destined broken ends are aligned through the partial pairing of their 5'-nTAn-3' extensions and joined after trimming of the 5' flaps.     

Developmentally regulated initiation of DNA synthesis by telomerase: evidence for factor-assisted de novo telomere formation.

Telomerase serves a dual role at telomeres, maintaining tracts of telomere repeats and forming telomeres de novo on broken chromosomes in a process called chromosome healing. In ciliates, both mechanisms are readily observed. Vegetatively growing cells maintain pre-existing telomeres, while cells undergoing macronuclear development fragment their chromosomes and form telomeres de novo. Here we provide the first evidence for developmentally regulated initiation of DNA synthesis by telomerase. In vitro assays were conducted with telomerase from vegetative and developing Euplotes macronuclei using chimeric primers that contained non-telomeric 3' ends and an upstream stretch of telomeric DNA. In developing macronuclei, chimeric primers had two fates: nucleotides were either polymerized directly onto the 3' terminus or residues were removed from the 3' end by endonucleolytic cleavage before polymerization began. In contrast, telomerase from vegetative macronuclei used only the cleavage pathway. Telomere repeat addition onto non-telomeric 3' ends was lost when developing macronuclei were lysed and the contents purified on glycerol gradients. However, when fractions from the glycerol gradient were added back to partially purified telomerase, telomere synthesis was restored. The data indicate that a dissociable chromosome healing factor (CHF) collaborates with telomerase to initiate developmentally programmed de novo telomere formation.     

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.     

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.     

Pheromone 4 gene of Euplotes octocarinatus.

We have cloned and sequenced a 1.7 kb macronuclear chromosome encoding the pheromone 4 gene of Euplotes octocarinatus. The sequence of the secreted pheromone is preceded by a 42 amino acid leader peptide, which ends with a lysine residue. The sequence coding for the leader peptide contains information for a putative signal peptide and is interrupted by a 772 bp intron as shown by comparison with a cDNA clone. A 64 bp intron and a 145 bp intron interrupt the sequence coding for the secreted pheromone. The three introns contain typical 5' and 3' splice junctions and a putative branch point site. The small introns have a low GC content. The large intron has a GC content similar to that of the pheromone 4 gene exons. The amino acid sequence of pheromone 4, deduced from both the genomic DNA and the cDNA of pheromone 4, shows that the secreted pheromone consists of 85 amino acids. One of its amino acids is encoded by a UGA codon. Since it has been shown for pheromone 3 of E. octocarinatus that UGA is translated as cysteine, it is assumed that the UGA codon encodes cysteine in pheromone 4 as well. The 164 bp noncoding region upstream of the leader peptide is AT-rich and contains an inverted repeat capable of forming a stem-loop structure with a stem of 11 bp. The 151 bp noncoding region at the 3' end of the chromosome contains a putative polyadenylation sequence and an inverted repeat. The macronuclear molecule is flanked by telomeres and carries the pentanucleotide motif TTGAA, located at a distance of 17 nucleotides from the telomeres. This motif has been suggested to be involved in the formation of macronuclear chromosomes.