Hawkins MG et al. (1993) Worm Breeder's Gazette "A New GATA-Factor Isolated from C. elegans"

McGhee JD, Hawkins MG

[Worm Breeder's Gazette, 1993]

Ges-1 ,a gut-specific esterase first expressed at the 4-8E stage of development is a convenient marker gene to study early intestine development in C. elegans. We are interested in the factors that bind to the control region of the ges-1 gene to bring about its regulation. Footprinting and band-shift experiments using nuclear extracts from embryos (V. Stroeher et al., submitted) have identified a number of protein factors that bind to a region of the ges-1 gene 1.0 -1.3 kB upstream of the ATG. Of particular interest is a region that contains two WGATAR motifs similar to the VPE2 elements identified in the C. elegans vitellogenin genes, which are also expressed in a gut specific manner (MacMorris et al, 1992. Mol. Cell Biol. 12, 1652-1662). Egan et al. (see abstracts of the 1993 International C. elegans meeting) have shown that these GATA motifs are important for the gut-specific expression of ges-1 .We therefore used multimerized GATA-containing double stranded oligonucleotides to screen approximately 300,000 plaques from a C. elegans, mixed stage cDNA expression library ( Okkema et a/., WBG Vol12 ,ii).One clone was identified on the criteria of DNA-protein binding. This clone was then used to isolate a number of apparently close to full length cDNA clones from an independent C. elegans mixed stage cDNA library. The full length cDNA is 1597 nucleotides in length, is SL1 trans-spliced and contains a putative open reading frame of 433 amino acids in length, ie. encoding a protein of approximately 47,000Mr. Sequence analysis has shown the protein to be similar to GATA-factors isolated from a diverse range of species including elt-1 (C. elegans, Spieth et al., 1991. Mol. Cell Biol. 11, 4651-4659), GATA-4 (Mice, Arceci et al., 1993. Mol. Cell Biol. 13, 2235-2246), GATA-3 (Xenopus, Zon etal., 1991. Proc. Natl. Acad. Sci. USA. 88, 10642-10646), GATA-2 (humans, Lee et al., 1991. J. Biol. Chem. 266, 16188-16192) and GATA-1 (chicken, Evans and Felsenfeld, 1989. Cell 58,877-885). The new GATA factor contains two putative zinc-fingers as shown below. The C-terminal zinc-finger shows a similarity of 76%-80% when compared to the C-terminal zinc-fingers of the GATA factors listed above. However, the N-terminal zinc-finger shows a lower degree of similarity when compared to all other known GATA-factors ie. it has the sequence C-V-K-C-N(16)-C-V-N-C instead of the conserved motif C-X-N-C-N(17)-C-N-X-C. Furthermore the inter-finger distance is different from other GATA-factors. Thus we do not know if the protein has two zinc-fingers or one zinc-finger and a pseudo zinc-finger. It has been shown with the other GATA-factors that the C-terminal finger is much more important for DNA binding. For example Ormichinski et al. (1993, Proc. Natl. Acad. Sci. USA. 90, 1676-1680) showed the C-terminal zinc-finger of Chick GATA-1 was necessary for sequence specific DNA-protein interaction. The important question is whether the new gene is involved in regulating ges-1 and we hope to approach this via Tc1 insertion mutants and studying expression patterns. We now have genomic clones but they have yet to be positioned onto the C. elegans physical map.

Vladimir V Bakaev et al. (2003) Worm Breeder's Gazette "An attempt to slow aging in C. elegans. 32. No positive effect of peroxide"

Vladimir V Bakaev, Lyudmila M Bakaeva

[Worm Breeder's Gazette, 2003]

The purpose of this study was to investigate the effect of hydrogen peroxide in water solutions on the nematode life span. In this experiment hydrogen peroxide was used in following dilutions: 100 mg/L, 10 mg/L, 1 mg/L, 0.1 mg/L, 0.01 mg/L and 0.001 mg/L. Three adult animals (3-5 days old) were kept in microtitre wells containing 0.5 ml of liquid medium (with E. coli and without hydrogen peroxide) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (without hydrogen peroxide in medium) every day (one worm in one well) beginning from third day. Then, beginning from 3rd day, these worms were transferred every day in next wells containing medium with hydrogen peroxide in any concentration. This investigation was carried out in temperature +21C and in the darkness. The obtained results are presented in the following table. Conclusion: If hydrogen peroxide solution was applied to C. elegans, it was not able to increase their mean longevity significantly in comparison with control.

