{"id":301,"date":"2023-07-11T13:27:15","date_gmt":"2023-07-11T20:27:15","guid":{"rendered":"https:\/\/live-usc-dornsife.pantheonsite.io\/pombenet\/?page_id=301"},"modified":"2023-08-03T13:19:55","modified_gmt":"2023-08-03T20:19:55","slug":"drugs","status":"publish","type":"page","link":"https:\/\/dornsife.usc.edu\/pombenet\/drugs\/","title":{"rendered":"Fission Yeast Drugs and DNA damaging agents"},"content":{"rendered":"\n\n  \n    \n\n\n\n\n\n\n<div\n  class=\"cc--component-container cc--rich-text \"\n\n  \n  \n  \n  \n  \n  \n  >\n  <div class=\"c--component c--rich-text\"\n    \n      >\n\n    \n      \n<div class=\"f--field f--wysiwyg\">\n\n    \n  <p>This page provides information on drugs and supplements for a variety of purposes, including counterselection agents 5-FOA, canavanine, and FUDR, selection agents including G418, the spindle poison thiabendazole, and\u00a0<a href=\"\/pombenet\/drugs\/\">The Forsburg Lab Guide to DNA damaging agents<\/a>. Please let us know if you have corrections, additions, or more information to include here!<\/p>\n\n\n\n<\/div>\n\n\n  <\/div><\/div>\n\n\n\n\n                \n  \n    \n\n\n\n\n\n\n<div\n  class=\"cc--component-container cc--stacking-cards \"\n\n  \n  \n  \n  \n  \n  \n  >\n  <div class=\"c--component c--stacking-cards\"\n    \n      >\n\n    \n            <div class=\"header-container\">\n                                \n<div class=\"f--field f--section-title\">\n\n    \n  <h2>\n          Counterselection\/selection Agents\n      <\/h2>\n\n\n<\/div>\n            \n                                \n<div class=\"f--field f--description\">\n\n    \n  <p>These agents can be used to select for or against the indicated marker on a plasmid or in the genome<\/p>\n\n\n\n<\/div>\n                    <\/div>\n    \n            <div class=\"cards-container\">\n            \n                <div class=\"card\">\n\n                    <div class=\"title-description\">\n\n                                                      \n<div class=\"f--field f--cta-title\">\n\n    \n  <h3>\n          EMM + 5&#8242; FOA\n      <\/h3>\n\n\n<\/div>\n                        \n                                                        \n<div class=\"f--field f--description\">\n\n    \n  <p>Have available sterile 2mg\/ml uracil (this will not crash), 7.5 mg\/ml stock solutions of ade, leu, and his. Use 3.75 mg\/ml uracil.<\/p>\n<ol>\n<li>Autoclave 15 minutes: 10g agar in 250 ml MQ H2O, with stir bar, in 1 liter bottle.<br \/>\nWeigh bottle before autoclaving<br \/>\nCorrect for loss of H2O by adding sterile MQ H2O.<br \/>\nStore at 50*C until step 2 is completed.<\/li>\n<li>(The liquid volume should add up to 250 ml total for this step). To 198.3 ml MQ H2O, add:\n<ul>\n<li>3.5 g Yeast Nitrogen Base with NH4 and without amino acids (Difco 0919-15-3)<\/li>\n<li>10 g dextrose<\/li>\n<li>0.5 g 5FOA (American Biorganics, Inc 07-00850; Toronto Research Chemicals cat. no. F595000)<\/li>\n<li>6.7 ml 3.75 mg\/ml uracil<\/li>\n<li>15 ml 7.5 mg\/ml ade<\/li>\n<li>15 ml 7.5 mg\/ml leu<\/li>\n<li>15 ml 7.5 mg\/ml his<\/li>\n<\/ul>\n<p>leave at 50*C, with occassional mixing until 5FOA is dissolved. Filter sterilize.<\/li>\n<li>YNB\/amino acids\/5FOA to sterile agar and mix carefully.<\/li>\n<li>Pour plates. Store at 4* in the dark. Note that standard cerevisiae media calls for anything from 0.1% to 1% FOA: for example, see\u00a0this discussion\u00a0from the bionet.yeast site. Your mileage may vary!