Developmentally controlled telomere addition in wild-type and mutant paramecia.

Abstract

We analyzed sites of macronuclear telomere addition at a single genetic locus in Paramecium tetraurelia. We showed that in homozygous wild-type cells, differential genomic processing during macronuclear development resulted in the A surface antigen gene being located 8, 13, or 26 kilobases upstream from a macronuclear telomere. We describe variable rearrangements that occurred at the telomere 8 kilobases from the A gene. A mutant (d48) that forms a telomere near the 5' end of the A gene was also analyzed. This mutant was shown to create simple terminal deletions; telomeric repeats were added directly to the truncated wild-type A gene sequence. In both the mutant and wild-type cells, the telomeric sequences (a mixture of C4A2 and C3A3 repeats) were added to various sequences within a specific 200-to 500-base-pair region rather than to a single site. No similarities were found in the primary sequences surrounding the telomere addition sites. The mutation in d48 changed the region of telomere addition at the A gene locus; this is the first example in ciliates of a mutation that affects the site of telomere addition.Developmentally controlled formation of new telomeres occurs in diverse eucaryotes. There is cytological evidence for de novo telomere formation in Ascaris and the crustacean Cyclops (reviewed in reference 29) and molecular evidence for its occurrence in ciliated protozoans. Ciliates contain two functionally and morphologically distinct types of nuclei within a single cell. Both types, micronuclei and macronuclei, arise from a single diploid fertilization nucleus during a sexual event. In the ciliate Paramecium tetraurelia, the micronuclei remain diploid and are transcriptionally inactive, while the DNA in the transcriptionally active macronucleus is amplified to about 800C. The formation of the new macronucleus involves the loss and rearrangement of sequences from the germ line DNA as well as chromosomal fragmentation (reviewed in reference 5). Fragmentation of the micronuclear chromosomes during macronuclear development has been well documented in two classes of ciliates, the holotrichous ciliate Tetrahymena (1, 7) and the hypotrichous ciliates (reviewed in reference 16). The resulting linear macronuclear DNA fragments, which we will refer to as macronuclear chromosomes, have telomeric sequences added to their ends. Work by Preer and Preer (20) suggested that fragmentation of micronuclear chromosomes also occurs in P. tetraurelia.The telomeric sequences of several ciliates have been determined. The linear macronuclear chromosomes of two holotrichs, Tetrahymena and Glaucoma, terminate with (C4A2 T2G4) tandem repeats (4,14,31). Hypotrichous ciliates such as Oxytricha have C4A4 repeats on their macronuclear DNA ends (17). In both cases the telomeric repeats are highly homogeneous, with no sequence variation. Several lines of evidence from both holotrichs and hypotrichs support the idea that during macronuclear development telomeric repeats are added de novo to the nontelomeric sequence at the ends of new...

Lack of telomere shortening during senescence inParamecium.

“…The sharp bands corresponded to the expected chromosomal internal genomic restriction fragment upstream of the A gene region; the fainter, cross-hybridizing bands are probably derived from related surface antigen genes (10). The disperse, fuzzy band has been shown to be the telomeric restriction fragment possessing a terminal structure of 100-500 bp of telomeric G4T2/G3T3 repeats (16 Are the shortened macronuclear DNAs healed by addition of new, full-length telomeres, as has been shown for DNA transfected into Paramecium macronuclei a few fissions after autogamy (17)? As shown above, mean telomere length remains essentially constant throughout clonal aging, while the mean macronuclear chromosomal molecular weight decreases by >10-fold.…”

“…This strain is deleted for macronuclear surface antigen A gene sequences near the start of A gene transcription (15). Telomeric sequences have been shown to be added at this point of deletion during macronuclear development (16).…”

Induction of specific macronuclear developmental mutations by microinjection of a cloned telomeric gene in Paramecium primaurelia.

“…A second class of heterogeneity can be defined, which involves smaller differences: Some chromosomes show multiple telomeres, 5-15 kb apart, at one of their ends. Such is the case of the chromosome bearing the A surface antigen gene of P. tetraurelia (Forney and Blackburn 1988). In contrast, the telomere located -5 kb downstream of the G gene of P. phmaurelia is unique.…”

“…The other ends of both the 250-and the 480-kb chromosomes, however, show the multiple telomere structure (F. Caron, unpubl.). Finally, a third class of heterogeneity is found within each of the telomeres, where the telomeric repeats are added at different nucleotides within a region spanning 200-800 bp (Baroin et al 1987;Forney and Blackburn 1988). All three types of heterogeneity are found within caryonidal clones, that is, vegetative clones arising from a single macronuclear differentiation event.…”

Interstitial telomeres are hotspots for illegitimate recombination with DNA molecules injected into the macronucleus of Paramecium primaurelia.

“…The exact number of macronuclear chromosomes is hard to assess because of the multiple levels of heterogeneity of chromosome ends (Baroin et al, 1987;Forney and Blackburn, 1988;Caron and Meyer, 1989), and the different estimations of the complexity of the haploid genome (Gibson and Martin, 1971;Cummings, 1975;MacTavish and Sommerville, 1980). Nevetheless, the average size of the macronuclear chromosomes being -300 kb (Preer and Preer, 1979;Caron and Meyer, 1989), an estimation of 300-1000 haploid chromosomes per macronucleus would be fairly accurate.…”