Vladimir V Bakaev et al. (2003) Worm Breeder's Gazette "An attempt to slow aging in C. elegans. 31. No positive effect of glycerol"

Lyudmila M Bakaeva, Vladimir V Bakaev

[Worm Breeder's Gazette, 2003]

The purpose of this study was to investigate the effect of glycerol in water solutions on the nematode life span. In this experiment glycerol was used in following dilutions: 100 g/L, 10 g/L, 1 g/L, 100 mg/L, 10 mg/L, 1 mg/L and 0.1 mg/L. Three adult animals (3-5 days old) were kept in microtitre wells containing 0.5 ml of liquid medium (with E. coli and without glycerol) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (without glycerol in medium) every day (one worm in one well) beginning from third day. Then, beginning from 3rd day, these worms were transferred every day in next wells containing medium with glycerol in any concentration. This investigation was carried out in temperature +21C and in the darkness. The obtained results are presented in the following table.

Vladimir V Bakaev et al. (2003) Worm Breeder's Gazette "An attempt to slow aging in C. elegans. 30. No positive effect of kudesan"

Lyudmila M Bakaeva, Vladimir V Bakaev

[Worm Breeder's Gazette, 2003]

The purpose of this study was to investigate the effect of kudesan (a water-soluble medicine, containing 30.0 mg of ubiquinone and 4.5 mg of a-tocopherol in 1 mL) in water solutions on the nematode life span. In this experiment kudesan was used in following dilutions: 1000, 100, 10, 1.0 and 0.1 mg/L. Three adult animals (3 - 5 days old) were kept in microtitre wells containing 0.5 ml of liquid medium (with E. coli and without kudesan) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (without kudesan in medium) every day (one worm in one well) beginning from third day. Then, beginning from 3rd day, these worms were transferred every day in next wells containing medium with kudesan in any concentration. This investigation was carried out in temperature +21C and in the darkness. The obtained results are presented in the following table.

Golden JW (1980) Worm Breeder's Gazette "C elegans dauer larvae, and cyclic Amp."

Golden JW

[Worm Breeder's Gazette, 1980]

Cyclic AMP levels have been assayed in N2 synchronous cultures and starvation-induced dauer larvae as a first step in the analysis of hormonal involvement in dauer larva formation, maintenance, and recovery. N2 worms are synchronized by egg purification (after the method of Ralph Hecht), hatching into M9 buffer, and resuspension in liquid worm medium containing an adenyl cylase deletion mutant of E. coli. The worms are harvested at various time intervals by immediately microwaving to boiling and washing in glass distilled water. This is followed by lyophillization after which the dry weight is determined. The assay uses a cAMP binding protein (Calbiochem) in a competitive binding assay with [3H]cAMP. After equilibrium is reached, the protein is filtered through a nitrocellulose filter and counted in a liquid scintillation counter. Initial results indicate that changes in cAMP concentration are correlated with the life cycle. Assays performed on well fed worms at various times in larval development show a linear decrease in cAMP levels from 25 pmol/mg dry weight in L2's to 15 pmol/mg dry weight in L3's, down to 1 pmol/mg dry weight in L4's. Starvation-induced dauer larvae have a cAMP level of 5 pmol/mg dry weight, or about one-third the level present at the L2 molt. Recovering dauers isolated 6 hours after the addition of food to starvation-induced dauers, show a dramatic drop in cAMP levels to less than 0.1 pmol/mg dry weight. The data collected to this point indicate that dauer larvae have a reduced level of cAMP compared to the L2 an' L3 larval stages and that recovery from the dauer stage results in cAMP levels below that found in L4's. These preliminary studies are to be followed by a more detailed study of cAMP and cGMP levels in N2 worms at all stages of the life cycle and in selected dauer-constitutive mutants. Cyclic GMP assays and pharmacological studies using theophylline as a phosphodiesterase inhibitor are currently being developed.

Starck J (1976) Worm Breeder's Gazette "RNA synthesis during oogenesis of C. elegans bergerac."

Starck J

[Worm Breeder's Gazette, 1976]

RNA synthesis has been studied during oogenesis by autoradiography. Allow incorporation to occur, ovary has been dissected out of gravid adults into Mg medium containing 3H uridine. RNA is synthesized actively until the end of meiotic prophase. This synthesis follows a classical model : label appears at first in the nucleus and after in the cytoplasm. This 'in vitro system' gives homogeneous results in each sample .