<\/li>\n<\/ol>\n\n\n\n<\/div>\n                        \n                    <\/div>\n\n\n                    \n                <\/div>\n\n            \n                <div class=\"card\">\n\n                    <div class=\"title-description\">\n\n                                                      \n<div class=\"f--field f--cta-title\">\n\n    \n  <h3>\n          FUDR\n      <\/h3>\n\n\n<\/div>\n                        \n                                                        \n<div class=\"f--field f--description\">\n\n    \n  <p><strong>Selects against<\/strong>: adh-tk+<br \/>\nViral thymidine kinase makes the cells sensitive to this halogenated thymidine derivative.<br \/>\n<strong>Stock<\/strong>\u00a05&#8242; fluoro-2&#8242;-deoxyuridine (Sigma F-0503) Add 10mg per 500ml agar.<br \/>\n<strong>Reference<\/strong>\u00a0<a class=\" RLC_Start RLC_Redirect\" title=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=10655214&amp;dopt=Abstract | https:\/\/pubmed.ncbi.nlm.nih.gov\/10655214\/?dopt=Abstract\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=10655214&amp;dopt=Abstract\">Genetics. 2000 154:599-607.<\/a><\/p>\n\n\n\n<\/div>\n                        \n                    <\/div>\n\n\n                    \n                <\/div>\n\n            \n                <div class=\"card\">\n\n                    <div class=\"title-description\">\n\n                                                      \n<div class=\"f--field f--cta-title\">\n\n    \n  <h3>\n          Canavanine\n      <\/h3>\n\n\n<\/div>\n                        \n                                                        \n<div class=\"f--field f--description\">\n\n    \n  <p><strong>Selects against<\/strong>: CAN1 (from S. cerevisiae) in a\u00a0<i>can1-<\/i>\u00a0strain.<br \/>\nThe can1+ gene encodes an arginine permease. The permease allows uptake of arginine. Thus, in the absence of the can1+ product, cells will be unable to import arginine, so the can1-1 strain must be Arg+. There also seems to be an effect on other positively charged amino acids&#8211;that is loss of the permease may affect uptake of histidine and lysine. Thus, for most utility, can1-1 strains should be His+ and Lys+. The can1 mutation in S. pombe renders cells resistant to the drug, but it has not yet been determined which gene is affected.<br \/>\n<strong>Stock<\/strong>\u00a060 mg\/ml (this is 1000X), dissolved in H2O and filter sterilized. Store at 4*C. Use at 60\u00b5g\/ml.<br \/>\n<strong>Reference:<\/strong>\u00a0<a class=\" RLC_Start RLC_Redirect\" title=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=2020549&amp;dopt=Abstract | https:\/\/pubmed.ncbi.nlm.nih.gov\/2020549\/?dopt=Abstract\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=2020549&amp;dopt=Abstract\">NAR 19:1150 (1991)<\/a><\/p>\n\n\n\n<\/div>\n                        \n                    <\/div>\n\n\n                    \n                <\/div>\n\n            \n                <div class=\"card\">\n\n                    <div class=\"title-description\">\n\n                                                      \n<div class=\"f--field f--cta-title\">\n\n    \n  <h3>\n          G418\n      <\/h3>\n\n\n<\/div>\n                        \n                                                        \n<div class=\"f--field f--description\">\n\n    \n  <p><strong>Selects for<\/strong>: kanMX cassette (G418 marker)<br \/>\n&gt;Only works on YES plates&#8211;selection does not work in EMM. Use at 100-200 mg per liter. Store plates at 4*C in the dark. Can use minimal PMG medium (see below) &gt; This is a difficult marker that can show substantial variations in expression, especially when integrated. Cells must be allowed phenotypic expression before selection; we plate on YES (non-seletive), then replica plate in a day to YES + G418. Use care in identifying true G418-resistant colonies. It is helpful to use a wild type (negative) and known kanR strain (positive) as controls every time. &gt; Recent conversation (2\/2008) on the pombelist mailing list suggest that\u00a0<a class=\" RLC_Start RLC_Ok\" href=\"\/pombenet\/media\/\">PMG medium<\/a>, which replaces the ammonium chloride in EMM with glutamate, will work fine with G418. Added bonus: Ura- cells grow more similarly to Ura+ cells on PMG.