Faculty of Medicine Department of Cancer Research and Molecular Medicine

Exam MOL3000 Introduction to Molecular Medicine

Friday December 5th 2008, 9.00 am - 1.00 pm

ECTS credits: 7.5 Number of pages (included front-page and MCQ answers sheets): .

Contact person during the exam: Marit Otterlei, 92889422

Exam results: 5th January 2009 Examination results are announced on http://studweb.ntnu.no/

Question 1 (10p) a) Describe the structure, localisation regulation and function of the LDLreceptor. (5p) b) Explain how inactivating mutations in the LDL receptor contribute to the clinical and biochemical phenotype resulting in cardiovascular disease (5p). Answer:
Domain structure of the LDL receptor
LDL-receptor is a transmembranal protein

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Sequential steps in LDL receptor pathway: Receptor mediated endocytosis

If intracellular cholesterol is low, more cholesterol is synthesized If LDL-receptor is defective, cholesterol is synthesized in abundance HMG-CoA reductase is the regulatory enzyme in cholesterol biosynthesis Cholesterol lowering drugs of the type statins inhibit HMG-CoA-reductase

THE LDL receptor is required for LDL uptake in cells. Defective LDL-receptor results in hypercholesterolemia
Tendon Xanthomas

Five classes of LDL receptor mutations

Question 2 (10p) In eukaryotic cells the regulatory regions of genes contain short stretches of DNA which serve as binding sites for proteins that regulate gene expression. The proteins that bind to these sites are termed transcription factors, which functionally can be divided into two classes: basal transcription factors and special transcription factors (activators/repressors). What are the functional roles of basal transcription factors and special transcription factors (activators/repressors) in the cell? Explain the main functional difference between these two classes of transcription factors.

Answer: Spesielle transkripsjonsfaktorer (aktivatorer) gjenkjenner og binder til korte DNA sekvenser i genenes proximale og distale promoter. De binder vanligvis som dimerer og kan vre sammensatt av to identiske subenheter (homodimerer) eller to ulike subenheter (heterodeimerer). De spesielle transkripsjonsfaktorene er sentrale i rekruttering av transkripsjonsmaskineriet (RNA polymerase og de generelle transkripsjonsfaktorene) til genets core-promoter, og dette er deres overordnede funksjon i cella. Spesielle transkripsjonsfaktorer kan inndeles i familier eller klasser basert p struktur. Eksempler er zink-finger proteiner, leucin-zipper, og helix-loop-helix proteiner.

Answer: De generelle transkripsjonsfaktorene utgjr et sett p minst fem proteiner som er ndvendig for at RNA polymerase II (RNA pol II) skal gjenkjenne og binde korrekt til genets promoter. Faktorene betegnes TFIID, TFIIB, TFIIF, TFIIE, og TFIIH. De generelle faktorene assosierer med RNA pol II og besrger korrekt plassering i forhold til startpunktet for transkripsjon. Bare en av faktorene, TFIIH, har enzymatisk aktivitet, bl.a. en kinaseaktivitet som er rettet mot den C-terminale halen til RNA polymerasen og som er viktig i selve initieringen. Man antar at disse faktorene er ndvendig av transkripsjon av de fleste, og kanskje alle, proteinkodende gener.

