The effects of Malathion and the comparison to the NTE1 gene in yeast Ashley Swift Mentor: David Singleton Introduction : Malathion is a widely used organophosphorous insecticide in the U.S. that causes acute toxicity by targeting the nervous system in animals Organophosphates (OP) have been found to affect the neuropathy target esterase (NTE) enzyme. NTE is found in the nervous system of adult mammals. NTE1 is the yeast homolog of NTE. The two genes for these comparable esterases share many domain similarities and demonstrate similar methods of regulation. There is experimental evidence that NTE1 plays a role in lipid turnover in yeast. The actual role of NTE in mammalian cells in not completely clear but seems to be related to this as well. (4) Since the mammalian NTE and the yeast NTE1 share similar protein function, determining the proteins and genes that interact with NTE1 in yeast may help show the mechanism of how malathion damages mammalian cells. These interactions in yeast can be determined by seeing if other genes related to NTE1 are up- or down- regulated in response to malathion exposure. The objective: Find genes that respond by up-regulating or down-regulating when treated with malathion Review of Literature : OPs and NTE A clinical study comparing patients with a genetic defect from the NTE gene, to the phenotype found in OP toxicity (spastic par paresis with distal weakness). It was found that the patients with OP toxicity overlap the predominant localization of neurological signs in subjects with NTE mutation. This shows a direct correlation with defects of OPs and the NTE gene. (2) NTE1 role in yeast Phophatidycholine (PtdCho) is a major phospholipid component of eukaryotic membranes and is found to deacylated by the NTE1 gene. This allows for lipid homeostasis to occur. This article also found that the role of mammalian and Drosophila homologues seem to be similar. Found that the NTE1 null mutant yeast was viable under all conditions analyzed but mice and Drosophila tested died. In an invitro posphatdyl choline deacylation assay the yeast NTE1 protein, the Drosophila and the C. elegans proteins all demonstrated similar enzymatic specificities and activities. (1) Fig. 1. Model of the regulation of the CDP–choline pathway for phosphatidylcholine synthesis and its turnover by Nte1p. Sec14p negatively regulates PC synthesis by inhibiting Pct1p, and positively regulates PC catabolism by activating Nte1p mediated PC deacylation. Cho molecules formed by Gde1p are released into the cytoplasm where they can be used for resynthesis of PC by the CDP–Cho pathway. Solid lines indicate metabolic steps and dotted lines regulatory interactions. Results: Figure 1-RT-PCR gel. It compares different genes in the BY4741 and 511 strains both with and with out Malathion. Different genes are lane 1- ACT1, 2-HAC1, 3-HNM1, 4- IRE1, 5-NTE1, 6- SCS2, 7-SCS3. Lane with the letter M is a 500 base pair marker. Primer pairs were chosen from the yeast genome database. They were genes that corresponded with NTE1 gene function based on mass genomic screening in yeast. We will be looking at the band intensities and this correlates with the mRNA levels. For example in lanes 1 it is noticed that the band for ACT1 is more intense when treated with malathion. Literature Cited: 1. Fernandez-Murray, J, & McMaster, C. (2007). Phosphatidylcholine synthesis and its catabolism by yeast neuropathy target esterase 1. Molecular and Cell Biology of Lipids, 1771(3), doi: Rainier, S, Albers, J, Dyck, P, Eldevik, O, & Wilcock, S. (2011). Motor neuron disease due to neuropathy target esterase gene mutation: clinical features of the index families. Muscle Nerve, 43(1), doi: Read, D, Chao, M, Cavanagh, J, & Glynn, P. (2010). Organophosphates induce distal axonal damage, but not brain oedema, by inactivating neuropathy target esterase. Toxicol Appl Pharmacol, 245(1), doi: Zaccheo, O, Dinsdale, D, Meacock, P, & Glynn, P. (2004). Neuropathy target esterase and its yeast homologue degrade phosphatidylcholine to glycerophosphocholine in living cells. Journal of Biological Chemistry, 279(23), doi: Future studies: 1. To optimize the PCR to get product for the primer pairs that did not amplify-HAC1, HNM1, and SCS2. 2. Determine the quantification ratio with drug/without drug This will see if any genes are up- or down- regulated in the presence of Malathion 3. Lastly, determine if these changes are still comparable in the NTE1 mutant. If they disappear or are different in the NTE1, this will suggest that the mutant is somehow compensating for the loss of NTE1. Methods: Growing Yeast- Plated strains 551 and BY4741 then inoculated into YPD broth Susceptibility Test: 1:1 serial dilution to determine optimal amount of Malathion for future tests. Addition of Malathion: 4 beakers- both strains with Malathion and both strains with out Malathion Samples taken at 0, 1, 2, and 3 hours RNA extraction PCR Primers from the following genes added: NTE1, ACT1, HAC1, HNM1, IRE1, SCS2, and SCS3 Gel Electrophoresis: use 0.7% gel and then image Analysis: Band intensities of the PCR are compared between cells treated with and with out malathion