MIT Biology Department Instructors: Professor Eric Lander, Professor Robert A Weinberg, Dr. Claudette Gardel your Name TA 7.012 Quiz 2 Answers A≥85 12% of test takers B272 w31.2% of test takers C260 w34.1% of test takers D≥50 16. 3% of test takers F249 w6.2%of test takers grade requests (with a note attached indicating the problem and part you want looked at)accepted until Thursday November 4, 5p Question alue Score 17 16 234-5 30 17 20 100
Your Name: _______________________________________TA:______________ 7.012 Quiz 2 Answers A≥85 ~12% of test takers B≥72 ~31.2% of test takers C≥60 ~34.1% of test takers D≥50 ~16.3% of test takers F≥49 ~6.2% of test takers Regrade requests (with a note attached indicating the problem and part you want looked at) accepted until Thursday November 4th, 5pm. Question Value Score 1 17 2 16 3 30 4 17 5 20 100 MIT Biology Department 7.012: Introductory Biology - Fall 2004 Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel
Question 1 In the bacterium Funditus fabricatus, the metabolism of the sugar ridiculose is dependent on the rid operon shown below P Control region ridU rid ridX rid The ridiculose operon encodes the enzymes shown in the following pathway Rid U Rid v Rid x Ridiculose Ridiculose Ridiculose-5P Seriose-5P (outside cell) (inside cell) (used by cell) The rid gene is constitutively expressed. The expression of ridU, rid, and ridX genes is off in the absence of ridiculose and is activated by the product of the ridw gene in the presence of ridicules There is an artificial inducer of ridUVX expression called GIG-L, and an artificial substrate for Rid X, called stRN that turns red in the presence of active Rid X protein. grown in the presence of STRN, with and without the addition of the inducer, GIG-L. 10 pts? a)Several specific Rid -mutants of F. fabricatus are shown below. Predict their phenotypes wh Fill in the chart with either red or WHITe) Strain of f. Fabricate + GIG-L GIG-L WT WHITE M1(deletion of Puvx) White White M2(control region that can,'t bind Ridw protein) White White M3(Ridw protein that can't bind ridiculose White White M4 (nonsense mutation early in ridX) White White M1337(RidW protein that always binds control region) Red Red
2 Question 1 In the bacterium Funditus fabricatus, the metabolism of the sugar ridiculose is dependent on the rid operon shown below. The ridiculose operon encodes the enzymes shown in the following pathway. Rid U Rid V Rid X Ridiculose Ridiculose Ridiculose-5P Seriose-5P (outside cell) (inside cell) (intermediate) (used by cell) The ridW gene is constitutively expressed. The expression of ridU, ridV, and ridX genes is off in the absence of ridiculose and is activated by the product of the ridW gene in the presence of ridiculose. There is an artificial inducer of ridUVX expression, called GIG-L, and an artificial substrate for Rid X, called STRN that turns red in the presence of active Rid X protein. a) Several specific Rid- mutants of F. fabricatus are shown below. Predict their phenotypes when grown in the presence of STRN, with and without the addition of the inducer, GIG-L. 10 pts (Fill in the chart with either RED or WHITE) Strain of F. Fabricatus + GIG-L - GIG-L _______________________________________________________________________ WT RED WHITE _______________________________________________________________________ M1 (deletion of PUVX) White White _______________________________________________________________________ M2 (control region that can’t bind RidW protein) White White _______________________________________________________________________ M3 (RidW protein that can’t bind ridiculose) White White _______________________________________________________________________ M4 (nonsense mutation early in ridX) White White _______________________________________________________________________ M1337 (RidW protein that always binds control region) Red Red ________________________________________________________________________ ridU ridV ridX ridW P PW UVX Control region
Name TA b)Predict whether the following F. fabricatus strains, that are merodiploid for the ridiculose operon, will grow on minimal media with or without ridiculose as the only carbon source Fill in the chart with yEs if the merodiploid will GROW or No if the merodiploid will NOT GROW 7 Pts (5 points for the first column, 2 pts for entire last column) eroalbIol Ridiculose Ridiculose WT/WT yES NO M1/M2 (deletion of Puvx NO control region that can't bind Ridw) M2/M3 ( control region that can't bind Ridw) yES NO (Ridw protein that can't bind ridiculose) M3/M1337 (Ridw protein that can't bind ridiculose) yES NO (RidW protein that always binds control region) M4/M1 sense mutation early in rid) (deletion of Puvx M1337/M4 (RidW protein that always binds control region) (nonsense mutation early in ridX)
Name:___________________________________ TA:_______________________ 3 b) Predict whether the following F. fabricatus strains, that are merodiploid for the ridiculose operon, will grow on minimal media with or without ridiculose as the only carbon source. Fill in the chart with YES if the merodiploid will GROW or NO if the merodiploid will NOT GROW 7 pts (5 points for the first column, 2 pts for entire last column) Merodiploid +Ridiculose - Ridiculose _______________________________________________________________ WT / WT YES NO _______________________________________________________________________ M1 / M2 (deletion of PUVX) NO NO (control region that can’t bind RidW) _______________________________________________________________________ M2/ M3 (control region that can’t bind RidW) YES NO (RidW protein that can’t bind ridiculose) _______________________________________________________________________ M3 / M1337 (RidW protein that can’t bind ridiculose) YES NO (RidW protein that always binds control region) _______________________________________________________________________ M4 / M 1 (nonsense mutation early in ridX) NO NO (deletion of PUVX) _______________________________________________________________________ M1337 / M4 (RidW protein that always binds control region) YES NO (nonsense mutation early in ridX) _______________________________________________________________
