Nhat Cao Fall 2015 7.012 Problem Set 5-20 Points total DUE FRIDAY NOVEMBER 6", at 9:50 AM ONLINE Question 1 (4 points) ' You are studying a rare autosomal recessive disease. You have no knowledge of where the gene associated with the disease (gene A) lies in the human genome. To clone this gene you perform SNP (single nucleotide polymorphism) mapping on the individuals from a family that suffers from this disease. a) You isolate samples trom all the family members and use PCR to individually amplify 3000 different SNP regions for each individual. Assuming that each amplified SNP is unique, how many different PCR primers would you need? b) You find that SNP 1 and SNP 2 are linked to the disease gene, but SNP 1 is more tightly linked than SNP 2. Explain which SNP you want to use to study the disease in this famil SW belli wiorleiy 4h Wank te ONDE ble Lentrtd om dive be tib let hlely to reembine ‘The pedigree shows the inheritance of this disease and the alleles of the SNP chosen in part b. The individuals that are shaded show the disease phenotype, and all affected individuals ate indicated, Cacticts aid unaffected individuals are both un-shaded. “Assume complete penetrance. Although the SNP and the disease gene may recombine in the population over time, assume that no recombination has occurred between the SNP and the disease gene in the pedigree below. AaThe two letters identify the two allel a6 indicates that 09 on of the chromosomes you °! vies ane wi eh a Gon: Genes base pai), and oP chromosome you would find a G (a Cagg- Chajum base pait) ©) Given this, answer the following questions. + Individuals 3 and 4 have an affected child. What is the genotype for individual 4 at the disease gene locus? Choose from AA, Aa, aa, or can't tell. A A nme 4 fo diet ene «Individuals 3 and 4 have an affected child, Whats the genotype for individual 4 at the chosen SNP? Choose from: C/C, C/G, G/C, G/G, or can‘ tell Can't toll ) Assume that individual 17 is nota carrier of the disease al that the son of individuals 16 and 17 inherits the disease 19% IIcle. What is the chance allele from his mother? Question 2 (2 points) has many applications. It can be used to determine how 1 types or to genotype individuals. Based on your in be prepared using genomic DNA or cDNA. DNA microarray technology gene expression differs between cel intended purpose, the microarray ca ‘tto use a microarray to look at the differential gene expression between would you use to prepare the microarray for this experiment? Explain your answer . 1 oo - He abi ble acwA ¢ in hAruetion’s sor wo leng wh lf) Je) te rrupant kucha’ af oti, w/Aedoltoule, bb) You want to use a microarray to look at the genomic differences.vetween two ODNA different individuals. What would you use to prepare the microarray for this . experiment? Explain your answer. : oy ' ) ies DNA ble went to 200 polpndiel replacement, acdlivev A a) You wan two tissues in an individual. What {os10% Om {! ol » whole or pos q Crow cewe.i } Question 3 (6 points) | Below is the schematic for a gene (gene A) that you decide to study using the gene editing methods covered in class. ; Region 1 Region2 — Region3 ——Region 4 Region S Région 6 4) In class we mentioned two gene editing strategies that allow you to do this. Using a maximum of two sentences, explain how these strategies differ from one another. You don't need to explain the specifics of each strategy. i. Traditional ge knockout strategy Vis gee lecleirg nanny ged gran iv «body Bo Hs plants t ‘dc fogs Sodae ii, Conditional gene knockout strategy A gre Ob bnadlrad ot tn a erase | ta ha the funohons In & Soe re L ch. O4aw b) Suppose you want to study the function Hees liver cells. Which strategy Would you use to delete the gene and study the Phenotype of fe tissue? Tee weemtvand to biel ad the gered Us trobbomnal § Aeslegy © fom dhe Vendive b ¢) What kind of genomic alteration is caused by the CRISPR/Cas9 strategy? n Edd A spective, DMA Cequenee 2 Svapee Unto AILAr Of Crore | Merve oud * tack rine i sportard aquere cfortale trav Bash yaller preter) d) Another method you could use to study the role of a specific gene is RNAi Fano! ae Suellen ders 1. Which step of the central dogma does RNAi interfere with? if ( Translation anna 0 oa why cells ut have wed the mechanisms for RNAi Ue ase conblautl clodied one bil ln ena cowl fe dees 1A nol Hdienal ie th alle old fat cle gradation” ols proeet alls 4 my Aad rid 3 Med gene 40 lain Sort -|ramst wn RiAiIIL. Suppose you decide to study this gene using RNAi, Which regions of the gene from the schematic in part a could you use in designing your RNA construct? Which regions could you not use? Explain - tows RNAI nad 40 te mm @Nnk, fo cxone can bufed / le. 04 6 ancl Mol Unboud, vm vt oa ov 2 jl, 6 . Lovwot W reg in 33) ©) Suppose you