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Tetrads with n genes

Tetrads with n genes. A B C D x a b c d. A/a B/b C/c D/d. ABCD AbCd aBcD abcd. ABCD AbCD aBcd abcd. ABCD Abcd aBCD abcd. How many types???. 2:2 segregation for each locus If no linkage: 1/(2 n ) spores. 3-5 x oversampling to ensure obtaining strain.

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Tetrads with n genes

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  1. Tetrads with n genes A B C D x a b c d A/a B/b C/c D/d ABCD AbCd aBcD abcd ABCD AbCD aBcd abcd 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

  2. Suppressor screens, examples Suppressor of Multivulva in C. elegans Activated Go-alpha in C. elegans Enhancer screens, examples Rough eye in Drosophila

  3. LET-23 EGFR C. elegans KINASE rasGAP LET-60 RAS KINASE LIN-45 RAF GNEF SEM-5 Grb2 KINASE SH2 Y~P MEK-2 LET-341 SOS RING + SH3 pro SH2 Y~P SH3 pro pro KINASE SLI-1 Cbl KINASE Y~P MPK-1 ARK-1 Ack-related kinase Vulval differentiation

  4. Sevenless RTK KINASE GAP RAS1 KINASE MAP-KKK GNEF Drk Grb2 KINASE Y~P MAP- KK SOS GNEF SH3 pro SH2 Y~P SH3 KINASE Y~P MAP K R7

  5. Drosophila Photoreceptor Development R8 induces R7

  6. Multiple Ommatida in each eye: a population assay

  7. 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 22.7oC (R7 is present). However, at 24.3oC R7 is absent

  8. X R7 present sev/sev; +/+; P[sev-ts]/Y at 22.7oC sev/sev; +/+; P[sev-ts]/Y at 24.3oC R7 absent sev/Y; */+; P[sev-ts]/+ sev/sev; */+; P[sev-ts]/+ sev/sev; */+; P[sev-ts]/Y at 22.7oC R7 absent Look for mutation (*) that confers dominant enhancement of sev phenotype sev/sev; +/+; P[sev-ts]/balancer sev/Y; +/+; +/+male Screen for absence of R7 in individual flies. Isolate these chromosomes by balancing.

  9. Sevenless RTK KINASE GAP RAS1 KINASE MAP-KKK GNEF Drk Grb2 KINASE Y~P MAP- KK SOS GNEF SH3 pro SH2 Y~P SH3 KINASE Y~P MAP K R7

  10. Receptor is “exchange factor” GPCR g b Effector GDP GTP b GTP a Effector a g GDP Pi RGS RGS is the GTPase Activating Protein

  11. GPCR g b Effector GDP GTP b GTP a Effector a g GDP Pi a GTPase- or RGS- RGS

  12. G proteins Gq and Go control movement C. elegans Genotype Phenotype Wild type wild-type egl-30(lf) paralyzed egl-30(gf)hyperactive goa-1(lf)hyperactive goa-1(gf)paralyzed egl-30(lf) goa-1(lf) paralyzed lf, loss-of-function; gf, gain-of-function

  13. Mutations that Suppress activated Goa syIs17 syIs17; sag-4(sy433) Before Heat Shock After Heat Shock Jane Mendel, Yvonne Hajdu-Cronin, Wen Chen

  14. Suppressors of Activated Go (Sag) CLASSI hyperactive (14 alleles) encodes diacylgycerol kinase dgk-1/sag-1 • eat-16(sy348) • (p.k.a. sag-2) encodes RGS7 homologue CLASSII wild type sag-4, 8sag-4 encodes cyclin L homologue • CLASSIII Egg-laying defective sag-3, 5 sag-3 encodes Heat Shock Factor • CLASSIV wild type •sag-6 CLASSV Egg-laying defective •sag-7 Yvonne Hajdu-Cronin & Wen Chen

  15. G Protein Coupled Receptors (GPCRs) EAT-16 RGS EGL-10 RGS GOA-1 Go EGL-30 Gq ? [IP3] EGL-8 PLCb [PIP2] DGK-1 [DAG] [PA] UNC-13 [DAG-binding] etc. Synaptic transmission: movement

  16. Extragenic suppression • many mechanisms--key issue is the genetic specificity of the suppressor

  17. 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?

  18. allele-specific suppression • null mutants are not suppressed, so not bypass suppressor • stabilization or altered processing of mutant gene product

  19. suppression by formation of new protein-protein interactions ACT SAC 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

  20. gene non-specific, allele specific • suppression at level of gene expression: ‘informational’ • Nonsense suppression • Frameshift suppression • Splicing machinery • stabilization of unstable mRNA or protein • suppression of transposon insertion alleles

  21. 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

  22. 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…

  23. 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

  24. mRNAs with premature stop codons produce truncated proteins. stop AUG AAAA Expression of these from many loci can be detrimental to the animal. Cells have mechanisms of removing aberrant mRNAs

  25. mRNAs with premature stop codons are recognized and destroyed by nonsense mediated decay stop AUG AAAA SMG factors stop AAAA decapping and exonucleolytic cleavage

  26. Screens for suppressors of nonsense mutations revealed smg genes • smg-1 phosphatidylinositol-3 kinase homolog • smg-2 Upf1 helicase homolog, phosphoprotein • smg-3 Upf2 homolog • smg-4 Upf3 homolog • smg-5 novel, binds SMG-7 • smg-6 -- • 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.

  27. mRNAs with premature stop codons have a low level of readthrough, these levels may be enough to rescue the mutant phenotype 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 stop AUG AAAA Short protein fragment is not functional or antimorphic

  28. suppression by stabilization of protein • E. colilon protease degrades aberrant proteins • mutations in lon suppress thermolabile mutations in many genes (RNA polymerase etc)

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