A H Jay Burr et al. (2001) International C. elegans Meeting "Light-increased reversal frequency and spontaneous reversal frequency are affected by starvation."

Ann Chan, Wendell Challenger, A H Jay Burr

[International C. elegans Meeting, 2001]

C elegans was shown previously to reverse direction in response to light (Burr 1985). In that study, 40-80% increases in reversal frequency over the background level varied with light intensity and wavelength in a normal way and were shown not to be due to radiant heating. Because large sample sizes are needed in order to detect small increases on top of the fluctuating frequency of spontaneous reversals, we have developed a computer automated tracking system that makes experimentation possible in a reasonable length of time. Under continuous near-infrared illumination, video images are digitized once each second and processed to find the new coordinates of up to 23 worms. Another program identifies reversals from the coordinates along the track. These are counted during a 10 s light period (1.6 x10 4 uW cm -2 blue-white broadband light) and a preceding 10 s dark period in each 40 s measurement cycle. There are 5 cycles per 200 s run. The number of reversals per 10 s measurement period, averaged over all worms and cycles during a run, counts as one independent measurement. Data are analyzed as a generalized randomized complete block design using the Proc Mixed procedure of SAS version 8. For detecting the effect of light, the difference in reversal frequency between the paired light and dark periods is analyzed in the same way. A new sample of worms was used for each run. About 30 synchronous six day-old, well-fed worms were washed off an OP50 culture plate and sedimented twice through behavior buffer (5.5 mM Tris + 34 mM NaCl). The worms were transferred in a drop of buffer to the edge of a 50-mm behavior plate and the plate was rotated to distribute the worms around the edge. The behavior plates were prepared by making 1.8% agar in 90% behavior buffer then drying to 90% of poured weight. Just before using, about 100 uL distilled water was distributed around the edge and allowed to soak in. The resulting lower osmolarity at the agar perimeter discouraged the worms from crawling near the edge. Worms were allowed to crawl into the 22x28 mm field of view and were tracked within 30-50 min of removal from the food. All procedures were carried out at 19 C. Sensitivity of the system under actual conditions was estimated by simulation, starting with data from an experiment with fed worms during which the light beam was blocked. Reversals were added to the data during the 'light' periods at frequencies of 0.01 to 0.10 per 10 s period (perP), and the data were reanalyzed. The increase in reversal frequency became significant at 0.050 perP (P = 0.037, n = 21 runs), a 45% increase over the background (dark period) frequency. A greater sensitivity would be expected at higher sample sizes. In all our experiments with well-fed worms, light had no significant effect on reversal frequency. To investigate the influence of starvation, we pooled worms from several food plates and put half on a food-free plate and half on a fresh food plate. After a starvation period, the fed and starved worms were tested in alternate runs. Light increased reversal frequency significantly in worms starved 20 h (+0.040 perP, +62%, P=0.049, n=51), but not significantly in worms starved 6 h (+0.013 perP, +12%, P = 0.4, n = 39). In both experiments, light had no significant effect on the fed controls (-0.023 perP, -15%, P = 0.2, n = 58 and -0.015 perP, -9%, P = 0.4, n = 42). In addition to being more responsive to light, starved worms had a significantly lower background (dark period) frequency. For the 0 h, 6 h and 20 h starved worms, average dark frequency (+/- SE) was 0.15 (0.013) perP, 0.11 (0.013) perP and 0.064 (0.012) perP, respectively. Thus, both background reversal frequency and responsiveness to light appear to be affected by starvation. In the previous study in which light responses were observed (Burr 1985), the worms may inadvertently have been starved.

Boule JB et al. (2005) Nature "The yeast Pif1p helicase removes telomerase from telomeric DNA."