Question 3 (5p) You want to do an iterative PSI-Blast search, but unfortunately you do not have access to PSI-Blast. However, you do have access to Pro-Blast, which can do a Blast search with a user-specified profile. You also have access to Perl, a programming language that can be used to automate various tasks, like reading and writing protein sequences and run software tools. Explain how you will combine this with ClustalW to make a PSI-Blast like tool. Answer: Denne oppgaven tester forstelsen av hvordan PSI-Blast fungerer. Essensen er at PSI-Blast bruker skeresultatet til lage en profil, basert p multippel alignment, og s sker med den profilen i neste iterasjon. Dette blir s gjentatt med oppdatering av profilen for hver iterasjon, basert p siste skeresultat. Dette kan man etterligne ved bruke Perl til hente ut sekvensene fra siste skeresultat, bruke ClustalW til lage et multippelt alignment av disse, omforme dette til en profil, og bruke den som input til Pro-Blast. Dersom man gjentar dette iterativt har man en PSI-Blast lignende prosess. Question 4 (10p) Describe the main actions of insulin Answer: Insulin hemmer glykogenolyse og glukoneogenese i leveren. Insulin hemmes ogs opptak av glucose i lever, muskel og fettvev. Insulin har effekter p fettsyremetabolism og hemmer lipolyse. Insulin har effekter p proteinsyntes gjennom ke opptak av aminsosyrer i forskjellige vev og ke syntes av proteiner. Insulin er et anabolt hormon. Mangel p insulin gir hyperglykemi og forhyede niver av fettsyer i blodet. Insulineffektene formiddles gjennom binding til insulinreseptorer i cellemembranen. Bindningen frer til autofosforylering av reseptoren hvilket via forskjellige kinasereaksjoner frer til aktivering av en rekke signalkjeder intracellulrt. Question 5 (5p) Many of the cancers that originate from hematopoietic cells have translocations involving genes that participate in immune defence reactions. Give examples of such genes and cancers. Why are these genes often involved in tumor development from hematopoietic cells? Answer: Gener for immunglobulin og T-celle reseptorer Eksempler: Neseten alle former for leukemier og lymfomer og myelomatose Pga. at disse genene gir opphav til spesifisitet i immunforsvaret som medieres ved hematopoetiske celler gjennom prosesser som omfatter kutting og ligering av DNA.

Det siste ker mulighetene for feil-ligering av DNA som kan medfre aktivering av onkogener.

MCQ, each question 1p. One answer is the most correct. Answer the questions on separate MCQ answers sheets enclosed. Only one cross for each question (the best alternative). 1. The cell cycle is divided into several phases and different biochemical event are characteristic of the different phases. In which phase is the DNA duplicated / copied ? A S-phase B M-phase C G0-phase D G1-phase E G2-phase Corrects answer: A 2. In the G1-S phase of the cell cycle the cells are checking the environment, growth conditions and presence of DNA damage. Many proteins are capable of detecting DNA damage and lead to cell cycle arrest, one protein is more important than others. This is also a tumor suppressor gene. Which protein is this? A c-myc B gp120 C Ras D p53 E hsp70 Corrects answer: D 3. DNA and proteins can be separated by size by gel-electrophoresis. Why do we use SDS (sodium dodecylsulfate) in gel-electrophoresis? A SDS denaturates the proteins, unfolds them and gives them an uniform charge B SDS is emitting light when it is bound to proteins C SDS leads to protein aggregates, thus SDS is making the aggregates easy to detect D SDS preserves the enzyme activity of the proteins Corrects answer: A 4. Latent / resting viruses do not produce much protein and can be hard to diagnose. Which techniques could be used in order to detect latent virus?

A SDS-polyacrylamid gelelectrophoresis B Polymerase chain reaction (PCR) and DNA gelelectrophoresis C Polymerase chain reaction (PCR) and iso-electric focusing D 2D-gelelectrophoresis, iso-electric focusing and western blotting Corrects answer: B 5. At each end of all chromosomes there are a few kilobases of DNA that contain a specific 6bp repeated sequence. This region is successive shortened during mitosis. What do we call these regions on the chromosomes? A Kinetochores B Telomers C Haplotypes D Centromers Corrects answer: B 6. DNA replication initiates at specific chromosomal sites. These sites are termed: A Okazaki fragments B Replication forks C Replication primers D Replication origins E Replication bubbles Corrects answer: D 7. A DNA polymerase cannot initiate DNA replication on its own but rely on the presence of short nucleotide fragments. What is the correct designation of these nucleotide fragments? A Okazaki fragments B RNA primers C Primases D Telomers E Centromers Corrects answer: B

