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by: Miss Maximo Barrows


Miss Maximo Barrows
GPA 3.57

Robert Duronio

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Robert Duronio
Class Notes
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This 2 page Class Notes was uploaded by Miss Maximo Barrows on Sunday October 25, 2015. The Class Notes belongs to BIOL 445 at University of North Carolina - Chapel Hill taught by Robert Duronio in Fall. Since its upload, it has received 27 views. For similar materials see /class/228826/biol-445-university-of-north-carolina-chapel-hill in Biology at University of North Carolina - Chapel Hill.




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Date Created: 10/25/15
We analyze the cell biology of oncogenes in model organisms II Unraveling the ras pathway in worms 1 Induction amp the single cell Cell interactions in the vulva a Many cell fate decisions require cell interactions b Example vulval development in the nematode C elegans vulva connection of gonad to outside Derived from somatic hypodermal epidermal cells In worms use laser to kill single cells ask if they39re required for neighbor39s development In normal development anchor cell sits at junction of hypoderm amp gonad Experiment 1 Ablate kill with laser anchor cell gt no vulva Ablate gonad leave anchor cell intac gt normal vulva Hypothesis anchor cell gt cell cell signal to hypodermal cells quotMake vulva quot 2 Instructions for vulval assembly a Anchor cell sits near 6 quotvulval precursorquot cells 2 Pnp cells ie P3p P4p P5p P6p P7p amp P8p Vulva normally made by descendants of 3 central cells Outside 3 normally contribute to hypoderm normal skin Hypothesis All 6 precursors have potential to respond to vulval inducing signal but only 3 are normally exposed to it The 6 vulval hypodermal cells thus form an quotequivalence groupquot Anchor cell signal gt 1 amp 2 cells gt turn off hypoderm program gt vulval fate b Genes must control these cell interactions To identify these genes Look for mutants that have an abnormal vulva Found gt100 mutations in gt40 genes that affect process Thought they had identi ed quotallquot genes that affect this pathway but see later 2 types muv multiple vulvas vul vulvaless e g lin 2 lin 3 lin 7 lin 10 and let 23 all vulval precursors gt 3 fate the animal is vul e g in15 all vulval precuIsors take on 1 or 2 fates animal is muv Also some dominant mutations e g dominant allele of lin 34 gtMuv c The search for the AC signal What would be expected properties of a mutant lacking AC signal If there is no induction all cells gt 3 fate So to look for mutations in signal or receptor look among vul mutations One such mutation that fits criteria let23 was cloned It encodes a receptor tyrosine kinase 2 receptor for the AC signal related to human EGF Receptor There is an important lesson in this gene Note gene name is let23 not inXX Why Because a null allele of let23 is lethal as an embryo Therefore if you set up a screen for quotallquot genes that affect a process you may miss those genes also required for other earlier and essential processes In this case the scientists were lucky or persistent Weak alleles of let23 live but are vul Clone more vul genes lin 3 EGFTGF alpha like protein 2 anchor cell signal Hypothesis signaling pathway in which lin 3 anchor cell signal and let 23 2 receptor 3 3 To extend this analysis scientists carried out screens for suppressors of other mutations a Start with lin 15 homozygous mutants Muv Feed EMS and look among progeny for non Muv animals Screened 100000 Isolated 10 extragenic ie outside of lin 15 gene suppressors two 2 recessive let 23 mutations which on their own have a Vul phenotype One 2 recessive mutation in new gene 2 lin 45 Recessive phenotype by itself 2 Vul U Other seven mutations dominant have Vul phenotype alone all map to same region Six of seven of these mutations are also recessive lethal Can use this phenotype for complementation tests all fail to complement thus are same gene Is this a gain of function or haploinsufficiency ie half is not enough Look at chromosome deficiency39s for this region DO NOT have a Vul phenotype 0 How do learn about loss of function phenotype Revert dominant phenotype Muv gtWT Found 3 revertants in 44000 animals screened Complete suppression of dominant phenotype loss of function recessive lethal let 60 0 Funny thing is dominant Vul mutations of let 60 have same phenotype as recessive loss of function mutations in let 60 Idea these are dominant negative mutations ie Mutant gene product poisons the function of the wild type gene product in same cell so dominant loss of function d Another mutation in same region had a dominant Muv phenotype lin 34 mentioned above Also mutations in let 60 gene lin 34 dominant gain of function let 60 gf e Double mutants used to place let 60 in pathway eg let 60 gf let 23 is Muv so Let 60 downstream e g let 60 gf lin 45 is Vul so lin 45 downstream 4 Clone let60 ras let 60 gf mutations cannot hydrolyze GTP similar to oncogenic mutations in mammalian ras Clone lin 45 Raf ie Raf in pathway downstream of Ras Continue screens for suppressors isolate suppressors of activated Ras let 60gf In this find MAP Kinase placing it downstream of Ras and Raf Later studies identify MAP kinase kinase and transcription factors downstream of MAP Kinase


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