Bi Lecture 8 Genetic Pathways and Genetic Screens
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1 Bi Lecture 8 Genetic Pathways and Genetic Screens
2 WT A 2X:2A her-1 tra-1 1X:2A her-1 tra-1 Female body Male body Female body Male body her-1(lf) B 2X:2A her-1(lf) tra-1 1X:2A her-1(lf) tra-1 Female body Male body Female body Male body tra-1(lf) C 2X:2A her-1 tra-1(lf) 1X:2A her-1 tra-1(lf) Female body Male body Female body Male body tra-1(lf; her-1(lf) D 2X:2A her-1(lf) tra-1(lf) 1X:2A her-1(lf) tra-1(lf) Female body Male body Female body Male body
3 confused epistasis with non-null alleles AR cell c + AR cell survives d + neuroar forms e + neuroar makes E and is functional 100% Functional neuroar AR cell c 2 AR cell survives d + neuroar forms Functional 80% neuroar e + neuroar makes E and is functional 20% no AR 50% Functional neuroar AR cell c + AR cell survives d 2 neuroar forms e + neuroar makes E and is functional 50% no neuroar AR cell c 2 AR cell survives d 2 neuroar forms e + neuroar makes E and is functional 40% Functional neuroar 20% no AR 40% no neuroar
4 confused epistasis with non-null alleles h + OM cell h 1 OM cell j 3 OM cell h 1 j 3 OM cell gene H gene J black pigment black h + OM cell h 1 OM cell j 3 OM cell h 1 j 3 OM cell gene J gene H black pigment
5 If a hypomorph is epistatic to a null, an inference can be made genotype f + g + phenotype white pigment f null g + black pigment f + f null g hypo g hypo white pigment white pigment gene F gene G black pigment white pigment
6
7 combining epistasis with site of action
8 Additivity and less than additivity with incompletely penetrant loss-of-function alleles. A. The r1 and s1 alleles display additive phenotypes. B. T he t1 and u1 alleles display less than additive phenotype. A r + s + r 1 s + r + s 1 r 1 s 1 All alive 30% alive 30% alive 10% alive B t + u + t 1 u + t + u 1 t 1 u 1 All alive 30% alive 30% alive 30% alive
9 2 vulval lineage polarity wild-type phenotype Reversed phenotype wild-type wild-type polarity Reversed Polarity cdh-3::cfp; ceh-2::yfp Takao Inoue et al (Cell 2004)
10 A ligand-1 receptor-1 100% viable penetrance 0% ligand-1 receptor-1 30% viable 70% ligand-1 receptor-1 30% viable 70% ligand-1 receptor-1 30% viable 70% B ligand-2 receptor-2 100% viable 0% ligand-2 receptor-2 70% viable 30% ligand-2 receptor-2 70% viable 30% ligand-2 receptor-2 70% viable 30% C
11 A ligand-1 receptor-1 100% viable penetrance 0% ligand-1 receptor-1 30% viable 70% ligand-1 receptor-1 30% viable 70% ligand-1 receptor-1 30% viable 70% B ligand-2 ligand-2 ligand-2 receptor-2 receptor-2 receptor-2 100% viable 70% viable 70% viable 0% 30% 30% ligand-2 receptor-2 70% viable 30% C ligand-1 ligand-2 receptor-1 receptor-2 0% viable 100% ligand-1 ligand-2 receptor-1 receptor-2 0% viable 100% ligand-1 ligand-2 receptor-1 receptor-2 0% viable 100% ligand-1 ligand-2 receptor-1 receptor-2 0% viable 100% D ligand-1 ligand-2 receptor-1 receptor-2 100% viable 0%
12 C. elegans programmed cell death A wild type B photo of dying cell(s) C no cell death D all cell death
