MIT Biology Department 7.012: Introductory Biology -Fall 2004 Instructors: Professor Eric Lander, Professor Robert A Weinberg, Dr. Claudette Gardel 7.012 Practice quiz 2 2004 Actual Quiz 2 (closed book) will be given Monday 10/25 at 10: 00 am No Sections on MONDAy or TUESDAy 10/25-10/26(No Kidding. NOTE THE ROOM MAy BE DIFFERENT THAN THE ROOM yOUR WERE ASSIGNED FOR QUIZ 1 Quiz Review Session Thursday, 10/21 7:00-9:00pm Tutoring Session Friday, 10/22 4:00-6:00pm
1 7.012 Practice Quiz 2 2004 Actual Quiz 2 (closed book) will be given Monday 10/25 at 10:00 am No Sections on MONDAY or TUESDAY 10/25-10/26 (No Kidding.) NOTE THE ROOM MAY BE DIFFERENT THAN THE ROOM YOUR WERE ASSIGNED FOR QUIZ 1 Quiz Review Session Thursday, 10/21 7:00 - 9:00 pm Tutoring Session Friday, 10/22 4:00 - 6:00 pm MIT Biology Department 7.012: Introductory Biology - Fall 2004 Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel
uestion 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 ODNA replication is initiated at promoter sequences in the dNA ii)RNA polymerase requires primers to initiate RNA synthesis ii) Okazaki fragments are the short fragments of dna that are produced on the leading strand at the dna replication fork iv)The 5 to 3 direction of DNA synthesis implies that deoxyribonucleotides are added to the 5 OH group on the growing strand v)Transcription is terminated at stop codons in the mRnA b)shown below is the DNA sequence of a gene from a virus that encodes a short viral peptide. Also hown is the sequence of the mRNa synthesized from this gene genomic DNA sequence 5 -AGCTCATGTGCGAGTCCTGACGCTGACTAGG-3 3 -TCGAGTACACGCTCAGGACTGCGACTGATCC-5 mature mRNA sequence(G = G cap) 5-G UCAUGUGCGAACGCUGACUAGGAAAAAAAA..-3 iIn the genomic DNA sequence shown above, draw a box around each of the two exons in the ii)In the mRNA above, some nucleotides are present that are not coded for in the genomic dNA sequence. Name the two processes that have occurred to add these nucleotides to the mrNA ii 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
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) DNA replication is initiated at promoter sequences in the DNA. ii) RNA polymerase requires primers to initiate RNA synthesis. iii) Okazaki fragments are the short fragments of DNA that are produced on the leading strand at the DNA replication fork. iv) The 5' to 3' direction of DNA synthesis implies that deoxyribonucleotides are added to the 5' OH group on the growing strand. v) Transcription is terminated at stop codons in the mRNA. b) Shown below is the DNA sequence of a gene from a virus that encodes a short viral peptide. Also shown is the sequence of the mRNA synthesized from this gene. genomic DNA sequence: 5'-AGCTCATGTGCGAGTCCTGACGCTGACTAGG-3' 3'-TCGAGTACACGCTCAGGACTGCGACTGATCC-5' mature mRNA sequence (G* = G cap): 5'-G*UCAUGUGCGAACGCUGACUAGGAAAAAAAA....-3' i) In the genomic DNA sequence shown above, draw a box around each of the two exons in the gene. ii) In the mRNA above, some nucleotides are present that are not coded for in the genomic DNA sequence. Name the two processes that have occurred to add these nucleotides to the mRNA. 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
U UUU phe(F UCU ser(S UAU UGU cys(c) UUc phe(F) UCc ser(s) UAc tyr (n) UGc cys( C) UUa leu(L) UAA STOP UGA STOP UUG leu L) UCG ser(s) UAG STOP UGG trp(w) cCU leu( L) pro(P) CGU arg (r) CUc leu l) Ccc pro(P) CAc his(H) cGc arg(R) CUA leu l) CCa pro(P) CAA CGa arg(R) CUG leu lL) (P) caG gIn( Q CGG (R) A| AUU ile(工) ACU thr(t) AAU asn(N) AUC ile (I) acc thr D) N AGUAGUcAG AUa ile(工 ACa thr D AAa lys(k) AGa arg(r) AUG AAG (K) Gau ala(a) GAU asp(D) GGu gly(G) GAc asp(D) gly GUA val (v Gca ala(a) gaa glu(e) GGa glyG) A gcg ala(a gag glu(e) Question 2 The term"central dogma"refers to the flow of biological information from dNa to RNA to protein RNA Prote 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 itWhat cellular machinery carries out each process? 1 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 RNA to protein. Streptomycin is a compound that affects the small ribosomal subunit in prokaryotes Streptomycin interferes with the binding of all Methionine -tRNAs to ribosomes. what two specific effects will streptomycin have on protein synthesis in prokaryotes?
