BIO1140 Final Exam 2020 Answer KEY PDF

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This exam deals asks you to consider a novel cell biology situation. Throughout the exam you will find pertinent background information about this scenario. You are expected to use the provided information AND apply the knowledge you have acquired throughout BIO1140 to answer the questions posed. Yo...

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This exam deals asks you to consider a novel cell biology situation. Throughout the exam you will find pertinent background information about this scenario. You are expected to use the provided information AND apply the knowledge you have acquired throughout BIO1140 to answer the questions posed. You must answer all of the questions below. Total = 60 Marks Melanocytes are a specialized cell type that gives rise to the colour in the skin, hair, and eyes of many organisms including humans. A melanocyte found in the skin has an ovoid cell body with dendritic processes that make contact with 30-40 keratinocytes, the major cell type of the skin (Figure 1). Melanocytes contain a membrane-bound, lysosome-related organelle known as a melanosome. Similar to lysosomes, melanosome use proton pumps to actively maintain an acidic internal pH. The acidic environment is required for the synthesis and storage of melanins, the most common family of light-absorbing pigments in the animal kingdom. Maturation of melanosomes occurs in stages (I to IV), characterized by progressive melanin synthesis and transport of the organelle toward the cell periphery. Once fully matured (stage IV), melanosomes are transferred to keratinocytes (see the zoomed-in region of figure 1). It is the melanin contained within the keratinocytes of the skin or hair that ultimately determine their colouration. Within melanocytes, mitochondria-melanosome tethering via 20-30 nm long fibrillar bridges has been observed and is depicted in the figure below. This tethering is more frequently observed in immature (stage I and II) melanosomes and evidence suggests that it helps to ensure a local and timely supply of ATP to immature melanosomes which have higher energy requirements associated with organelle transport, melanin synthesis, or maintenance of intramelanosomal pH.

Figure 1

1. You have identified an individual with a genetic mutation that prevents the actin protein from polymerizing in their melanocytes. Would you expect the structure of their melanocytes to differ from individuals who do not have this mutation? If so, describe how they would differ. (2 marks) Yes (1 mark) They would lack the dendritic projections (or torsion 0.5 marks) we typically see on melanocytes. (1 mark) 2. Considering what you know about transport across the cell membrane, describe all of the types of transport used in the transfer of mature melanosomes from a melanocyte to a keratinocyte as depicted in the zoomed-in region of figure 1. (2 marks) Melanosome leaves the melanocyte by exocytosis (0.5marks for active transport) (1 mark) Melanosome enters the keratinocyte by phagocytosis (1 mark) (0.5 marks if endocytosis) 3. What type of microscope would you use to study and observe mitochondria-melanosome tethering? Using no more than two sentences, explain your reasoning. ( 2 marks) Electron microscopy (I will accept either scanning or transmission) (1 mark) Rationale – has adequate resolution to observe individual organelles and smaller structures (1 mark) 4. You identify an individual who does not express a functional microtubule-associated motor protein, dynein, within their melanocytes. Would you expect to find mature melanosomes in the keratinocytes of this individual? Using no more than two sentences, explain your reasoning. (2 marks) No, melanosomes would not be transported to the cell periphery in these individuals and would therefore not be exported to the keratinocytes (1 mark – all or nothing) Yes, kinesin moves melanosomes in the peripheral direction (2 marks – all or nothing) 5. You are studying melanosome maturation in cultured melanocytes, predict the effect of each of the following drugs on this process. ( 9 marks) 1 mark per box Drug

2,4-dinitrophenol a drug that increases the permeability of the inner mitochondrial membrane to protons Protease that cleaves mitochondria - melanosome bridges Competitive inhibitor of the melanosome H+ pump

ATP production (increase/ decrease /no change) decrease

Melanin production (increase/ decrease /no change) decrease

Melanosome maturation (increased/ decreased /no change) decrease

no change

Decrease or no change

decrease

No change

decrease

Decrease or no change

Most melanocytes of the skin are fully differentiated and have long lifespans with low proliferative capacity, similar to neurons. Under certain circumstances, melanocytes can begin to hyperproliferate (divide at a higher than normal rate) leading to melanoma, a common type of skin cancer. Many treatments for cancer seek to disrupt DNA replication since having 2 intact and error-free copies of the genome is required for progression through the cell cycle. 6. As part of your summer research internship, you isolate 100 melanocytes from healthy noncancerous skin and determine the stage of the cell cycle they were currently in . What stage of the cell cycle would you expect to observe most frequently? How many copies of the genome are present in this cell stage? (2 marks) G0 (1 mark)

G0/G1 (0.5 marks)