<\/p>\n\n\n\n<\/div>\n                        \n                    <\/div>\n\n\n                    \n                <\/div>\n\n            \n        <\/div>\n    \n\n  <\/div><\/div>\n\n\n\n\n                            \n  \n    \n\n\n\n\n\n\n<div\n  class=\"cc--component-container cc--stacking-cards \"\n\n  \n  \n  \n  \n  \n  \n  >\n  <div class=\"c--component c--stacking-cards\"\n    \n      >\n\n    \n            <div class=\"header-container\">\n                                \n<div class=\"f--field f--section-title\">\n\n    \n  <h2>\n          Forsburg lab Guide to DNA damaging agents\n      <\/h2>\n\n\n<\/div>\n            \n                                \n<div class=\"f--field f--description\">\n\n    \n  <div class=\"html-content\">\n<p><strong>General Information:<\/strong><\/p>\n<ul>\n<li>Damaging agents cause DNA breaks and are therefore toxic and carcinogenic. Use caution in handling these reagents: wear gloves and a lab coat, pour plates in the hood and dispose of waste in the chemical containers in the hood.<\/li>\n<li>Use plates immediately (by the next morning) and\/or store at 4*C in the dark once dry. Plates still retain damage-inducing ability for a few weeks if stored this way.<\/li>\n<\/ul>\n<\/div>\n\n\n\n<\/div>\n                    <\/div>\n    \n            <div class=\"cards-container\">\n            \n                <div class=\"card\">\n\n                    <div class=\"title-description\">\n\n                                                      \n<div class=\"f--field f--cta-title\">\n\n    \n  <h3>\n          Bleomycin\n      <\/h3>\n\n\n<\/div>\n                        \n                                                        \n<div class=\"f--field f--description\">\n\n    \n  <p><strong>effect\u00a0<\/strong>Supposed to be a mimetic of gamma irradiation (double-strand breaks)<br \/>\n<strong>Liquid:<\/strong>\u00a02.5 &#8211; 5mU\/ml<br \/>\n<strong>S. pombe references:<\/strong>\u00a0<a class=\" RLC_Start RLC_Redirect\" title=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=9180692&amp;dopt=Abstract | https:\/\/pubmed.ncbi.nlm.nih.gov\/9180692\/?dopt=Abstract\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=9180692&amp;dopt=Abstract\">Mol Gen Genet 1997 254:389-99<\/a>\u00a0<a class=\" RLC_Start RLC_Redirect\" title=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=10637286&amp;dopt=Abstract | https:\/\/pubmed.ncbi.nlm.nih.gov\/10637286\/?dopt=Abstract\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=10637286&amp;dopt=Abstract\">MBC 11:1-11 (2002)<\/a>,\u00a0<a class=\" RLC_Start RLC_Redirect\" title=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=11238887&amp;dopt=Abstract | https:\/\/pubmed.ncbi.nlm.nih.gov\/11238887\/?dopt=Abstract\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=11238887&amp;dopt=Abstract\">MCB 21:1499-1508 (2001)<\/a><\/p>\n\n\n\n<\/div>\n                        \n                    <\/div>\n\n\n                    \n                <\/div>\n\n            \n                <div class=\"card\">\n\n                    <div class=\"title-description\">\n\n                                                      \n<div class=\"f--field f--cta-title\">\n\n    \n  <h3>\n          BrdU\n      <\/h3>\n\n\n<\/div>\n                        \n                                                        \n<div class=\"f--field f--description\">\n\n    \n  <p><strong>effect\u00a0<\/strong>easily visualized thymidine analogue. Requires thymidine kinase and thymidine uptake mechanism (strains typically carry HSV-tk+ and hENT; S. pombe cells can&#8217;t take up BrdU on their own). Eventually leads to DNA damage.