Hypothesis 3: C21/C22 ceramides and sphingomyelins are essential second messengers that confer resistance to anoxia
IX. MATERIAL AND METHODS
IX.10. ENU mutagenesis
Synchronized young adults Po were mutagenized in M9 containing 10mM of N-nitroso-N-ethylurea (ENU) for 4 h at 20°C, then were let to recover into seeded plate O/N at 15°C. 5 mutagenized Po animals were places into 10cm plates containing 600µl of OP50 for a total of 33 plates. Each Po was let to lay about 100 F1 embryos (about one night at 20°C) and then removed of the plates. The F1 were let to lay about 100 embryos and then removed with M9. The pseudo synchronized F2 populations were then let to growth for 3 days at 20°C and then tested for 96 h of anoxia. We did
three independent mutagenesis testing about 59’600 haploid genomes (29’800 F1), representing about 3’000’000 of F2 animals.
X. ABBREVIATIONS
AC Acid ceramidase
hyl-1 and 2 Homolog of yeast longevity genes
HYP Hypoxia at high temperature (28°C)
KSR Kinase suppressor of RAS
lagr-1 LAG1 related gene
Lass gene = CerS Longevity assurance gene
LDHA Lactate dehydrogenase A
Ptdlns(3,4,5)P3 Phosphatidylinositol-3,4,5-trisphosphate
PTK Protein tyrosine kinase
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