3 U C A G U UUU phe (F) UUC phe (F) UUA leu (L) UUG leu (L) UCU ser (S) UCC ser (S) UCA ser (S) UCG ser (S) UAU tyr (Y) UAC tyr (Y) UAA STOP UAG STOP UGU cys (C) UGC cys (C) UGA STOP UGG trp (W) UC A G C CUU leu (L) CUC leu (L) CUA leu (L) CUG leu (L) CCU pro (P) CCC pro (P) CCA pro (P) CCG pro (P) CAU his (H) CAC his (H) CAA gln (Q) CAG gln (Q) CGU arg (R) CGC arg (R) CGA arg (R) CGG arg (R) UC A G A AUU ile (I) AUC ile (I) AUA ile (I) AUG met (M) ACU thr (T) ACC thr (T) ACA thr (T) ACG thr (T) AAU asn (N) AAC asn (N) AAA lys (K) AAG lys (K) AGU ser (S) AGC ser (S) AGA arg (R) AGG arg (R) U C A G G GUU val (V) GUC val (V) GUA val (V) GUG val (V) GCU ala (A) GCC ala (A) GCA ala (A) GCG ala (A) GAU asp (D) GAC asp (D) GAA glu (E) GAG glu (E) GGU gly (G) GGC gly (G) GGA gly (G) GGG gly (G) U C A G Question 2 The term "central dogma" refers to the flow of biological information from DNA to RNA to protein. DNA RNA Protein 1 2 3 a) i) In the spaces below, indicate the process that corresponds to each arrow. 1. ___________ 2. ___________ 3. ___________ ii) Name the initiation site for each processes, and on which molecule this site exists. 1. ________________2. ____________________3. ________________ iii) What cellular machinery carries out each process? 1. ___________ 2. ___________ 3. ___________ 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 RNA to protein. Streptomycin is a compound that affects the small ribosomal subunit in prokaryotes. Streptomycin interferes with the binding of all Methionine-tRNAs to ribosomes. What two specific effects will streptomycin have on protein synthesis in prokaryotes?
Question 3 The primer shown below is used to sequence the following template dNA template dna 5 -ACTGAC-3' 5 -ACCACTAACGTCAGT-3' Draw the resulting DNA 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 DNA length G T C (nts) 111111 54321098765432 B Polio has been practically eliminated from the American population, however, in countries have little or no access to vaccinations, it is still prevalent. As a biologist with a global seek to create a transgenic banana that produces the protein used in the vaccine against 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 dna sequences that will allow the gene to be incorporated into banana DNA. These sequences are contained in the pBan plasmid, which carries a gene for ampicillin resistance. Maps of these two plasmids are shown on the next page, including mportant restriction sites and distances (in base pairs) between the sites
4 Question 3 A. The primer shown below is used to sequence the following template DNA. primer: template DNA: 5'-ACTGAC-3' 5'-ACCACTAACGTCAGT-3' Draw the resulting DNA 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. G A T C + DNA length (nts) 15- 14- 13- 12- 11- 10- 9- 8- 7- 6- 5- 4- 3- 2- 1- B. Polio has been practically eliminated from the American population, however, in countries where people have little or no access to vaccinations, it is still prevalent. As 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 DNA sequences that will allow the gene to be incorporated into banana DNA. These sequences are contained in the pBAN 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
Question 3 continued Banana specific BamHI promoter 120 EcoRI Bglll gene for polio antigen pBAN KR-polio 280 8900bp 4500bp kan gene banana insertion sequences which allow dna to integrate into na genome Bg1I工 BamH工: ECORI 5AVG A T GG A A ATT C. C a)An end generated by digestion with BamHI can be ligated to an end generated by digestion with BgIII. Why is this possible? b)you want to insert the gene encoding the polio antigen into pBAN. Devise a strategy to accomplish this. Identify the enzyme(s)you would use to cut pBAN, the enzyme (s) you would use to cut pkR olio, 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) both ampicillin and kanamycin neither ampicillin nor kanamycin Why?
