7.012 Practice Quiz

Transcription

1 7.012 Practice Quiz ctual Quiz 2 (closed book) will be given Monday 10/24 at 10:00 am No Sections on MONDY or TUESDY 10/24-10/25 NOTE THT THE ROOM MY BE DIFFERENT THN THE ROOM YOUR WERE SSIGNED FOR QUIZ 1 Student's last name Location of Quiz boukhalil - Gupta Room Han - Zorich Walker Gym -Rm Quiz Review Session Thursday, 10/20 7:00-9:00 pm Tutoring Session Friday, 10/21 4:00-6:00 pm Weekly Discussion Groups Thurs noon, 2pm, 4pm in and on next Fri 10/21 at noon in Tutors: Christopher Hemond chemond@mit.edu driana Tajonar atajonar@mit.edu Yiyan Jiang y_jiang@mit.edu Xiaoyu (Sylvia) Yang syang7@mit.edu John Cassady jcass@mit.edu lbert Kwon alkwon@mit.edu Raj Bhalla rbhalla@mit.edu my Lee missamy@mit.edu Jennifer Huang jen5720@mit.edu Office Hours. Each office hour listed lasts one hour. Eager helpers Margaret ckerman Palowski mack7@mit.edu Office Hour Wed 5 pm, Stata Cafe Ericka nderson erickaa@mit.edu Wed 9 pm, 1st fl Student Center Garrett Frampton frampton@mit.edu Tue 4 pm, Emily Miller esmiller@mit.edu Tues 8 pm, 1st fl Student Center Shlomo Meislin shm@mit.edu Mon 5:30 pm, Hung Nguyen nguyenhn@mit.edu Wed 4 pm, Renuka Sastry renuka@mit.edu Wed 6 pm, Sebastian Treusch STR@mit.edu Tues 5 pm, Bio Café, Blg 68 Claudette Gardel cgardel@mit.edu Wed 2:30, d 1

2 Question 1 a) Indicate whether each of the following statements is true or false. If false, correct the statement or provide a brief explanation for why it is false. i) DN replication is initiated at promoter sequences in the DN. ii) RN polymerase requires primers to initiate RN synthesis. iii) Okazaki fragments are the short fragments of DN that are produced on the leading strand at the DN replication fork. iv) The 5' to 3' direction of DN synthesis implies that deoxyribonucleotides are added to the 5' OH group on the growing strand. v) Transcription is terminated at stop codons in the mrn. b) Shown below is the DN sequence of a gene from a virus that encodes a short viral peptide. lso shown is the sequence of the mrn synthesized from this gene. genomic DN sequence: 5'-GCTCTGTGCGGTCCTGCGCTGCTGG-3' 3'-TCGGTCCGCTCGGCTGCGCTGTCC-5' mature mrn sequence (G * = G cap): 5'-G*UCUGUGCGCGCUGCUGG...-3' i) In the genomic DN sequence shown above, draw a box around each of the two exons in the gene. ii) In the mrn above, some nucleotides are present that are not coded for in the genomic DN sequence. Name the two processes that have occurred to add these nucleotides to the mrn. iii) How many amino acids are in the viral peptide encoded by this gene? iv) Is this virus more likely to replicate in prokaryotic or eukaryotic cells? Briefly explain your reasoning. 2

3 U UUU phe (F) UUC phe (F) UU leu (L) UUG leu (L) C CUU leu (L) CUC leu (L) CU leu (L) CUG leu (L) UU ile (I) UC ile (I) U ile (I) UG met (M) G GUU val (V) GUC val (V) GU val (V) GUG val (V) U C G UCU UCC UC UCG CCU CCC CC CCG CU CC C CG GCU GCC GC GCG pro (P) pro (P) pro (P) pro (P) thr (T) thr (T) thr (T) thr (T) ala () ala () ala () ala () UU tyr (Y) UC tyr (Y) U STOP UG STOP CU CC C CG U C G GU GC G GG his (H) his (H) gln (Q) gln (Q) asn (N) asn (N) lys (K) lys (K) asp (D) asp (D) glu (E) glu (E) UGU cys (C) UGC cys (C) UG STOP UGG trp (W) CGU CGC CG CGG GU GC G GG GGU GGC GG GGG gly (G) gly (G) gly (G) gly (G) UC G UC G U C G U C G Question 2 The term "central dogma" refers to the flow of biological information from DN to RN to protein. 2 3 DN RN Protein 1 a) i) In the spaces below, indicate the process that corresponds to each arrow ii) Name the initiation site for each processes, and on which molecule this site exists iii) What cellular machinery carries out each process? b) What is a gene? Please answer in one sentence. The first sentence written will be considered as your answer. c) Many antibiotics are compounds that interfere with the transfer of genetic information from RN to protein. Streptomycin is a compound that affects the small ribosomal subunit in prokaryotes. Streptomycin interferes with the binding of all Methionine-tRNs to ribosomes. What two specific effects will streptomycin have on protein synthesis in prokaryotes? 3