13 ced-3(lf) NO cell death ced-4(lf) NO cell death ced-1(lf), 2, etc No engulfment ced-9(gf) NO cell death ced-9(gf+lf) EXTRA cell death nuc-1 No DNA degradation ced-3(lf) ced-9(lf) NO cell death ced-3 (lf) nuc-1(lf) NO cell death
14 A ced-4(gf) or ced-3(gf) B ced-3(gf); ced-4(lf) C ced-3(lf); ced-4(gf) D pathway ced-9 ced-4 ced-3 cell death
15
16
17 Genetic Screens
18 Positive Modulation E A B C
19 Negative Modulation D A B C
20 A B gf C D Extra Product E A B gf C D Extra Product E A B gf C D lf Less Product E A B gf C D Product E lf
21
22 C. elegans EGF-receptor signaling: a branched signaling pathway
23 C. elegans Vulval Development Screen viable lethal/ste unknown dominant lin-1 16 lin-3 2 lin-25 2 lin-12 7 lin-2 13 lin-8 1 n300 1 lin-24 2 lin-7 13 lin-9 1 lin-33 2 lin-10 3 lin-13 2 let-60(gf) 1 lin-18 2 lin-26 1 lin-14 2 lin let-23 1 unc unc let-60 0 lin-15 5 lin-45 0 lin-4 1 sem-5 0 lin-11 4 mpk-1 0 lin-17 5 lin-44 0
24 m=0.1 m=0.3 m=1 m=3
25 Empirical Saturation
26 Why no mutations? 1. Redundancy Do an enhancer screen to see if you can bias the threshhold. 2. Pleiotropy Use tissue specific promoters or mosaic screens 3. maternal rescue of phenotype/epigenetics go the extra generation 4. bad target (size, place in chromosome, refractory to mutagen, missing from collection) molecular biology
27 LET-23 EGF-R [IP2] PLCγ [IP3] [PIP2] ITR-1 IP3 Receptor SEM-5 Grb2 LET-341 SOS LET-60 RAS Ovulation Vulval development
28 Extragenic suppressors of partially defective LET-23 activated: LET-60 Ras loss of: UNC-101 = clathrin adaptor medium chain SLI-1 = cbl proto-oncogene homolog GAP-1 = GAP homolog Gregg Jongeward, Junho Lee & Charles Yoon
29 Enhancers of sli-1 mutations loss of: UNC-101 = clathrin adaptor medium chain ARK-1 = Ack-related Kinase GAP-1 = GAP homolog Junho Lee, Chris Lacenere & Neil Hopper
30
31 LET-23 EGF-R [IP2] PLCγ [IP3] [PIP2] ITR-1 IP3 Receptor SEM-5 Grb2 LET-341 SOS LET-60 RAS Ovulation Vulval development
32 let-23(lf) LET-23 EGF-R [IP2] PLCγ [IP3] [PIP2] SEM-5 Grb2 LET-341 SOS ITR-1 IP3 Receptor LET-60 RAS Ovulation Vulval development
33 let-23(lf) let-60(gf) LET-23 EGF-R [IP2] PLCγ [IP3] [PIP2] SEM-5 Grb2 LET-341 SOS ITR-1 IP3 Receptor LET-60 RAS** Ovulation Vulval development
34 let-23(lf) itr-1(gf) LET-23 EGF-R [IP2] PLCγ [IP3] [PIP2] SEM-5 Grb2 LET-341 SOS ITR-1 IP3 Receptor** LET-60 RAS Ovulation Vulval development
35 let-23(lf) let-60(gf) itr-1(gf) [IP2] LET-23 EGF-R PLCγ [IP3] [PIP2] SEM-5 Grb2 LET-341 SOS ITR-1 IP3 Receptor** LET-60 RAS** Ovulation Vulval development
36 mutagenize! +! Balancer A! X! Balancer A! Balancer B! m! Balancer B! X! Balancer A! Balancer B! F1! B non-a! m! F2! Balancer B! X! Non-B non-a! m! F3! m! m! Balancer B! m/bal! X!!
Bi Lecture 9 Genetic Screens (cont.) Chromosomes
Bi190-2013 Lecture 9 Genetic Screens (cont.) Chromosomes C. elegans EGF-receptor signaling: a branched signaling pathway LET-23 EGF-R [IP2] PLCγ [IP3] [PIP2] ITR-1 IP3 Receptor SEM-5 Grb2 LET-341 SOS LET-60
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