Yoon CH et al. (1996) Worm Breeder's Gazette "Genetic interactions between sli-1 and let-60 ras."

Sternberg PW, Yoon CH

[Worm Breeder's Gazette, 1996]

sli-1 is a negative regulator of let-23-mediated vulval induction and encodes a protein similar to the mammalian proto-oncoprotein c-Cbl (1). Reduction-of-function (rf) mutations in sli-1 suppress the vulvaless (Vul) phenotype associated with severe reduction-of-function, but not null, mutations of let-23 (2). let-60 ras acts downstream in the signal transduction process initiated by LET-23. Initial studies have shown that sli-1 has only a weak suppression of the slight Vul phenotype associated with the let-60(rf) mutation, n2021 (2). We have furthered this study to more exactly determine how sli-1 interacts with let-60. We used four severe let-60(rf) alleles defined by Beitel et al.(3): n1876, n2022, n2034, n2035. Animals bearing any of these four let-60(rf) alleles display partial lethality as segregants from heterozygous mothers. Lethality is essentially 100 % for F2 homozygotes of these four alleles, indicating that the partial penetrance of lethality in F1 homozygotes is due to maternal rescue from the let-60(+) wild-type copy in the heterozygous mothers. We scored vulval induction in F1 homozygotes of these four let-60(rf) alleles in sli-1(+) and sli-1(rf) backgrounds. We used the sli-1(rf) reference allele, sy143. We find that sli-1(sy143) does not suppress the Vul phenotype associated with severe let-60(rf) mutations. For example, n2035 is suppressed from 0.016 induced VPCs/animal (n=62) in a sli-1(+) background to 0.034 induced VPCs/animal (n=87) in a sli-1(sy143) background.

Magnenat L et al. (1995) Worm Breeder's Gazette "IN VITRO EVIDENCES FOR TELOMERASE-MEDIATED CHROMOSOME HEALING IN NEMATODES"

Mueller F, Tobler H, Nilsen TW, Magnenat L

[Worm Breeder's Gazette, 1995]

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.

Riddle DL et al. (1982) Worm Breeder's Gazette "a few notes on dauers."

Riddle DL, Golden JW

[Worm Breeder's Gazette, 1982]

To be or not to be (a dauer)? We previously reported that the decision between formation of an L3 or a dauer is determined by the relative concentrations of a Caenorhabditis-specific, fatty acid-like pheromone and a competitive 'food-signal.' Dauer pheromone shift experiments were performed by starting with synchronized L1's and shifting into or out of pheromone at various points in the life cycle ( worms are always on a lawn of OP50). Commitment to the dauer occurs a few hours before the L2 molt when removal of the worms from pheromone fails to rescue them from entering the dauer stage. The committed worms enter the L2-dauer molt and progress through at least the dauer- shrinkage stage before recovering. Larvae grown in the absence of added pheromone become committed to the L3 by the end of the L1 molt, after which dauer larva formation cannot be induced by a shift into pheromone. Growth in the presence of pheromone causes a specific 6 hour (at 25 C) elongation of the L2 intermolt which, along with subtle morphological differences, indicates that an L2 destined to become a dauer is undergoing a distinct developmental program. N2 dauer formation is temperature-dependent. Pheromone-induced dauer formation of N2 on a lawn of OP50 is enhanced at higher temperatures in the normal growth range with a transition centered around 22.5 C. Temperature-shift experiments of synchronized populations show transitions of both up-shifts and down-shifts centered at the L1 molt. This suggests that temperatures above 22 C are an environmental cue favoring dauer formation, or that the decision to enter the dauer developmental pathway has a temperature- dependent component. Dauer recovery in the presence of pheromone is also temperature-dependent; 85% recovery was induced by a 24 hour temperature down-pulse (25 C to 15 C ). Putative null alleles with a temperature-sensitive phenotype. The information given above along with other evidence suggested to us that some of our ts dauer-constitutive mutants might be null alleles which uncover and enhance the wild-type ts pheromone response. All of the dauer-constitutives, with the possible exception of daf-2, are hyperresponsive to the dauer pheromone. The ts phenotype of many of the dauer-constitutives closely resembles the ts characteristics of N2 dauer formation. One hypothesis explaining these data is that some of the dauer constitutives are unable to smell 'food' and, therefore, overrespond to pheromone, and this defect could be the result of null alleles. We have tested all of the dauer-constitutive reference alleles and all alleles of daf-4 and daf-7 for suppression by sup-7( st5). We found 2 ts alleles, daf-4(m72) and daf-7(m62), which are suppressed. No non-ts alleles of daf-4 or daf-7 have been found. A mutant which fails to secrete the dauer pheromone. Mutant DR476, daf-22(m130)II produces the pheromone, but fails to secrete it. The mutant was obtained by assaying medium from F2 clones for the absence of pheromone. Pheromone concentration in liquid cultures of the mutant is less than 1% of N2 levels up to and including the day of starvation, but then increases to near wild-type levels a few days after starvation, presumably because of worm Iysis. DR476 obtained from nonstarved plates was homogenized and found to contain an amount of pheromone similar to N2. Pheromone bioassays of single worms were used to show that the mutation is recessive. In both liquid and plate cultures the mutant is dauer-defective, producing only a few dauers which appear later than normal. While the dauer-defective phenotype of DR476 is completely reversed by the addition of exogenous pheromone, no other dauer-defective can be induced to form dauers in this way. The dauer-pheromone is not a single compound. Thin-layer chromatography of pheromone, partially purified by organic extraction of starved liquid medium followed by chromatography of the extract on DEAE-cellulose and silicic acid columns, consistently results in a 'streak' of activity. However when portions of the streak are rerun in the same solvent system they run at their original Rf without restreaking. The activity of different fractions is additive without synergistic effects. Hypotheses which would explain the heterogeneity are (1) that the pheromone may be a mixture of variable length fatty- acids modified in the same way or (2) variably modified forms of a fatty acid may have roughly equivalent biological activities.