4 Question 3. The primer shown below is used to sequence the following template DN. primer: template DN: 5'-CTGC-3' 5'-CCCTCGTCGT-3' Draw the resulting DN fragments that would be produced from each of the 4 sequencing reactions at the correct position (length in nucleotides) as they would appear on the diagram of the sequencing gel below. DN length (nts) G T C + B. Polio has been practically eliminated from the merican population, however, in countries where people have little or no access to vaccinations, it is still prevalent. s a biologist with a global vision, you seek to create a transgenic banana that produces the protein used in the vaccine against polio. By consuming these bananas, individuals will develop immunity against the disease. The gene for this protein has already been cloned into a plasmid with a kanamycin-resistance gene (pkr-polio). You need to attach to the gene a banana-specific promoter and DN sequences that will allow the gene to be incorporated into banana DN. These sequences are contained in the pbn plasmid, which carries a gene for ampicillin resistance. Maps of these two plasmids are shown on the next page, including important restriction sites and distances (in base pairs) between the sites. 4

5 Question 3 continued Banana specific promoter BamHI 120 BglII start codon BamHI pbn 8900 bp ori 1120 pkr-polio bp gene for polio antigen stop codon BglII R amp gene ori R kan gene banana insertion sequences which allow DN to integrate into banana genome BglII: BamHI: : 5'... G T C T...3' 5'...G G T C C...3' 3'...T C T G...5' 3'...C C T G G...5' 5'...G T T C...3 3'...C T T G...5 a) n end generated by digestion with BamHI can be ligated to an end generated by digestion with BglII. Why is this possible? b) You would like to insert the gene encoding the polio antigen into pbn. Devise a strategy to accomplish this. Identify the enzyme(s) you would use to cut pbn, the enzyme(s) you would use to cut pkr-polio, and the steps necessary to generate the intact plasmid. c) You next transform E. coli with the plasmids you have made. You grow the transformed cells on media containing (circle one): ampicillin both ampicillin and kanamycin kanamycin neither ampicillin nor kanamycin Why? 5

6 Question 3 continued You isolate plasmid DN from three colonies that pass your antibiotic resistance test. You digest the DN with the restriction enzyme. You size separate the resulting fragments from each plasmid on an agarose gel. You find the following results. DN fragment sizes are indicated to the left. 10 kb 9 kb kb.4 kb d) i) Draw plasmids associated with the colonies 1, 2, and 3. Indicate all relevant features such as the promoter, the origin of replication, the genes for ampicillin resistance and the polio antigen, ii) Which of the three plasmids would allow synthesis of the protein in a banana? Explain your reasoning. 6

7 Question 4 In E. coli, the fictitious B operon is induced by the presence of Compound W. diagram of the operon, its regulatory proteins and regulatory sites is shown below: PX X P S B P X gene X gene P S promoter for the regulatory protein gene for the regulatory protein of the B operon promoter for the B genes sequence shown to be important for regulation by W structural gene for enzyme structural gene for enzyme B gene B The following table shows the genotypes of different E. coli strains with a wild-type B operon and various mutant B operons, and the number of molecules of protein per cell in the absence or presence of Compound W ( W or +W, respectively). a) In the table below, the symbol "+" indicates that the gene or control element is functional (wt) and " " indicates that the gene or control element is non-functional. ssume the genes not listed are wild type. Molecules of Strain X P S -W +W Expression Wild type inducible M M M M i) For each strain on the table above, label the expression as either inducible, uninducible or constitutive. ii) Based on the data shown above, does the regulatory protein X act as a repressor or an activator of the B operon? Explain your reasoning. 7

8 Question 5 You are studying a family with hemophilia, a sex-linked recessive disease, caused by mutations in the Factor VIII gene. The Factor VIII gene contains 35 exons. The complete sequence and exon/intron structure of this gene are known. The start codon is in exon 3; the stop codon is in exon 34. partial restriction map and a diagram showing the location of exons 12, 13 and 14 is shown below. You synthesize two PCR primers, which anneal to sequences located within exons 12 and 14, as shown. Wild-type(normal) llele: HindIII PstI HindIII EagI HindIII 280 bp 180 bp 150 bp 100 bp exon 12 exon 13 exon 14 PCR primer #1 PCR primer #2 Using these PCR primers, you amplify DN from a normal male and his hemophiliac brother. You determine the restriction map for the PCR product from these two individuals, shown below: PCR-amplified DN fragment from normal male: HindIII PstI HindIII EagI HindIII 20 bp 280 bp 180 bp 150 bp 100 bp 30 bp PCR-amplified DN fragment from hemophiliac brother: HindIII PstI HindIII 20 bp 280 bp 100 bp 30 bp Briefly describe the likely DN alteration in the hemophiliac. 8