1 copy (2N or 23 pairs of chromosomes) of the genome (1 mark) 7. For another part of your research project, you are tasked with investigating potential treatments for melanoma. You have developed a drug that inactivates DNA ligase in treated cells and are observing cells undergoing replication in its presence. 7a) Fill in the following parts of the growing replication fork shown for cells treated with this novel compound. (8 marks) ▪ Using a star(s) indicate the location of the origin(s) of replication. ▪ Using an arrow which identifies the direction of replication, add in the newly synthesized nucleic acids, representing at least 2 Okazaki fragments wherever appropriate ▪ Label the 5’ and 3’ ends of all nucleic acid strands that you draw ▪ Label the new strands as “leading” or “lagging” ▪ Draw and label an oval representing the location of topoisomerase at this replication fork (do not include any other proteins in your drawing)

• Labeling the origin of replication on both strands (0.5 x 2 = 1 mark) • Arrows on nucleic acids showing correct direction of synthesis (0.5 x 2 = 1 mark) • Correctly labeling both leading and lagging strand X 2 (1 x 2 = 2 marks) • Labeling 5’ and 3’ ends correctly (1 mark for leading and 1 mark for lagging strands – only need to label one fragment for the point) • Continuous DNA on leading strands (0.5 marks) • Okazaki fragments on lagging strand, not connected (0.5 marks) • Oval showing topoisomerase (must be in the double stranded region past the fork - 1 mark)

7b) When you look at the replicated DNA at the end of the experiment described in part “a” (i.e. – once replication is complete), do you expect to see any RNA present on either strand? Using no more than two sentences, explain your reasoning. ( 2 marks) NO (1 mark) Reasoning - the DNA polymerase I or RNAse will still be able to remove the RNA primers (laid down by primase), and replace them with DNA (1 mark). 7c) Will a cell treated with this drug that inactivates DNA ligase, be able to pass through the S phase checkpoint and continue into G2? Do you predict that this will be an effective cancer treatment? Using no more than 2 sentences describe why or why not? (3 marks) Pass through S-phase: NO, they cannot pass through the S phase checkpoint (1 mark) Effective cancer treatment: YES, effective (1 mark) because replication remains incomplete since the Okazaki fragments cannot be joined. (1 mark) 7d) Your lab mates are investigating several other compounds that affect DNA replication as part of the search for an effective treatment for melanoma. Complete the following table for each one, state whether you believe it will be an effective cancer treatment and describe why of why not. (6 marks) 1 point yes/no 1 point explanation Treatment

#1 - Replacing the deoxynucleotide triphosphates (dNTPs) in the melanocytes with dideoxynucleotide triphosphates (ddNTPs) which lack both a 2’ and 3’ OH group #2 - A compound that promotes DNA supercoiling

#3 - A protease that degrades the single strand DNA binding proteins

Effective Why or why not? (no more than 2 sentences) melanoma treatment (YES/NO) YES Nucleotides can’t be joined together (no replication)

NO

Despite increased coiling topoisomerase is present and functioning so the cell can overcome this and still replicate the DNA.

YES

DNA will be kinked ahead of the DNA pol impairing progress preventing replication

Human melanocytes synthesize both eumelanin (a dark brown-black pigment) and pheomelanin (a red-yellow pigment). Ultimately, it is the ratio of eumlanin to pheomelanin that determines the colour of skin and hair. The main regulator of eumelanin synthesis is a G protein coupled receptor, the melanocortin 1 receptor (MC1R). MC1R is expressed on the surface of melanocytes and acts through a Gs protein to stimulate production of eumelanin (figure 2). 8. The sequence of the beginning of the MC1R gene is as follows: +1 5’ CAGGAGACAG AGGCCAGGAC GGTCCATAGGT GTCGAAATGTC CTGGGGACCT GAGCAGCAGC CAC 3’

3’ GTCCTCTGTC TCCGGTCCTG CCAGGTATCCA CAGCTTTACAG GACCCCTGGA CTCGTCGTCG GTG 5’

8a) What is the sequence of the first 10 nucleotides of the primary transcript of this gene? (1 mark) 5’ __ __ __ __ __ __ __ __ __ __ 3’ 5’ AGGACGGUCC3’ 8b) The MC1R gene has only one exon. Describe the processing steps its primary transcript must undergo before becoming a mature mRNA. (3 marks – needs option for 0.5 marks) Addition of the 5’ cap (1 mark) and poly-A tail (1 mark) Splicing not mentioned or does not occur (1 mark) 8c) What are the first 5 amino acids encoded by this protein? ( 1 mark) Note - there is a codon table available on the final page. N’ _____ _____ _____ _____ _____ C’ MET SER TRP GLY PRO

