Screens Suppressor genetics 1. Direct screen: phagocytosis and apoptotic in C. elegans Reading: lecture notes 2. Mosaic screen: tumor suppressor genes

3. Revertant screens

Forward and reverse A revertant contains the original and a forward reverse suppressor mutation. mutagen mutagen The suppressor can be: wild type Intragenic: in the same mutant revertant (wild type or more wild type) Extragenic: in another gene Donʼt confuse reverse mutations with reverse genetics

Intragenic suppressors: true Intragenic suppressors: partial revertants revertants

missense mutation missense mutation DNA ..ATTCTTCAT.. ..ATTTTTCAT.. DNA ..ATTCTTCAT.. ..ATTTTTCAT.. RNA ..AUUCUUCAU.. ..AUUUUUCAU.. RNA ..AUUCUUCAU.. ..AUUUUUCAU.. .. Ile Leu His .. .. Ile Phe His .. Protein .. Ile Leu His .. .. Ile Phe His ..

..ATTATTCAT.. ..ATTTTGCAT.. ..ATTCTTCAT.. ..AUUAUUCAU.. ..AUUUUGCAU.. ..AUUCUUCAU.. .. Ile Ile His .. .. Ile Leu His .. .. Ile Leu His ..

1 Intragenic suppressors: second site Extragenic suppressors

1. Informational suppressors: allele CTT UUU Leu Phe specific, gene nonspecific 2. Bypass suppressors (parallel pathways): allele nonspecific, gene specific

CTT UUU TAT ATT Leu Phe Tyr Ile 3. Bypass suppressors (same pathway): allele nonspecific, gene specific

Second mutation compensates for the first 4. Interaction suppressors: allele specific, gene specific

Informational suppressors

T4 amber Two types that we will discuss: grow on E. coli strain CR63 but not strain K. Nonsense suppressors

smg suppressors

How amber mutants got their name. Amber mutations can be in many different T4 genes. (and E. coli genes)

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 The T4 head protein gene. 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

2 Amber mutations are nonsense mutations So if amber mutations are nonsense mutations, why do nonsense mutants grow on strain CR63?

Genetic map of amber mutations in T4 head protein gene Genetic map of mutations in T4 head protein gene

H11 C140 B17 B272 C137 H36 A489 C208 H11 C140 B17 B272 C137 H36 A489 C208

Protein size wild type on strain B C208 wild type A489 C137 Because strain CR63 H36 makes full length T4 C137 on strain CR63 head protein! B272 wild type B17 C137 C140 H11

The supF change in tRNATyr is in The difference between the two the anticodon loop. E. coli strains can be mapped.

The difference, known as supF, mapped to a tRNATyr gene.

Genetic map of mutations in T4 head protein gene H11 C140 B17 B272 C137 H36 A489 C208 Genetic map of the E. coli phoA gene UAG UAG UAG UAG UAG UAG UAG UAG A B C D UAG UAG UAG UAG

UAA stop F to G Genetic map of the E. coli phoA gene missense A B C D UAG UAG UAG UAG But supF will not suppress other nonsense The supF suppressor will suppress UAG amber mutations (opal and ochre), missense mutations, mutations in the T4 head protein gene and in the phoA frameshift mutations or deletions. Therefore supF gene and in many other genes. Therefore the is allele specific in its suppression. suppressor is gene nonspecific.

3 smg suppression A couple of final questions. (Suppressors with morphological defects in the genitalia) Informational suppressors Will supF suppress all UAG nonsense mutations? Allele specific, gene nonspecific

Why do supF E. coli live? Mutations in seven genes isolated as allele-specific suppressors of tra-2, lin-29, and unc-54.

The unc-54 gene encodes unc-54 mutants are paralyzed muscle myosin 5ʼUTR 3ʼUTR 5ʼUTR 3ʼUTR

5ʼ 3ʼ nonsense mRNA

Myosin heavy chain No myosin--animals paralyzed

Suppression of the Unc-54 locomotion defect Suppression caused by stabilizing abnormal mRNA identified recessive smg suppressors 5ʼUTR 3ʼUTR

EMS nonsense deletion unc-54(del)/unc-54(del) hermaphrodites Northern blot probed with labeled unc-54 DNA

F1 unc-54(del) unc-54(del) wild type smg

F2 suppressed animals that move unc-54 mRNA (unc-54/unc-54; smg/smg)

4 Smg mutations can also suppress mRNA instability caused by nonsense mutations (but not Unc ).

5ʼUTR 3ʼUTR Model nonsense deletion smg genes encode components of a mRNA surveillance system that degraded abnormal. Northern blot probed with labeled unc-54 DNA

unc-54(nonsense) wild type unc-54(nonsense) smg nonsense mediated mRNA decay unc-54 mRNA

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