decide to study the gere using the traditional gene krockoumeshod. Your lb partner has designed three constructs below he says could work in deleting the gene. For each for the constructs indicate whether they would be successful in knocking out the gene of interest. If the construct would not work, indicate why. Construct 1 Intron 2 ntibrotiC Negative resistance gene Selection marker \ 7 ant vol Aeally 43 Exod and inboud are uot fe ‘ rnd Cp Lard Evol wak Hed eae qt intron 1 Aabrow Bone Tegative 1 dW Ae so resistance gene Selection marker wd Lesheu de YD d Intron 1 Construct 2 Construct 3 Exon 1 os “Antibiotic Negative resistance gene _| Selection marker | No } { | 1) Is there a way for you to study the function of gene A by altering its expression levels? If yes, explain. If no, explain why not j [ye Rnlat ble Wa omted reduc gene Coy while Battin geentact, 4Quention a (2 pains) | nih yagees ae fwcewor . Woot Ailend even yelane Innere Ga yriiain Phen peniige 1 mg 8p” mmm 09 so ome natin beng eyebens ae wwe e © fntton pan pean nents smnan vee i Countess Above | 4 schematic of « signaling cascade that 16 stimulated by hormone A. Hormone A binds to the receptor, which activates the G protein, ‘Tie activated G protein, in turn, activates Adenylyl cyclase, Activated Adenylyl eyelase stimulates the farmation of CAMP from ATP, CAMP binds to and activates Protein Kinase A, which stimulates 4 kinase sigaaing cascade and results in the cellular responses, a) The cellular responses Indicated usually occur only tn the presence of the hormone However, mutations that eliminate the need for the hormone oF Cause af activation of the signaling pathway result In an increased Cellular response, Kut an % nent ty wach of the following mutations if they result In an increased cellular response, ‘A mutation such that Adenylyl eyclase produces chMA? independent of the G protein 26 A cannot be activated ___ A mutation such that the proteins after Protein Kir ___ A mutation such that the receptor cannot bind the hormone ZX A mutation such that the G-protein fails to hydrolyze GTY A mutation such that the receptor activates the G protein independent of hormone binding A mutation such that the protein on which Protein Kinase A acts cannot be phosphorylated,b) Protein Kinase A adds phosphate groups to its target proteins, What molecule does Protein Kinase A use as a donor for the phosphate group? ! — 4 ~ phosohat f La i slod to aww aac ales g phosp ole ofl ofr TP anclranst0d Yo ann accel nt de Mara of Cubelale profecind Question 5 (6 points) f The following is the schematic of a signal transduction pathway that is activated by the binding of Epidermal growth factor (EGF), produced by one cell type, to its specific membrane receptor on a target cell. The major steps involved in this pathway are outlined below: + EGF ligand binds to the EGF receptor. + Ligand-bound EGF receptors become acti phosphorylation. Active EGF receptor causes Ras to exchange its bound GDP for GTP and become active. Active Ras activates the kinase cascade (RAF, MEK and MAPK) through phosphorylation. This cascade increases the expression of the c-myc gene which results in cell proliferation through homodimerization and subsequent EGF Receptor ® @ “—— @ FGF Plasma membrane par" —— emyc transeription Nucleus Cell Froliferation a) What would be the effect of each of the following treatments/mutations on cell s effect. ‘liferation? Explain why you would see this et — eoaeneree nutation that results in the constitutive homodimerization of the EGF receptor. oliforahsy Yi ae tecephor B aluvagh arch Jii, Treatment of cells with a Raf-specific phosphatase. Prolf J ble Loaf achvedss tha. pol “a lil, ‘Treatment of cells with PD9809, an inhibitor of MAPK that blocks its phosphorylation, Sof d ble no espn of cme b) Consider the following cells that have mutations in different components of the EGF signal transduction pathway, Mutant 1 (m1); Kas protein that continues to stay in its GDP-bound form. + Mutant 2 (m2): RAF protein that lacks its kinase domain, + Mutant 3 (m3); MAPK that lacks a nuclear localization sequence. Mutant 4 (m4): EGF receptor that lacks its extracellular domain. Mutant 5 (m5): MAPK that is constitutively phosphorylated at its active site. Mutant 6 (m6); c-myc gene that has a constitutively active promoter. Complete the table for each of the following homozygous double mutants in the presence of EGF relative to wild type (wt), Also explain the change in cell proliferation relative to wt cells. [ Mutations iw-[emye 7 Call proliferation increased / decreased /unchanged? Explain. | he cell expressed (Yes/No?) wtTwt Yes fmi/mt and | : m2/m2 Trachve (as 4 No Rat can't phosphorylee mo Tind and A Pc , ee Ya Tnorreccl. © me syotvesi eh aecprlemaric 47m4 and | ) | mms | Ye [Ineroaded, 7 |