<br \/>\n<strong>Stock solution<\/strong>\u00a010 mg\/ml in water. Use at 1:50 in liquid cultures Wrap flasks in aluminum foil to protect from light.<\/p>\n\n\n\n<\/div>\n                        \n                    <\/div>\n\n\n                    \n                <\/div>\n\n            \n                <div class=\"card\">\n\n                    <div class=\"title-description\">\n\n                                                      \n<div class=\"f--field f--cta-title\">\n\n    \n  <h3>\n          Camptothecin (CPT)\n      <\/h3>\n\n\n<\/div>\n                        \n                                                        \n<div class=\"f--field f--description\">\n\n    \n  <p><strong>effect\u00a0<\/strong>Inhibitor of topoisomerase I; causes strand breaks in S phase<br \/>\n<strong>Plates:<\/strong>1uM to 20 uM<br \/>\n<strong>S. pombe references:<\/strong>\u00a0<a class=\" RLC_Start RLC_Redirect\" title=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=10407262&amp;dopt=Abstract | https:\/\/pubmed.ncbi.nlm.nih.gov\/10407262\/?dopt=Abstract\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=10407262&amp;dopt=Abstract\">Yeast 15:821-828 (1992);\u00a0<\/a>;\u00a0<a class=\" RLC_Start RLC_Redirect\" title=\"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/16641370 | https:\/\/pubmed.ncbi.nlm.nih.gov\/16641370\/\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/16641370\">Mol Biol Cell. 2006 Jul;17(7):2976-85.<\/a><\/p>\n\n\n\n<\/div>\n                        \n                    <\/div>\n\n\n                    \n                <\/div>\n\n            \n                <div class=\"card\">\n\n                    <div class=\"title-description\">\n\n                                                      \n<div class=\"f--field f--cta-title\">\n\n    \n  <h3>\n          Hydroxyurea (HU)\n      <\/h3>\n\n\n<\/div>\n                        \n                                                        \n<div class=\"f--field f--description\">\n\n    \n  <p><strong>effect\u00a0<\/strong>Causes a range of lesions. Most often used to deplete the supply of nucleotides, leading to an S-phase arrest. Note that pombe is sensitive to HU at much lower levels than cerevisiae!<br \/>\n<strong>Plates:<\/strong>\u00a05mM to 10mM. Wild type will eventually form colonies; sensitive strains will not.<br \/>\n<strong>Liquid:<\/strong>\u00a012-15mM to arrest; e.g, for block and release. Reversable.<br \/>\n10mM will delay S phase.<br \/>\nFor permanent arrest (irreversable), use 20-25mM<br \/>\n<strong>Stock:<\/strong>\u00a01.5M, good for several days if stored at 4* in the dark.<br \/>\n<strong>S. pombe references:<\/strong>\u00a0<a class=\" RLC_Start RLC_Redirect\" title=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=1427071&amp;dopt=Abstract | https:\/\/pubmed.ncbi.nlm.nih.gov\/1427071\/?dopt=Abstract\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=1427071&amp;dopt=Abstract\">Genes Dev. 6:2035-2046 (1992)<\/a><\/p>\n\n\n\n<\/div>\n                        \n                    <\/div>\n\n\n                    \n                <\/div>\n\n            \n                <div class=\"card\">\n\n                    <div class=\"title-description\">\n\n                                                      \n<div class=\"f--field f--cta-title\">\n\n    \n  <h3>\n          Methyl Methanesulfonate (MMS)\n      <\/h3>\n\n\n<\/div>\n                        \n                                                        \n<div class=\"f--field f--description\">\n\n    \n  <p><strong>effect\u00a0<\/strong>Alkylating agent (causes a range of DNA damaged structures)<br \/>\n<strong>Plates<\/strong>\u00a00.0025% to 0.0050%<br \/>\n<strong>Liquid:<\/strong>\u00a00.007%<br \/>\nMMS plates should be kept for only a few days. Also, the fresher the MMS stock the better: apparently it becomes generally cytotoxic over time, but paradoxically loses some of its genotoxic effects. The nature of this chemical change is not clear. The same is true of EMS and may explain why some people consider EMS to be an inefficient mutagen in pombe. It&#8217;s probably just fine as long as its very fresh, before other compounds develop.