5 Question 3 continued ori amp gene R Banana specific promoter banana insertion sequences which allow DNA to integrate into banana genome BamHI EcoRI pBAN 8900 bp 120 pKR-polio 4500 bp ori kan gene R BglII BglII BamHI start codon stop codon gene for polio antigen EcoRI 280 1120 BglII: BamHI: EcoRI: 5'...A G A T C T...3' 5'...G G A T C C...3' 5'...G A A T T C...3' 3'...T C T A G A...5' 3'...C C T A G G...5' 3'...C T T A A G...5' a) An end generated by digestion with BamHI can be ligated to an end generated by digestion with BglII. Why is this possible? b) You want to insert the gene encoding the polio antigen into pBAN. Devise a strategy to accomplish this. Identify the enzyme(s) you would use to cut pBAN, the enzyme(s) you would use to cut pKRpolio, 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 kanamycin both ampicillin and kanamycin neither ampicillin nor kanamycin Why?
Question 3 continued you isolate plasmid DNA from three colonies that pass your antibiotic resistance test. you diges DNA with the restriction enzyme EcoRI. you size separate the resulting fragments from each ple on an agarose gel. you find the following results. DNA fragment sizes are indicated to the left 1.2kb 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 Question 3 continued You isolate plasmid DNA from three colonies that pass your antibiotic resistance test. You digest the DNA with the restriction enzyme EcoRI. You size separate the resulting fragments from each plasmid on an agarose gel. You find the following results. DNA fragment sizes are indicated to the left. 10 kb 9 kb .4 kb 1.2 kb 1 2 3 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
Question 4 In E. coli, the fictitious AB operon is induced by the presence of Compound w. a diagram of the operon, its regulatory proteins and regulatory sites is shown below P gene a gene B PX promoter for the regulatory protein x gene for the regulatory protein of the AB operon P promoter for the aB genes sequence shown to be important for regulation by w structural gene for enzyme A structural gene for enzyme B The following table shows the genotypes of different E. coli strains with a wild-type AB operon and various mutant AB operons, and the number of molecules of protein a 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. Assume the genes not listed are wild Molecules of Strain E xpression Wild type 0200 inducible M1 200200 M2 0 200200 0 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 AB operon? Explain your reasoning
7 Question 4 In E. coli, the fictitious AB operon is induced by the presence of Compound W. A diagram of the operon, its regulatory proteins and regulatory sites is shown below: gene X gene A P P S X gene B PX promoter for the regulatory protein X gene for the regulatory protein of the AB operon P promoter for the AB genes S sequence shown to be important for regulation by W A structural gene for enzyme A B structural gene for enzyme B The following table shows the genotypes of different E. coli strains with a wild-type AB operon and various mutant AB operons, and the number of molecules of protein A 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. Assume the genes not listed are wild type. Molecules of A Strain X P S A -W +W Expression Wild type + + + + 0 200 inducible M1 - + + + 200 200 M2 + - + + 0 0 M3 + + - + 200 200 M4 + + + - 0 0 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 AB operon? Explain your reasoning
Question 4, continued b)you make partial diploids of various E. coli mutant strains using a single-copy plasmid introduced into the E. coli cell. In cells of the following genotypes, predict the number of molecules of enzyme A per cell (0, 200, 400) produced in the absence or presence of Compound w(w or +w respectively). Put the total number of molecules of enzyme a on the lines provided #f of molecules of enzymes a per cell 200 X+ P+ S+A+ X* P* S A X+ P+ St A X P+ S+ A X* P* S A