9 NSWERS Question 1 a) i) FLSE. DN replication is initiated at the origin of replication. RN polymerases bind to promoter sequences to initiate transcription. ii) FLSE. RN polymerase does not require primers to initiate RN synthesis. DN polymerase requires primers to initiate DN replication. iii) FLSE. Okazaki fragments are made on the lagging strand at the replication fork. iv) FLSE. DN synthesis occurs by addition of dntps to the 3' OH group of the nucleotide at the end of the growing strand. v) FLSE. Transcription terminates at the transcription termination sites in the DN. Translation terminates at stop codons in the mrn. b) genomic DN sequence: 5'-GCTCTGTGCGGTCCTGCGCTGCTGG-3' 3'-TCGGTCCGCTCGGCTGCGCTGTCC-5' mature mrn sequence (G * = G cap): start codon stop codon 5'-G*UCUGUGCGCGCUGCUGG...3' met cys glu arg i) see DN sequence above ii) 1) 5' capping 2) 3' polyadenylation iii) There are four amino acids in this viral peptide: NH3 + -met-cys-glu-arg-coo - iv) In eukaryotic cells because the RN processing and splicing machinery is only present in eukaryotes. Question 2 The term "central dogma" refers to the flow of biological information from DN to RN to protein. 2 3 DN RN Protein 1 a) i) In the spaces below, indicate the process that corresponds to each arrow. 1. replication 2. transcription, 3. translation 9

10 ii) Name the initiation site for each processes, and on which molecule this site exists. 1. Origin of replication, DN 2. Promoter, DN 3. Start codon (first UG), mrn iii) What cellular machinery carries out each process? 1. DN polymerase 2. RN polymerase 3. ribosome b) What is a gene? Please answer in one sentence. The first sentence written will be considered as your answer. gene is a segment of DN containing information for directing the synthesis of a protein (or RN). c) What two specific effects will streptomycin have on protein synthesis in prokaryotes? Streptomycin will prevent the correct initiation of protein synthesis since it prevents association of the met-trn with the ribosome. Streptomycin will also lead to inaccurate translation (insertion of incorrect amino acids) in those proteins that were in the process of being translated. Question 3. primer: template DN: 5'-CTGC-3' 5'-CCCTCGTCGT-3' DN length (nts) G T C + a) n end generated by digestion with BamHI can be ligated to an end generated by digestion with BglII. Why is this possible? Digestion with BglII or BamHI produce the same overhangs, or sticky ends. Thus the ends produced by cutting with BglII are complementary to the ends produced with BamHI, base pairing and ligation can occur. b) You want to insert the gene encoding the polio antigen into pbn. Devise a strategy to accomplish this. Identify the enzyme(s) you would use to cut pbn, the enzyme(s) you would use to cut pkr-polio, and the steps necessary to generate the intact plasmid. Cut pbn with BamHI to linearize. Cut pkr-polio with BglII, this gives a 1400 bp fragment containing the gene for the polio antigen and a 3100 bp fragment. Size select the DN from the pkr-polio plasmid to obtain the 1400 bp fragment. Ligate the 1400 bp fragment together with the cut pbn plasmid. c) You next transform E. coli with the plasmids you have made. You grow the transformed cells on media containing (circle one): ampicillin kanamycin both ampicillin and kanamycin neither ampicillin nor kanamycin 10

11 Why? The media should contain ampicillin only, because the plasmid with the promoter and banana insertion sequences has a gene for ampicillin resistance. d) i) Draw plasmids 1, 2, and 3, indicating all relevant features such as the promoter, the origin of replication, the genes for ampicillin resistance and the polio antigen, restriction sites and distances (in base pairs) between the sites. Banana specific BamHI/BglII promoter stop codon 280 polio antigen gene 1120 BamHI start codon plasmid BamHI/BglII Banana specific promoter plasmid 2 BamHI/BglII start codon BamHI polio antigen gene stop codon BamHI/BglII R amp gene R amp gene ori ori Banana specific promoter BamHI 120 plasmid 3 pbn B. R amp gene ii) Which of the three plasmids would allow synthesis of the protein in a banana? Explain yor reasoning. Only plasmid two would allow synthesis of functional protein. The polio antigen gene must be correctly oriented with respect to the promoter..question 4 i) Strain X P S -W +W Expression ori Wild type inducible M constitutive M uninducible M constitutive M uninducible ii) Protein X acts as a repressor. In mutants 1 and 3 that lack either the repressor or the repressor binding site, expression is constitutive (high with or without W). Question 5 There is a deletion of 330 bp in the DN, including exon 13 and possibly part of exon 14, which also removes a Hin d III site and an Eag I site. 11