9. There are as many as 200 common variations in the MC1R gene that are associated with differences in skin and hair color and several others associated with pathological conditions associated with a lack of (albinism) or loss of (vitiligo) pigmentation. 9a) Red hair and fair skin is associated with a variant located in the second intracellular loop of the receptor (depicted as a red star on the light side of figure 2). Compare the most common, wildtype, mRNA sequence for the MC1R gene with that of the redhead variant. i)

Translate the mRNAs below to identify the mutation and its effect. Clearly identify the difference by circling the affected amino acid(s). Answer the subsequent questions to identify how this might result in a change in pigmentation. (5 marks)

Wildtype mRNA

478 – GCG

CGG

CGA

GCC

GUU – 493

N’ ____ ____ ____ ____ ____ C’ Ala Arg Arg Ala Val (1 mark)

Redhead mRNA

478 – GCG UGG CGA GCC GUU - 493 N’ ____ ____ ____ ____ ____ Ala

-

Trp

Arg

Ala

Val

C’ (1 mark)

1 mark for circling the Trp

ii)

Does this mutation affect the chemistry of the amino acid(s)? If so, how? YES Goes from a polar, positively charge AA to a nonpolar, non charged AA. (1 mark)

iii)

How could this result in a change in pigmentation in affected individuals? *Hint - think about the mechanism of action of this receptor.

The intracellular region of the receptor is involved in contact with and activation of the coupled G protein, changes to the sequence will change the efficiency with which this happens. ( 1 mark)

9b) You are studying the genome of a family that experiences albinism (lack of pigmentation). You do not observe any of the known mutations that cause albinism but identify a tRNA gene that has undergone a mutation such that its anticodon now recognizes the stop codon of the MC1R gene. Describe the impact this would have on the MC1R protein produced and why this might lead to an absence of pigmentation. (2 marks) Instead of stopping translation at the usual codon, it (the ribosome) will continue and create an elongated protein. (1 mark) Extra nucleotides mean that the protein fold and functions differently therefore signalling required to generate pigmentation will not proceed as it normally does. Most likely won’t work at all – they don’t need to specifically say this but it should be accepted as an answer. (1 mark)

10. There are a total of five different melanocortin receptors that are expressed in various tissues of the human body. MC1R is, however, the only family member expressed in melanocytes. Using no more than one short paragraph (max 5 sentences) describe, from a molecular perspective, how MC1R but not the other MCRs are expressed in melanocytes. (2 marks) Each gene requires a unique set of transcription factors to allow its transcription to proceed. We can assume that: - the set of TFs needed by MC1R expression ARE present in melanocytes (1mark) AND - that the MC1R TFs are different from those required for the other MCRs (2-5) and that this group of TFs are NOT found in melanocytes – therefore they are not expressed. (1mark) In addition to normal variations in pigmentation, melanin deposition is responsible for tanning following sun exposure in some individuals. Exposure to UV rays leads to the synthesis and secretion by α-melanocyte stimulating hormone (α-MSH) by keratinocytes. α-MSH signals in a paracrine fashion, binding to MC1R and activating the transduction pathway depicted in figure 2. This pathway directly produces the expression of the microphthalmia-associated transcription factor (MITF) which is, in turn, promotes the expression of tyrosinase-related protein 1 (TRP1). TRP1 is a biosynthetic enzyme required for eumelanin (black/brown pigment) production which gives the skin a darker or tanned appearance (figure 2 – dark side). Figure 2

11. Why is MITF depicted as exiting and re-entering the nucleus before activating expression of the TRP1 gene in figure 2? (1 mark) The RNA must first leave the nucleus to be transcribed by a ribosome which are located in the cytoplasm. (0.5 marks) The protein then returns to its site of activity the nucleus. (0.5 marks)

12. Over the years many researchers have developed treatments that affect the activity of this pathway in a way that allows individuals to get a tan without exposure to harmful UV rays. Complete the table below indicating the behaviour of melanocytes treated with each of the compounds described below in the absence of UV exposure or α-MSH. You can assume that cells being treated in these experiments have the most common (or wildtype) variant of the MC1R gene (8 marks) 0.5 marks per square Treatment

NDP-MSH - a potent analog that mimics α-MSH db cAMP - a cyclic nucleotide derivative that mimics cAMP Forskolin - an activator of adenylyl cyclase Allosteric activator of TRP1

G protein activation (yes/no) Yes

cAMP production (yes/no) Yes

MITF expression (yes/no) Yes

Eumelanin production (yes/no) Yes

No

No

Yes

Yes

No

Yes

Yes

Yes

No

No

No

No...