uestion 2 you believe that a disruption in a gene, soks, may contribute to an interesting disease phenotype in cardinals. you wish to PCR amplify and sequence soks from both wild type and diseased cardinals. the sok s gene is shown below. Exons are represented by numbered boxes and the terminal sequences are depicted in bold Start of End of 5'CTCGAGGTAT (1) (3)GTAATCGATA 3 3 GAGCTCCATA (2) 4)CATTAGCTAT 5 a)For PCR amplification, the primers should be identical to which of the following sequences?(Circle one. )3 pts 1 and 3 2 and 4 2 and 3 b)Which primer(s) should you use in one sequencing reaction to obtain sequence that looks most similar to the mRNA (the coding strand)? 2 pts you use the dideoxy sequencing method to sequence your PCR products. you see the following pattern in the sequencing gel representing the sequence spanning the end of the first intron and the beginning of exon 2 Wild Type ( WT) Diseased (D) dda ddc ddg ddT dda ddc ddg ddT c)These gels correspond to which WT and diseased sequences respectively?(WT, D)(Circle i, ii, iii, or iv i)5′- ACTGGAAC-3’,5′- ACTTGAAC-3 i1)5′- GACCTTG-3’,5′- IGAACTG-3 [iii)5'-CAAGGTCA-3,5'-CAAGTTCA-3 iv)5′- GTTCCAGT-3′,5′-5′- GTCAAGT-3
4 Question 2 You believe that a disruption in a gene, sokS, may contribute to an interesting disease phenotype in cardinals. You wish to PCR amplify and sequence sokS from both wild type and diseased cardinals. The sokS gene is shown below. Exons are represented by numbered boxes and the terminal sequences are depicted in bold. 5'CTCGAGGTAT (1) (3) GTAATCGATA 3' 3'GAGCTCCATA (2) (4) CATTAGCTAT 5' a) For PCR amplification, the primers should be identical to which of the following sequences? (Circle one.) 3 pts 1 and 3 2 and 4 1 and 4 2 and 3 b) Which primer(s) should you use in one sequencing reaction to obtain sequence that looks most similar to the mRNA (the coding strand)? 2 pts 1 2 3 4 You use the dideoxy sequencing method to sequence your PCR products. You see the following pattern in the sequencing gel representing the sequence spanning the end of the first intron and the beginning of exon 2. Wild Type (WT) Diseased (D) ddA ddC ddG ddT ddA ddC ddG ddT c) These gels correspond to which WT and diseased sequences respectively? (WT, D) (Circle i, ii, iii, or iv.) 4 pts i) 5’-ACTGGAAC-3’, 5’-ACTTGAAC-3’ ii) 5’-TGACCTTG-3’, 5’-TGAACTTG-3’ iii) 5’-CAAGGTCA-3’, 5’-CAAGTTCA-3’ iv) 5’-GTTCCAGT-3’, 5’-5’-GTTCAAGT-3’ 1 2 3 Start of gene End of gene
Name: TA To determine how this mutated sequence might affect the mRNa and the protein product, you obtain tw cDNA libraries: one derived from wild type cardinal RNa and one derived from diseased cardinal rna d)you choose to use a radioactively labeled DNa probe to screen these cDNA libraries for clones containing soks. Which region of soks would make the best probe for screening the available cDNA libraries?( Circle either Region A, Region B, or Region C )4 pts Region Region B Region C 100 200 400 Distances in base pairs your probe hybridizes to cDNA clones in both cDNA libraries. you purify the plasmids from single clones and cut them with the restriction enzyme used for cloning to verify insert size. The gel is shown below Ladder WT Diseased 1000bp ← Vector 700bp 500b 300 e)Based on all evidence above, what is the most likely explanation for the difference in restriction enzyme digestion patterns of the soks cDNA clones? 3 pts i)A mutation in the soks cDNa from the diseased cardinal cDNA library gives rise to an additional restriction enzyme site i The mRNA encoded by the soks gene from the diseased cardinal is incorrectly spliced ii There is likely a problem with the gel and it should be rerun. iv) The soks gene from the diseased cardinal acquired a spontaneous insertion v The mRNa encoded by the soks gene from the diseased cardinal poly A tail
Name:___________________________________ TA:_______________________ 5 To determine how this mutated sequence might affect the mRNA and the protein product, you obtain two cDNA libraries: one derived from wild type cardinal RNA and one derived from diseased cardinal RNA. d) You choose to use a radioactively labeled DNA probe to screen these cDNA libraries for clones containing sokS. Which region of sokS would make the best probe for screening the available cDNA libraries? (Circle either Region A, Region B, or Region C.) 4 pts Region Region B Region C 100 100 40 200 400 50 Distances in base pairs Your probe hybridizes to cDNA clones in both cDNA libraries. You purify the plasmids from single clones and cut them with the restriction enzyme used for cloning to verify insert size. The gel is shown below. Ladder WT Diseased e) Based on all evidence above, what is the most likely explanation for the difference in restriction enzyme digestion patterns of the sokS cDNA clones? 3 pts i) A mutation in the sokS cDNA from the diseased cardinal cDNA library gives rise to an additional restriction enzyme site. ii) The mRNA encoded by the sokS gene from the diseased cardinal is incorrectly spliced. iii) There is likely a problem with the gel and it should be rerun. iv) The sokS gene from the diseased cardinal acquired a spontaneous insertion. v) The mRNA encoded by the sokS gene from the diseased cardinal has no poly A tail. 1000 bp 700 bp 500 bp 300 bp 50 bp Vector 1 2 3