An unusual sequence arrangement in the telomeres of the germ-line micronucleus in Tetrahymena thermophila.

Abstract

The ciliated protozoan Tetrahymena thermophila contains two nuclei that differ dramatically in function, chromosome size and number, chromatin structure, and mode of division. It is possible that the telomeres of the two nuclei have different functions. Although macronuclear telomeric DNA has been well characterized and consists of tandem G4T2/C4A2 repeats that are synthesized by the enzyme telomerase, micronuclear telomeres have not been isolated previously. Here, we report the identification and cloning of micronuclear telomeres and demonstrate that although they contain the same terminal tandem G4T2 repeats as macronuclear telomeres, they are strikingly different in three respects. First, the tracts of G/C-rich telomeric repeats are approximately seven times longer in the micronucleus than in the macronucleus (approximately 2.0-3.4 vs. approximately 0.3-0.5 kb, respectively) from the same cell population. Second, the immediate telomere-associated sequences (TASs) from six different micronuclear chromosome ends have an unusually high G/C content and degree of homology to one another, unlike macronuclear TASs. The TAS from at least one micronuclear chromosome is unique to micronuclear telomeres and is not present in the macronucleus. Finally, and unexpectedly, all micronuclear telomere clones contain an inner homogeneous tract of a variant G4T3 repeat adjacent to the distal tract of G4T2 repeats. The native micronuclear telomeric DNA is composed of approximately 30% G4T3 repeats, corresponding to 0.6-1.0 kb per average telomere, positioned centromere-proximally to most or all of the G4T2 repeats. Neither the G4T3 sequence nor any other variant repeat is found in macronuclear telomeres. Furthermore, such a homogeneous tract of a variant repeat has not been found in the telomeres of any eukaryote.

Telomeric repeats (TTAGGC)n are sufficient for chromosome capping function in Caenorhabditis elegans.

“…This is in contrast to the subtelomeric sequences identified from six cloned Tetrahymena micronuclear telomeres, which are 55-87% identical to each other (20), and to the subtelomeric regions of Saccharomyces cerevisiae chromosomes, which often contain two types of subtelomeric repeats, X and Y' (30 Whereas the subtelomeric regions of the telomeric clones cTel3X, cTel54X, cTel55B, and cTell7X contain single-copy DNA sequences, the sequences adjacent to the TTAGGC repeats in cTel4X, cTel5.3B, cTel7X, cTel33B, and cTel29B are repeated once to several times at nontelomeric sites. The telomeres corresponding to the clones cTel4X, cTel79B, and cTel52S have tandemly repeated sequences in their subtelomeric regions (see Fig.…”

“…However, the number of TTAGGC sequences in the different clones varied from only 20 to 53 repeats, likely the result of deletion events during the cloning process in E. coli. (20). The G-rich strand of the telomeric repeats was oriented 5' to 3' toward the end of the insert, corresponding to the defined orientation of eukaryotic telomeric sequences The second column indicates the number of independently isolated clones from the three end libraries and in brackets the number of partially sequenced clones.…”

A functional telomerase RNA swap in vivo reveals the importance of nontemplate RNA domains

“…Libraries were screened first with the G 4 T 3 -specific oligonucleotide probe and reprobed with the G 4 T 2 -specific probe. Plasmids containing G 4 T 3 and G 4 T 2 repeat tracts provided positive controls (17). Extent of G 4 T 3 probe hybridization was Ͼ10 fold in Tt.ter1-43A telomere clones compared with Gc.ter1-43A telomere clones, likely resulting from better probe hybridization to longer variant repeat tracts present in the Tt.ter1-43A telomeric clones vis-à-vis shorter tracts in Gc.ter1-43A clones.…”

“…Southern blot detection of contiguous G 4 T 3 and G 4 T 2 telomeric repeat tracts from Glaucoma and Tetrahymena telomerase RNA transformant genomic DNAs (PstI-cut) was performed as described (17). The G 4 T 3 repeat probe used was 5Ј-TTT(GGGGTTT) 3 , and the G 4 T 2 probe was 5Ј-GTT(GGGGTT) 3 G. TER1 gene dosage studies were determined using duplicate Southern blots of PstI-cut genomic DNA prepared from Tetrahymena and Glaucoma TER1 and ter1-43A transformants probed with Tt.TER1 or Gc.TER1 gene probes.…”

“…PstI-cut transformant genomic DNAs were separated by 0.7% agarose gel electrophoresis and subjected to Southern blot analysis. Transformant macronuclear rDNA telomeres were analyzed using either a G4T3-(lanes 1-4) or a G4T2-specific (lanes 5-8) oligonucleotide probe (17). Lanes 5-8 are duplicate lanes to lanes 1-4.…”