Worm Breeder's Gazette 14(1): 89 (October 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Institute of Zoology, University of Fribourg, Perolles, CH-1700 Fribourg, Switzerland|
|2||School of Medicine, C.W.R.U., Cleveland, Ohio 44106, USA|
Telomerases are ribonucleoproteins that act as RNA-dependent DNA polymerases. Their function is to maintain the protecting telomeric extremities of the chromosomes after each round of replication and to heal broken chromosomes by adding telomeric repeats to them. Strong indirect evidences indicate that telomerase activity is involved in the process of chromatin diminution in Ascaris suum. This mechanism involves developmentally-programmed chromosomal cleavage at specific breakage regions (CBRs), new telomere formation (chromosomal healing) and degradation of the eliminated chromatin (F. Mueller et al., 1991. Cell 67: 815-822). Spontaneous chromosomal healing has been described for the C. elegans X-chromosome mutation me8, which disrupts meiotic crossing over and segregation (A. M. Villeneuve, 1994. Genetics 136: 887-902). Molecular analysis of the me8 mutation revealed that it represents a terminal chromosomal truncation healed by de novo addition of telomeric repeats directly to the site of breakage, most likely by the action of a telomerase (C. Wicky et al., submitted). Chromosomal healing in both nematodes share the same molecular characteristics, arguing for a similar molecular mechanism (S. Jentsch, pers. comm.; C. Wicky et al., in prep.) Developmentally-programmed healing in A. suum , however, is highly efficient and occurs in all presomatic cells, whereas the spontaneous chromosomal healing in C. elegans takes place randomly in germ cells with low efficiency. Thus, both nematode species A. suum and C. elegans represent interesting model systems to study telomerase activity and chromosome healing processes in metazoans. The aim of the present project is to investigate an in vitro telomerase activity in both nematode species. Therefore, the cell-free extracts were incubated with dNTPs and an oligonucleotide primer designed to serve as substrate for the telomerase enzyme. The elongated reaction products were detected by PCR, according to a modified TRAP (telomere repeat amplification protocol) assay (N.W. Kim et al., 1994. Science 266:2011-2015), using an oligonucleotide complementary to the telomeric repeats. The PCR products were separated on a denaturing polyacrylamide gel and visualised by autoradiography. In our Ascaris 4-cell stage extracts, the telomeric primer was elongated by up to 30 repeats of 6 bases. Because this activity was sensitive to heat inactivation, proteinase K or RNase A digestions, and depends on the time of incubation and the concentration of the extract in the reaction, we propose that it represents telomerase activity. Our data thus provide further strong evidence that telomerase is responsible for the developmentally-programmed healing process during chromatin diminution in Ascaris. Currently, different C. elegans extracts are tested for telomerase in vitro and preliminary results indicate a similar activity. Fractionation of the telomerase extracts will be useful to purify and characterize the protein and RNA components of the telomerase enzyme and specific co-factors involved in developmentally-programmed or spontaneous chromosomal healing. The cloning of the corresponding genes will accelerate the research on telomere function in multicellular organisms.