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Sample_Exam_1 - MCB 121 2010 Sample Exam I Questions...

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1 MCB 121 2010 Sample Exam I Questions I) (Questions 1-4) Consider the following (simplified) pathways for head and tail formation for bacteriophage T4: One new temperature sensitive mutant (Gene X) of T4 has been isolated. All indications are that this mutant represents a new gene that has not been implicated before in T4 assembly. This mutant produces a complete and functional virus following infection of E. coli at 20°C or 30°C but not at 40°C. Following infection of E. coli at 20°C and 40°C, the following structures are observable in the Electron Microscope (EM): 1) Based upon the EM data alone, what possible steps might Gene X be involved in, with respect to viral assembly? a) Head maturation b) Tail maturation c) Head-tail joining d) Any of the above
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2 A number of double mutant viruses have been formed that contain the mutant gene X and several cold sensitive (cs) mutations in known genes that are involved in T4 head or T4 tail assembly. Remember that cs mutants produce viable phage at 40°C but not 20°C. Below Table I shows results of reciprocal temperature shift experiments performed using these double mutant viruses. For comparison, some infections are shown using either wild type virus or single mutant viruses. PFU stands for “Plaque Forming Units” and is a measure of the success of the infection. A PFU above 95 is considered a productive infection and a PFU below 5 is considered unproductive. Table I: Reciprocal temperature shift experiments Genotype of Phage T4 20°C shifted to 40°C (PFU) 40°C shifted to 20°C (PFU) Wild type 100 101 Gene X (ts) 99 100 Gene 21 (cs) 100 99 Gene X (ts) + Gene 21 (cs) 3 98 Gene X (ts) + Gene 4 (cs) 2 99 Gene X (ts) + Gene 13 (cs) 4 97 Gene X (ts) + Gene 14 (cs) 98 3 Gene X (ts) + Gene 5 (cs) 99 97 Gene X (ts) + Gene 54 (cs) 98 100 Gene X (ts) + Gene 18 (cs) 99 97 Gene X (ts) + Gene 15 (cs) 100 99 2) Based upon both the EM data as well as the data in Table I, how would you revise your answer to question 1? a) Head maturation b) Tail maturation c) Head-tail joining d) Any of the above 3) To which In Vitro Extract Group(s) is Gene X most likely to belong? 4) Gene X is likely to function following which other T4 Gene?
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3 II) (Questions 5-6) You have taken a job in a biotech company that studies the use of ± / ² tubulin filaments for use in clothing manufacturing. Your first assignment is to express the TUB2 gene from yeast in coli and purify the protein for further study. At your disposal is a PCR machine, a DNA oligonucleotide synthesizer, yeast genomic DNA, and an expression vector for E. coli that uses the promoter from bacteriophage T7. Shown below is a portion of the multiple cloning site (MCS) in which you will insert the coding region of TUB2 .
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