Your employment contract at a Biotechnology Inc. is nearing its end. Before you move on to your next job, you have several unfinished projects that you need to complete. Your department has been busy of the last few days characterizing an interesting strain of bacteria that was discovered growing on some food that was served at the company’s annual holiday party.
The bacteria that your department has isolated can synthesize a molecule called sweetose and use it as a source of carbon from which it can derive its energy when glucose is unavailable. The sweetose operon contains 4 genes (A, B, C and D) that encode proteins needed for the biosynthesis of sweetose. The operon is only transcribed when the protein “sugarfree” (which is encoded by the SGF gene) is bound to a region of DNA located between -50 and -75 from the operon’s transcriptional start site. There are no other transcriptional regulators that control the expression of the sweetose operon.
Is the sweetose operon under positive or negative regulation? Briefly explain your choice. (2 pts)
Would the sweetose promoter be an example of a strong promoter or a weak promoter? Your answer must include a brief explanation of what dictates the strength of a promoter and an explanation of what distinguishes a strong promoter from a weak one. Provide one piece of supporting information from the question that helps explain your choice. (5 pts)
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If the expression of sugarfree is constitutive (it is always produced in the cell, no matter the conditions), how can the transcription of the sweetose operon be dependent on the availability of glucose? What is the role of glucose in this case? (3 pts)
The bacteria that your department has isolated contains another interesting operon. This operon encodes two enzymes (enzyme A and enzyme B) that help the bacteria breakdown complex carbohydrates so that it can survive in the human gut. There are four DNA sequences associated with this operon: 1, 2, 3 and 4. These sequences could be either protein-coding genes or regulatory sequences. You also know that the operon is under the control of the effector Y. You have introduced mutations in each of the regions 1-4 (assume that the mutations prevent the normal functioning of each region) and measured the expression of the proteins that are encoded by the operon genes A and B. Your results are shown below (“+” means that a functional protein is produced and “-” means that a functional protein is absent).
Effector Y is absent
Effector Y is present
Enzyme A
Enzyme B
Enzyme A
Enzyme B
No mutation
–
–
+
+
1
–
–
–
–
2
–
–
+
–
3
–
–
–
+
4
+
+
+
+
Match each of the sequences (1 to 4) to the correct element. (4 pts)
Element
Sequence #
Promoter (operon)
Operator (operon)
operon protein-coding gene A
operon protein-coding gene B
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What is the role of effector Y in this scenario? (2 pts)
Draw a picture of the organization of this operon and its associated sequences. You must include all cis-regulatory regions, their names, and the relative position. It is not necessary to draw the trans-acting proteins. (2 pts)
Excited by your preliminary results, you return your bacterial cells to the incubator and head home for the weekend. On Monday morning you make the unfortunate discovery that some of your bacterial samples in the incubator have been infected by pathogens.
You have managed to isolate and sequence the nucleic acids from the first pathogen of interest. The results revealed that they are composed of 40% A, 25% U, 15% G, 20% C. What does this reveal about the pathogen’s genome? Explain your thinking. (4 pts)
The second pathogen is bacteriophage lambda which has integrated into the host cell genome (prophage).
Identify the promoter(s) that is (are) active during this phase of the bacteriophage’s life cycle. (1 pt)
Which bacteriophage lambda gene would you need to turn off it order to switch to the lytic life cycle? Briefly explain your answer. (4 pts)
(do not write below this line)
You have less than three weeks left before you leave your position at Coutu Biotechnology Inc., so you push ahead with your experiments so that you can finish the second project that you are currently working on. This project involves the study of yeast cells and their response to DNA damaging agents.
You generate loss-of-function mutations in four proteins that are involved in the nucleotide excision repair pathway (the proteins are now non-functional). Match the mutated proteins below (loss of function) with the correct phenotype in the table. Briefly justify your choices. (6 points)
UvrC
UvrD
DNA Polymerase
DNA Ligase
Phenotype
Mutated proteins
Very brief justification
Damaged nucleotides are repaired but there is a larger number of nicks in the DNA.
Lots of damaged nucleotides that are unrepaired, but no nicks in the DNA.
Increase in the amount of single-stranded DNA regions.
Damaged nucleotides are not repaired but there is an increase in the amount of nicked DNA.
**Nick = a break in a single strand of the DNA double helix.
a) Very briefly explain the difference between an endogenous factor that causes mutations and an exogenous factor that causes mutations. (2 pts)
b) Provide one example of a mutagen that would be considered endogenous and one example of a mutagen that would be considered exogenous. (2 pts)
c) Give one example of a type of mutation that could be produced by an endogenous factor and an example of one type of mutations that could be produced by an exogenous factor. (2 pts)
Success! You have managed to complete all of you project before the end date of your contract. Since your supervisor was key in helping you obtain your next position, you have decided to create her a unique, genetically engineered population of yeast (eukaryotic) cells. With the holidays quickly approaching, you’ve thought it fitting to genetically engineer a cell that can fluorescence green under specific conditions.
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