PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Suppressor geneticsReading: lecture notesScreens1. Direct screen: phagocytosis and apoptotic genes in C. elegans2. Mosaic screen: tumor suppressor genes3. Revertant screenswild typemutantmutagenForward and reverse mutationsmutantrevertant(wild type or more wild type)mutagenforward reverseDon’t confuse reverse mutations with reverse geneticsA revertant contains the original mutation and a suppressor mutation.The suppressor can be:Intragenic: in the same geneExtragenic: in another geneIntragenic suppressors: true revertantsDNA ..ATTCTTCAT..RNA ..AUUCUUCAU..Protein .. Ile Leu His ..missense mutation..ATTTTTCAT....AUUUUUCAU.... Ile Phe His .. ..ATTCTTCAT....AUUCUUCAU.... Ile Leu His .. ..ATTTTGCAT....AUUUUGCAU.... Ile Leu His ..Intragenic suppressors: partial revertantsDNA ..ATTCTTCAT..RNA ..AUUCUUCAU..Protein .. Ile Leu His ..missense mutation..ATTTTTCAT....AUUUUUCAU.... Ile Phe His .. ..ATTATTCAT....AUUAUUCAU.... Ile Ile His ..Intragenic suppressors: second siteCTT UUULeu PheTAT ATTTyr IleCTT UUULeu PheSecond mutation compensates for the firstExtragenic suppressors1. Informational suppressors: allele specific, gene nonspecific2. Bypass suppressors (parallel pathways): allele nonspecific, gene specific3. Bypass suppressors (same pathway): allele nonspecific, gene specific4. Interaction suppressors: allele specific, gene specificInformational suppressorsTwo types that we will discuss:Nonsense suppressorssmg suppressorsT4 amber mutants grow on E. coli strain CR63 but not strain K.Charley agreed to help look for the mutants, picking 2000 plaques in the first try. We also managed to convince Harris Bernstein (then a graduate student working on Neurospora genetics) to help and offered him the dubious reward of naming the mutants after him. Harris had the nickname Immer Wieder Bernstein ("Forever Amber" in German). That night we isolated several of the desired mutants and named them "amber mutants.”Dick Epstein letter to Frank StahlHow amber mutants got their name.Amber mutations can be in many different T4 genes.(and E. coli genes)The T4 head protein gene.Amber mutations are nonsense mutationsGenetic map of amber mutations in T4 head protein geneH11 C140 B17 B272 C137 H36 A489 C208wild typeC208A489H36C137B272B17C140H11Protein sizeSo if amber mutations are nonsense mutations,why do nonsense mutants grow on strain CR63?Genetic map of mutations in T4 head protein geneH11 C140 B17 B272 C137 H36 A489 C208on strain Bwild typeC137on strain CR63wild typeC137Because strain CR63 makes full length T4 head protein!The difference between the two E. coli strains can be mapped.The difference, known as supF, mapped to a tRNATyr gene.The supF change in tRNATyr is in the anticodon loop.H11 C140 B17 B272 C137 H36 A489 C208UAG UAG UAG UAG UAG UAG UAG UAGGenetic map of mutations in T4 head protein geneGenetic map of the E. coli phoA gene A B C DUAG UAG UAG UAGThe supF suppressor will suppress UAG amber mutations in the T4 head protein gene and in the phoA gene and in many other genes. Therefore the suppressor is gene nonspecific.Genetic map of the E. coli phoA gene A B C DUAG UAG UAG UAGUAA stop F to G missense But supF will not suppress other nonsense mutations (opal and ochre), missense mutations, frameshift mutations or deletions. Therefore supF is allele specific in its suppression.A couple of final questions.Will supF suppress all UAG nonsense mutations?Why do supF E. coli live?smg suppression(Suppressors with morphological defects in the genitalia)Informational suppressorsAllele specific, gene nonspecificMutations in seven genes isolated as allele-specific suppressors of tra-2, lin-29, and unc-54.5’UTR 3’UTRThe unc-54 gene encodesmuscle myosin5’ 3’mRNAMyosin heavy chain5’UTR 3’UTRunc-54 mutants are paralyzednonsenseNo myosin--animals paralyzeddeletionEMSunc-54(del)/unc-54(del) hermaphroditesF1F2 suppressed animals that move(unc-54/unc-54; smg/smg)Suppression of the Unc-54 locomotion defectidentified recessive smg suppressors5’UTR 3’UTRnonsensedeletionNorthern blot probed with labeled unc-54 DNAunc-54(del) smg unc-54mRNAwild typeunc-54(del)Suppression caused by stabilizing abnormal mRNA5’UTR 3’UTRnonsensedeletionNorthern blot probed with labeled unc-54 DNAunc-54(nonsense) smg unc-54mRNAwild typeunc-54(nonsense)Smg mutations can also suppress mRNA instabilitycaused by nonsense mutations (but not Unc phenotype).Modelsmg genes encode components of a mRNA surveillance system that degraded abnormal.nonsense mediated mRNA
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