\u00a0<strong>S. pombe references:<\/strong>\u00a0<a class=\" RLC_Start RLC_Redirect\" title=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=12052858&amp;dopt=Abstract | https:\/\/pubmed.ncbi.nlm.nih.gov\/12052858\/?dopt=Abstract\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=12052858&amp;dopt=Abstract\">MCB 22:4477-4490 (2002)<\/a>,\u00a0<a class=\" RLC_Start RLC_Redirect\" title=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=11971984&amp;dopt=Abstract | https:\/\/pubmed.ncbi.nlm.nih.gov\/11971984\/?dopt=Abstract\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=11971984&amp;dopt=Abstract\">MCB 22:3537-3548 (2002)<\/a><\/p>\n\n\n\n<\/div>\n                        \n                    <\/div>\n\n\n                    \n                <\/div>\n\n            \n                <div class=\"card\">\n\n                    <div class=\"title-description\">\n\n                                                      \n<div class=\"f--field f--cta-title\">\n\n    \n  <h3>\n          4-Nitroquinoline N-Oxide (4-NQO)\n      <\/h3>\n\n\n<\/div>\n                        \n                                                        \n<div class=\"f--field f--description\">\n\n    \n  <p><strong>effect\u00a0<\/strong>Supposed to be a UV mimetic; induces primarily G to A transitions<br \/>\n<strong>References:<\/strong>\u00a0<a class=\" RLC_Start RLC_Redirect\" title=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=1561085&amp;dopt=Abstract | https:\/\/pubmed.ncbi.nlm.nih.gov\/1561085\/?dopt=Abstract\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;list_uids=1561085&amp;dopt=Abstract\">NAR 20:1283-1287 1992<\/a><\/p>\n\n\n\n<\/div>\n                        \n                    <\/div>\n\n\n                    \n                <\/div>\n\n            \n                <div class=\"card\">\n\n                    <div class=\"title-description\">\n\n                                                      \n<div class=\"f--field f--cta-title\">\n\n    \n  <h3>\n          Ultraviolet light (UV)\n      <\/h3>\n\n\n<\/div>\n                        \n                                                        \n<div class=\"f--field f--description\">\n\n    \n  <p><strong>effect\u00a0<\/strong>Causes a range of mutations.<br \/>\n<strong>Plates<\/strong>\u00a0expose to 50-300 J\/m2. Wrap plates in aluminum foil to prevent photoreactivation repair (may not be an issue in pombe)<\/p>\n\n\n\n<\/div>\n                        \n                    <\/div>\n\n\n                    \n                <\/div>\n\n            \n        <\/div>\n    \n\n  <\/div><\/div>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":354,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"page-content-detail.php","meta":{"_acf_changed":false,"footnotes":""},"class_list":["post-301","page","type-page","status-publish","hentry"],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.1.1 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Fission Yeast Drugs and DNA damaging agents - PombeNet Forsburg Lab<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/dornsife.usc.edu\/pombenet\/drugs\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Fission Yeast Drugs and DNA damaging agents - 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