Burglin TR (1991) Worm Breeder's Gazette "A comment on ceh-"

Burglin TR

[Worm Breeder's Gazette, 1991]

Recently Nancy Hawkins and Jim McGhee published their four homeobox- containing genes from C. elegans (1). One of their genes, ceh-9, did not have an obvious homologue in any other organism. However, a newly discovered sea urchin homeobox gene, TgHbox5, published in a relatively obscure journal (2) is similar to ceh-9. Comparison of the ceh-9 and TgHbox5 homeodomains showed that they are 65% identical, and that they have additional amino acids outside the homeodomain conserved. Clearly this type of homeodomain constitutes a novel class that has not yet been detected in vertebrates or flies, but certainly must be present in these organisms. Further sequencing should reveal if this new class has additional sequence motifs conserved outside the homeodomain. [See Figure 1]

Vladimir V Bakaev et al. (2002) Worm Breeder's Gazette "An attempt to slow aging in C. elegans. 27. A positive effect of acetylsalicylic acid"

Alexey V Shabalin, Lyudmila M Bakaeva, Vladimir V Bakaev

[Worm Breeder's Gazette, 2002]

The purpose of this study was to investigate the effect of acetylsalicylic acid in water solutions on the nematode life span. In this experiment acetylsalicylic acid was used in following dilutions: 1000, 100, 10, 1.0 and 0.1 mg/L. Three adult animals (3 - 5 days old) were kept in microtitre wells containing 0.5 ml of liquid medium (with E. coli and without acetylsalicylic acid) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (without acetylsalicylic acid in medium) every day (one worm in one well) beginning from third day. Then, beginning from 3rd day, these worms were transferred every day in next wells containing medium with acetylsalicylic acid in any concentration. This investigation was carried out in temperature +21C and in the darkness. The obtained results are presented in the following table. Conclusion: If acetylsalicylic acid solution was applied to C. elegans in concentration of 10 mg/L, it was able to increase significantly (P>0.05) their mean longevity in comparison with control to 83.96 percent.

Coles GC et al. (1980) Worm Breeder's Gazette "Use of C. elegans for drug detection"

Simpkin KG, Coles GC

[Worm Breeder's Gazette, 1980]

By maintaining C. elegans on ampicillin treated E. coli a high throughput inexpensive screen for detecting potential anthelmintics is obtained. Method: 5 mg of ampicillin and 1.7 mg nystatin are added to 10 mls of E. coli broth (Uracil requiring mutant) and 100 mls of M9 buffer and the mixture stood for 2 hrs. Multi well plates are filled with 2 mls of the mixture and substances to be tested are added in the minimum volume of water, ethanol or DMSO. Between 12 and 20 worms of mixed age are added per well and after 7 days incubation at 20 C experimental wells are compared to controls for numbers of larvae and adults and normal movement. Most benzimidazoles are detected at 1 p.p.m. with the exceptions of oxfendazole (10 p.p.m.) and thiabendazole (2.5 p.p.m.). Levamisole is detected down to 0.5 p.p.m. and avermectin Bla to 0.0005 p.p.m. At 50 p.p.m. less than 10% of random compounds are active. Although older drugs such as piperazine , bephenium, diethylcarbamazine, methyridine and santonin would be missed this is not considered to be of significance as modern drugs must be both broadspectrum and very active. The test can also probably be used to detect potential plant nematocides.

Dew M et al. (1975) Worm Breeder's Gazette "DNase"

Sulston JE, Dew M

[Worm Breeder's Gazette, 1975]

E1392 (nuc-1,X) lacks the principal endodeoxyribonuclease. When determined on crude sonicates by an assay sensitive to endolytic cleavage of DNA (e.g. the spectrophotometric method of Kunitz, reviewed in Prog. in Nucl. Acid Res. 1 85 (ed Cantoni and Davies, 1967)), the DNase activity is well under 1% of that in N2. Feulgen staining reveals stranded material in the gut, persistent DNA from programmed cell deaths, and extruded polar bodies accumulating in the gonads of old animals; this suggests that a single endo-DNase is used both in the gut and in the tissues of C. elegans. There is no visible phenotype. The map position is: [See Figure 1] The endo-DNase of N2 is equally active in the presence of Mg++ and of EDTA