8 Question 4, continued b) You make partial diploids of various E. coli mutant strains using a single-copy plasmid introduced into the E. coli cell. In cells of the following genotypes, predict the number of molecules of enzyme A per cell (0, 200, 400) produced in the absence or presence of Compound W (–W or +W, respectively). Put the total number of molecules of enzyme A on the lines provided. # of molecules of enzymes A per cell Genotype –W +W X+ P+ S+ A+ 0 400 X+ P+ S+ A+ X+ P+ S+ A+ 0 200 X+ P+ S+ A– X+ P+ S+ A+ X– P+ S+ A+ _____ _____ X+ P+ S– A+ X+ P+ S+ A+ _____ _____ X– P+ S+ A+ X+ P+ S+ A– _____ _____ X+ P+ S– A+ X+ P+ S+ A– _____ _____ X+ P+ S– A– X+ P+ S+ A+ _____ _____
Question 5 structure of this gene are known. the start codon is in exon 3 the stop codon is in exon y 3,3 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 exor A 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) Allele HindIII PstI HindIlI Eagl HindIII 280bp180bp150bp100bp exon 12 lexon 13 [ exon 14 PCR rimer PCR primer Using these PCR primers, you amplify dna 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 DNA fragment from normal male Hindlll E Psti HindIn Eag Hindlll 80 bp 180 bp 30b PCR-amplified DNA fragment from hemophiliac brother Hindm Pstl HindI 20 bp 280 bp 100 bp 30 bp Briefly describe the likely dNa alteration in the hemophiliac
9 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. A 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. exon 12 exon 13 exon 14 Wild-type(normal) Allele: PCR primer #1 PCR primer #2 HindIII PstI HindIII EagI HindIII 280 bp 180 bp 150 bp 100 bp Using these PCR primers, you amplify DNA 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 DNA fragment from normal male: HindIII PstI HindIII HindIII EagI 20 bp 280 bp 180 bp 150 bp 100 bp 30 bp PCR-amplified DNA fragment from hemophiliac brother: HindIII PstI HindIII 20 bp 280 bp 100 bp 30 bp Briefly describe the likely DNA alteration in the hemophiliac
Question 6 Use this diagram of a cell as a guide when answering the following questions membrane transport vesicle nucleus secretory endoplasmi reticulum(er) Golgi extracellular a)Consider the following receptor protein ligand-binding signaling domain COO signal sequence transmembrane i Where would this receptor protein be localized if it's the cell was lacking SRP? Would it be functional? iiWhere would this receptor protein be localized if its signaling domain were deleted? Would it be functional? iii what common characteristic do the side chains of the amino acids found in a transmembrane domain share. and wh iv)Which domain (ligand binding transmembrane, or signaling) of the wild-type receptor protein is inserted into the ER lumen during translation? Why must the receptor protein be inserted in this fashion to become functional? b)The mature mRNA encoding a protein destined for secretion specifies a polypeptide chain of 497 amino acids; yet the protein that is actually secreted by the cell is 475 amino acids long i How can you account for this size difference? ii)would a transmembrane protein also show this disparity? Briefly explain your answer
10 Question 6 Use this diagram of a cell as a guide when answering the following questions. nucleus endoplasmic reticulum (ER) plasma membrane Golgi transport vesicle secretory vesicle cytosol extracellular space a) Consider the following receptor protein: H3N + COO ligand-binding domain transmembrane signaling domain – domain signal sequence i) Where would this receptor protein be localized if it’s the cell was lacking SRP? Would it be functional? ii) Where would this receptor protein be localized if its signaling domain were deleted? Would it be functional? iii) What common characteristic do the side chains of the amino acids found in a transmembrane domain share, and why? iv) Which domain (ligand binding, transmembrane, or signaling) of the wild-type receptor protein is inserted into the ER lumen during translation? Why must the receptor protein be inserted in this fashion to become functional? b) The mature mRNA encoding a protein destined for secretion specifies a polypeptide chain of 497 amino acids; yet the protein that is actually secreted by the cell is 475 amino acids long. i) How can you account for this size difference? ii) Would a transmembrane protein also show this disparity? Briefly explain your answer