Transcript act
Tetrads with n genes ABCD x abcd A/a B/b C/c ABCD AbCd aBcD abcd ABCD AbCD aBcd abcd D/d ABCD Abcd aBCD abcd How many types??? 2:2 segregation for each locus If no linkage: 1/(2n) spores 3-5 x oversampling to ensure obtaining strain Suppressor screens, examples Suppressor of Multivulva in C. elegans Activated Go-alpha in C. elegans Enhancer screens, examples Rough eye in Drosophila LET-23 EGFR C. elegans rasGAP KINASE LET-60 KINASE LIN-45 RAS RAF RING + SLI-1 Cbl pro SH2 Y~P SEM-5 Grb2 GNEF SH3 SH2 pro Y~P SH3 pro Y~P KINASE LET-341 SOS KINASE MEK-2 KINASE MPK-1 ARK-1 Ack-related kinase Vulval differentiation Sevenless RTK GAP KINASE Y~P Drk Grb2 SH3 SH2 Y~P SH3 Y~P pro GNEF RAS1 SOS GNEF KINASE MAPKKK KINASE MAPKK KINASE MAP K R7 Drosophila Photoreceptor Development R8 induces R7 Multiple Ommatida in each eye: a population assay An enhancer screen for essential genes required for R7 development The fly eye consists of approximately 800 20-cell repeating units known as ommatidia. Each ommatidium consists of eight photoreceptor neurons (R1-R8), four lens secreting cone cells and eight additional accessory cells. The ommatidia arise from an undifferentiated epithelium by a series of cell interactions. We will only consider an interaction between the R8 and presumptive R7 cells that determines the fate of R7. The R7 photoreceptor detects light in the UV range. Screens for mutants with ommatidia that lack R7 cells identified three genes: sevenless (sev), bride of sevenless (Boss) and seven-in-abstentia (sina). Adult flies homozygous for mutations in any of these genes have ommatidia that lack an R7 cell and contain an additional cone cell. In the absence of R7 differentiation, the presumptive R7 cell becomes a cone cell. sev and sina are a receptor tyrosine kinase and a nuclear protein, respectively, and both genes act in R7 to specify R7's fate. boss appears to encode the ligand for the Sev receptor tyrosine kinase, and in contrast to sev and sina, acts in R8 cell to specify R7's fate. Now consider the problem that many genes functioning downstream of receptor tyrosine kinse receptor activation are likely to be required for other tyrosine kinase signaling pathways that are required for the viability of the organism. How can one use the fly eye to identify such mutations in such genes. Make a partially active mutant version of sev and introduce it into a sev mutant background. These flies have a temperature-sensitive phenotype. A fly carrying one copy of this transgene is wildtype at 22.7 oC (R7 is present). However, at 24.3oC R7 is absent sev/Y; +/+; +/+male sev/sev; X */+; P[sev-ts]/+ sev/sev; +/+; P[sev-ts]/balancer sev/Y; */+; P[sev-ts]/+ Screen for absence of R7 in individual flies. Isolate these chromosomes by balancing. R7 present R7 absent R7 absent sev/sev; +/+; P[sev-ts]/Y at 22.7oC sev/sev; +/+; P[sev-ts]/Y at 24.3oC sev/sev; */+; P[sev-ts]/Y at 22.7oC Look for mutation (*) that confers dominant enhancement of sev phenotype Sevenless RTK GAP KINASE Y~P Drk Grb2 SH3 SH2 Y~P SH3 Y~P pro GNEF RAS1 SOS GNEF KINASE MAPKKK KINASE MAPKK KINASE MAP K R7 Receptor is “exchange factor” GPCR g GTP GDP Effector a Effector GDP b a b GTP g Pi RGS RGS is the GTPase Activating Protein GPCR g GTP GDP Effector a Effector GDP b a b GTP g Pi RGS a GTPaseor RGS- G proteins Gq and Go control movement C. elegans Genotype Phenotype Wild type egl-30(lf) egl-30(gf) goa-1(lf) goa-1(gf) egl-30(lf) goa-1(lf) wild-type paralyzed hyperactive hyperactive paralyzed paralyzed lf, loss-of-function; gf, gain-of-function Mutations that Suppress activated Goa syIs17 syIs17; sag-4(sy433) Before Heat Shock After Heat Shock Jane Mendel, Yvonne Hajdu-Cronin, Wen Chen Suppressors of Activated Goa (Sag) CLASS I hyperactive • dgk-1/sag-1 (14 alleles) encodes diacylgycerol kinase • eat-16(sy348) (p.k.a. sag-2) encodes RGS7 homologue CLASS II wild type • sag-4, 8 sag-4 encodes cyclin L homologue CLASS III Egg-laying defective sag-3 encodes Heat Shock Factor • sag-3, 5 CLASS IV wild type • sag-6 CLASS V Egg-laying defective • sag-7 Yvonne Hajdu-Cronin & Wen Chen G Protein Coupled Receptors (GPCRs) EAT-16 RGS EGL-30 Gq ? EGL-10 RGS GOA-1 Go EGL-8 PLCb [IP3] [PIP2] DGK-1 [DAG] [PA] UNC-13 [DAG-binding] etc. Synaptic transmission: movement Extragenic suppression • many mechanisms--key issue is the genetic specificity of the suppressor gene-specific allele-nonspecific gene-specific allele-specific gene-nonspecific allele-specific epistasis (bypass suppression) direct interaction? ‘informational’ suppression suppression by compensatory change in direct interactor? • ‘Lock and Key’ model: binding site is restored • in general a very rare event as target size is 1(or a few) bp-need screens of >106 genomes • RNA-RNA interactions: – restoration of base pairing (nonsense suppression) – splice site suppression e.g. Lesser + Guthrie 1993 Science 262: 1982 • protein-DNA interactions – lac operon: oC mutations suppressed by mutations in repressor that bind more tightly to operator (Pfahl 1981, J. Mol. Biol. 147: 1-10) • protein-protein interactions? allele-specific suppression • null mutants are not suppressed, so not bypass suppressor • stabilization or altered processing of mutant gene product suppression by formation of new protein-protein interactions Adams + Botstein 1989. suppressors of ts actin mutants – get sac mutants. sac6 is fimbrin, actin-binding – sac6 mutations are missense in actin binding domain, increase affinity for mutant actin – But the affinity for wild type actin is also increased ACT SAC act SAC act sac ACT sac gene non-specific, allele specific • 1. 2. 3. 4. 5. suppression at level of gene expression: ‘informational’ Nonsense suppression Frameshift suppression Splicing machinery stabilization of unstable mRNA or protein suppression of transposon insertion alleles nonsense suppression • conditional ‘amber’ mutations in many T4 genes (Epstein et al) – grow on one E coli strain (CR63) but not on B – cause premature termination – suppression due to mutant tRNA that can recognize amber codon UAG and insert amino acid (usually Trp; codon is UGG) – amber suppressor strains are a bit sick because of readthrough frameshift suppression • extragenic suppression of frameshifts by two mechanisms – limitation of Trp-tRNA – other tRNAs loosely bind to codon (mismatch) and allow frameshifting – also mutant tRNA with 4-base anticodon now ‘reads’ frameshift as a 3-base codon… suppression by stabilization of message • mRNAs with ‘premature’ stop codons are recognized and degraded – nonsense mediated decay/ ‘mRNA surveillance’ – Upf pathway (yeast), SMG pathway (worms) – get rid of aberrant mRNAs before they get to ribosome • some nonsense mutations can be suppressed if partially functional protein can be made mRNAs with premature stop codons produce truncated proteins. AUG stop AAAA Expression of these from many loci can be detrimental to the animal. Cells have mechanisms of removing aberrant mRNAs mRNAs with premature stop codons are recognized and destroyed by nonsense mediated decay AUG stop AAAA SMG factors stop AAAA decapping and exonucleolytic cleavage Screens for suppressors of nonsense mutations revealed smg genes • • • • • • • smg-1 smg-2 smg-3 smg-4 smg-5 smg-6 smg-7 phosphatidylinositol-3 kinase homolog Upf1 helicase homolog, phosphoprotein Upf2 homolog Upf3 homolog novel, binds SMG-7 -novel, binds SMG-5 Mutations in the proteins required for nonsense mediated decay suppress nonsense mutations by allowing stabilizing mRNAs with premature stop codons. Functional proteins are made since low levels of readthrough make some normal protein or because expression of the truncated protein can suppress the phenotype Hodgkin J, Papp A, Pulak R, Ambros V, Anderson P. A new kind of informational suppression in the nematode Caenorhabditis elegans. Genetics. 1989 Oct;123(2):301-13. In the absence of SMG proteins mRNAs with premature stop codons will persist Expression of these from many loci can be detrimental to the animal AUG stop AAAA Short protein fragment is not functional or antimorphic mRNAs with premature stop codons have a low level of readthrough, these levels may be enough to rescue the mutant phenotype suppression by stabilization of protein • E. coli lon protease degrades aberrant proteins • mutations in lon suppress thermolabile mutations in many genes (RNA polymerase etc)