High incidence of rapid telomere loss in telomerase-deficient Caenorhabditis elegans

Abstract

Telomerase is essential to maintain telomere length in most eukaryotes. Other functions for telomerase have been proposed but molecular mechanisms remain unclear. We studied Caenorhabditis elegans with a mutation in the trt-1 telomerase reverse transcriptase gene. Mutant animals showed a progressive decrease in brood size and typically failed to reproduce after five generations. Using PCR analysis to measure the length of individual telomere repeat tracks on the left arm of chromosome V we observed that trt-1 mutants lost approximately 125bp of telomeric DNA per generation. Chromosome fusions involving complex recombination reactions were observed in late generations. Strikingly, trt-1 mutant animals displayed a high frequency of telomeres with many fewer repeats than average. Such outlying short telomeres were not observed in mrt-2 mutants displaying progressive telomere loss very similar to trt-1 mutants. We speculate that, apart from maintaining the average telomere length, telomerase is required to prevent or repair sporadic telomere truncations that are unrelated to the typical 'end-replication' problems.

Figure 1

Telomere length heterogeneity in wild-type C.elegans. (A) Telomere length fluctuations in the wild-type strain N2. Telomere length of VL was measured by STELA at F1, F5, F9, F13 and F17. DNA was extracted from five reproductive-stage adult sampled at each of the generations. DNA was ligated to the telorette and 0.1 worm equivalent was used in each PCR. Marker lane is shown on the left and the corresponding telomere length is indicated on the right. Actual telomere length was 1.1 kb shorter than the size of the PCR product because 1.1 kb of subtelomeric sequences were also amplified. (B) Telomere length heterogeneity in a clonal population. A N2 parent and 10 of its progeny were analyzed by STELA. DNA was extracted from each single worm, ligated to telorette, and the entire DNA sample was used as template in PCR. Each lane represents a single worm.

Figure 2

Progressive telomere shortening in trt-1. Trt-1 was outcrossed to N2 males. From 2 heterozygous parents, 16 homozygous trt-1 lines were set up separately. For each generation, the parent (post-reproductive stage) was analyzed by STELA, starting from F2 and ending at the generation that became sterile. Generation numbers are indicated on top of each panel; the heterozygous parent is considered as Po. A number was assigned for each line (1–16) and it is shown at the top right corner of each panel.

Figure 3

Short outlying telomeres are readily observed in the DNA from single trt-1 mutants but not in the DNA from five mrt-2 mutants. (A–D) Eight to sixteen reproductive-stage individual progeny from four separate trt-1 parents were analyzed by STELA. Each lane represents a single worm. The two clusters of bands in most worms likely represent the two alleles of VL. In order to maintain the strain, trt-1 has to be routinely outcrossed. The longer VL telomere could be derived from the wild-type parent and the shorter VL telomere from the trt-1 parent. (E) DNA was extracted from five worms from the same mrt-2 parent in each of F2 (lane 1), F5 (lane 2) and F9 (lane 3) and subsequently used in STELA. A DNA equivalent of one worm was used as the template in each PCR.

Figure 4

Structure of telomere fusions isolated from trt-1. Eight clones of telomere fusions amplified from nested PCR were sequenced and classified into three categories. Category I consists of simple end-to-end fusions. White block arrow denotes the sequence between the inner primer and the beginning of telomeric DNA (represented by the wiggly line). Hatched block arrow denotes sequence between the inner primer and the outer primer. The presence of microhomology or insertion at the fusion junction (shown by an arrow head) is also indicated. In clone 3, 25 telomeric repeats were present at the fusion junction. Category II consists of complicated rearrangements in which a fragment from an internal VL site (denoted by a black block arrow) was inserted between the two ends. In clone 4, the black block arrow represents a 3.8 kb fragment found within the cosmid Y39D8B (∼360 kb from the VL telomere). In clone 5, the black block arrow represents a 0.47 kb fragment found within the cosmid B0348 (∼16 kb from the VL telomere). In clone 6, it represents a 0.73 kb fragment from cosmid B0348 that overlaps with the 0.47 kb fragment inserted in clone 5. The inserted fragments in clones 4–6 all contained imperfect telomeric repeats around the region where it joined the telomeric repeats (represented by wiggly line). In clone 7, the black block arrow represents a 50 bp fragment found in the cosmid C39F7 (1.2 Mb from the VL telomere). This fragment is interspersed with imperfect telomeric repeats. It was inserted in an opposite orientation as the other clones in this category. Category III consists of a clone which involved complicated rearrangement of the VL subtelomeric sequence. Within the fragment between the inner primer and the beginning of telomeric DNA, a 172 bp sequence is replicated 2.5 times (indicated by the division of the block arrow into three parts in gradient) but the replicates carry several mismatches. In clone 8, a sequence (denoted by the small orange block arrow) that overlaps with two of the replicates was inserted between two chromosome ends, one of which was truncated within the 10 bp fragment as in clones 1–3 in category I. Although only the nested primer was used in secondary PCRs, most clones (1–3, 7 and 8) were flanked by the outer primer sequence and the nested primer sequence, indicating that the low concentration of the outer primer in the secondary PCRs was enough to prime amplification with the more abundant nested primer.