Boule JB, Zakian VA, Vega LR

[Nature, 2005]

Telomeres are the physical ends of eukaryotic chromosomes. Genetic studies have established that the baker''s yeast Pif1p DNA helicase is a negative regulator of telomerase, the specialized reverse transcriptase that maintains telomeric DNA, but the biochemical basis for this inhibition was unknown. Here we show that in vitro, Pif1p reduces the processivity of telomerase and releases telomerase from telomeric oligonucleotides. The released telomerase is enzymatically active because it is able to lengthen a challenger oligonucleotide. In vivo, overexpression of Pif1p reduces telomerase association with telomeres, whereas depleting cells of Pif1p increases the levels of telomere-bound Est1p, a telomerase subunit that is present on the telomere when telomerase is active. We propose that Pif1p helicase activity limits telomerase action both in vivo and in vitro by displacing active telomerase from DNA ends.

Melissa M Harrison et al. (2004) East Coast Worm Meeting "Analysis of synMuv Protein Complexes in vivo and Characterization of the Class B synMuv Gene lin-61"

Melissa M Harrison, Bob Horvitz, Xiaowei Lu

[East Coast Worm Meeting, 2004]

Vulval induction in C. elegans is negatively regulated by at least three redundant functions provided by the synthetic Multivulva (synMuv) class A, B, and C genes. Loss-of-function mutations in members of any single class do not result in a Multivulva (Muv) phenotype, but animals with mutations in any two synMuv classes are Muv. The molecularly characterized synMuv class A genes encode novel proteins. Many of the class B and class C synMuv genes, including lin-35 Rb, dpl-1 DP, efl-1 E2F, lin-53 RbAp48, hda-1 HDAC, let-418 Mi-2, trr-1 TRRAP, mys-1 HAT, and epc- 1 E(Pc), have homologs in other species that are involved in chromatin remodeling and transcriptional modulation. Some of the proteins that act in the synMuv B pathway have been shown to physically interact in vitro (1,2), in yeast two-hybrid assays (3), or in vivo based on co-immunoprecipitation experiments (4). These results along with homology to proteins in other species suggest that many synMuv proteins may function together in transcriptional regulatory complexes. We are using co-immunoprecipitation experiments to further explore in vivo physical interactions among the synMuv proteins. Such studies may allow us to better understand the functions of novel synMuv proteins, to analyze the effects of various synMuv mutations on physical interactions, and to identify potential sub-complexes among the synMuv proteins. We have focused initially on the gene products of the class B synMuv genes lin-37 and lin-61 as well as the class A synMuv gene lin-56, since null mutants in these genes are viable and can serve as negative controls for the immunoprecipitation experiments. LIN-37 is a small hydrophobic protein with no known homologs outside of nematodes. LIN-61 contains four MBT ( m alignant b rain t umor) repeats, which are loosely defined sequences of approximately 100 amino acids found in a number of nuclear proteins including the Drosophila Polycomb group protein Sex Comb on Midleg. LIN-56 is a novel acidic protein with no canonical motifs. We have surveyed the interactions of these proteins with a number of class A and B synMuv proteins by immunoprecipitation followed by western blots and shown that a subset of class B proteins co-immunoprecipitate from embryo extract. We are developing reagents to perform immunoprecipitation studies of other synMuv proteins. We also hope to identify new proteins involved in vulval development on the basis of their interaction with the synMuv proteins by co-immunoprecipitations followed by mass spectrometry. We are also characterizing the class B synMuv gene lin-61 in detail as mutations in this gene cause a number of pleiotropic defects that differ from those of most other class B synMuv mutants. lin-61 loss-of-function alleles cause GFP transgene misexpression (see abstract by Schwartz, Wendell, and Horvitz), and Pothof et al. have shown that RNAi of lin-61 results in an increased mutation rate (5). We have made polyclonal antibodies that recognize LIN-61 and have shown that it is a ubiquitously expressed nuclear protein that is localized to condensed chromosomes in the germline. We have also shown that this localization remains unchanged in a large number of synMuv mutant backgrounds. (1) Ceol and Horvitz. Mol. Cell 7 : 461-473, 2001. (2) Lu and Horvitz. Cell 95 : 981-991, 1998. (3) Walhout et al. Science 287 : 116-122, 2000. (4) Unihavaithaya et al. Cell 111 : 991-1002, 2002. (5) Pothof et al. Genes Dev. 17 :443-448, 2003.