Suppressor genetics Reading lecture notes Screens 1 Direct screen phagocytosis and apoptotic genes in C elegans 2 Mosaic screen tumor suppressor genes 3 Revertant screens Forward and reverse mutations forward mutagen reverse mutagen wild type mutant mutant revertant wild type or more wild type Don t confuse reverse mutations with reverse genetics A revertant contains the original mutation and a suppressor mutation The suppressor can be Intragenic in the same gene Extragenic in another gene Intragenic suppressors true revertants DNA ATTCTTCAT RNA AUUCUUCAU Protein Ile Leu His missense mutation ATTTTTCAT AUUUUUCAU Ile Phe His ATTTTGCAT ATTCTTCAT AUUUUGCAU AUUCUUCAU Ile Leu His Ile Leu His Intragenic suppressors partial revertants DNA ATTCTTCAT RNA AUUCUUCAU Protein Ile Leu His missense mutation ATTTTTCAT AUUUUUCAU Ile Phe His ATTATTCAT AUUAUUCAU Ile Ile His Intragenic suppressors second site CTT Leu UUU Phe CTT Leu UUU Phe TAT Tyr ATT Ile Second mutation compensates for the first Extragenic suppressors 1 Informational suppressors allele specific gene nonspecific 2 Bypass suppressors parallel pathways allele nonspecific gene specific 3 Bypass suppressors same pathway allele nonspecific gene specific 4 Interaction suppressors allele specific gene specific Informational suppressors Two types that we will discuss Nonsense suppressors smg suppressors T4 amber mutants grow on E coli strain CR63 but not strain K How amber mutants got their name 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 Stahl Amber mutations can be in many different T4 genes and E coli genes The T4 head protein gene Amber mutations are nonsense mutations Genetic map of amber mutations in T4 head protein gene H11 C140 B17 wild type C208 A489 H36 C137 B272 B17 C140 H11 B272 Protein size C137 H36 A489 C208 So if amber mutations are nonsense mutations why do nonsense mutants grow on strain CR63 Genetic map of mutations in T4 head protein gene H11 C140 B17 on strain B wild type C137 on strain CR63 wild type C137 B272 C137 H36 A489 C208 Because 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 Genetic map of mutations in T4 head protein gene H11 C140 B17 UAG UAG UAG B272 UAG C137 H36 UAG UAG A489 UAG C208 UAG Genetic map of the E coli phoA gene A UAG B UAG C UAG D UAG The 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 UAG B UAG C UAG UAA stop D UAG 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 suppressors Allele specific gene nonspecific Mutations in seven genes isolated as allele specific suppressors of tra 2 lin 29 and unc 54 The unc 54 gene encodes muscle myosin 5 UTR 3 UTR 5 3 mRNA Myosin heavy chain unc 54 mutants are paralyzed 5 UTR 3 UTR nonsense No myosin animals paralyzed deletion Suppression of the Unc 54 locomotion defect identified recessive smg suppressors EMS unc 54 del unc 54 del hermaphrodites F1 F2 suppressed animals that move unc 54 unc 54 smg smg Suppression caused by stabilizing abnormal mRNA 5 UTR 3 UTR nonsense deletion Northern blot probed with labeled unc 54 DNA wild type unc 54 del unc 54 mRNA unc 54 del smg Smg mutations can also suppress mRNA instability caused by nonsense mutations but not Unc phenotype 5 UTR 3 UTR nonsense deletion Northern blot probed with labeled unc 54 DNA unc 54 nonsense wild type unc 54 nonsense smg unc 54 mRNA Model smg genes encode components of a mRNA surveillance system that degraded abnormal nonsense mediated mRNA decay
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