8. Eukaryotic cells contain different RNA polymerases, which transcribe different sets (types) of genes. 7

Which polymerase transcribes the protein coding genes? A RNA polymerase I B RNA polymerase II C RNA polymerase III Corrects answer: B 9. In eukaryotic cells approximately 180-200 base pairs of DNA is twisted around an octamer of basic proteins. What is the correct term of this octamer? A Histone B Chromatin C Chromosome D Nucleosome E Chromatid Corrects answer: D 10. DNA methylation is a modification of one of the DNAs four bases. In human cells methylation occurs usually on Cytosine (C) in the context of: A 5-CpA-3 B 5-GpC-3 C 5-CpT-3 D 5-CpG-3 E 5-ApC-3 Corrects answer: D 11. Many DNA polymerases have the possibility to remove mis-incorporated nucelotides during DNA-replication. What do we call this proof reading activity of the polymerases? A 5- 3- endonucleaseactivity B 3- 5- endonucleaseactivity C 5- 3- exsonucleaseactivity D 3- 5- exsonucleaseactivity E Topoisomeraseactivity Corrects answer: D

12. Meselson and Stahls experiment from 1958 demonstrated that DNA is replicated in a semi-conservative manner. They showed this by growing cells for a long period of time with heavy isotop of nitrogen, followed by culturing for a specific number of generations in light isotop of nitrogen. When they isolated the DNA from these cells at different time point they found combinations of light and heavy as well as hybrid DNA. Which forms of DNA (light- heavy- or hybrid) did Meselson and Stahl find after incubation in light nitrogen for 2 cell generations? A Only light DNA B Only heavy DNA C Equal amount light DNA and hybrid-DNA D Equal amount of heavy DNA and hybrid-DNA E Equal amount light DNA and heavy DNA Corrects answer: C 13. Gluconeogenesis is the body`s own synthesis of glucose. Many organs are dependent of glucose from the blood as the main energy source, e.g. the brain. Which organ is the main site for gluconeogenesis? A Liver B Brain C Kidney D Milt E Pancreas Corrects answer: A 14. The body have a storage of energy, it fills up during good times and is used during bad times. When you fast, your body breaks down fatty acids via beta-oxydation. Where in the cells do we have beta-oxydation? A Endoplasmic reticulum B Cytosol C Mitochondria D The Golgi-apparatus Corrects answer: C 15. Several risk factors for cardiovascular disease (CVD) have been identified. High blood pressure (BP) is one such risk factor Which statement is the more correct one among those below? A High BP is a risk factor for coronary heart disease, but not for brain stroke.

B High BP is a risk factor for coronary heart disease and brain stroke. C High BP is mainly a risk factor for cardiovascular disease in elderly persons. D High BP is a risk factor for CVD, but not an independent risk factor. Corrects answer: B 16. Cardiovascular disease is an important cause of death. Which of the following statements about cardiovascular disease (CVD) is most correct? A CVD is a leading cause of death world wide, but with regional differences. B CVD is a leading cause death only in the western world and Japan. C CVD was a leading cause of death world wide, but is now surpassed by cancer. D CVD is a major cause of disease in men, but not in women. Corrects answer: A 17. Extracellular communication is often considered as biochemical signalling between cells. Which is the best definition of extracellular signalling molecules? A Different proteins which regulate the function of other cells B Very small molecules which are transported via blood vessels C All molecules released from a cell and which are able to induce response in another cell D A specific hormone for intracellular communication E All molecules necessary for growth in different tissues Corrects answer: C 18. Nitroglycerin is a medication used for treatment of angina pectoris. The drug leads to expansion (relaxation) of blood vessels and increased supply of blood and oxygen to the heart. How does this drug induce relaxation of the blood vessels? A By degradation to the signalling molecule nitric oxide (NO) B By activation of the enzyme nitric oxide (NO) synthase C By inhibiting the enzyme nitric oxide (NO) synthase D By activation of protein kinase G Corrects answer: A 19. Pathologic changes in the production of neurotransmitters may lead to hypo or hyper function of the extracellular signalling system.

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What kind of disease listed below is caused by loss of dopamine? A Huntingtons disease B Neuropathy C Parkinsons disease D Multiple sclerosis (MS) Corrects answer: C 20. The most common form of haemochromatosis among Caucasians is caused by mutation in the HFE gene. The HFE protein acts by binding to: A Hepcidin and inhibit the iron uptake B Beta-2-microglobulin and increases the secretion of iron C Beta-2-microglobulin and inhibit the iron uptake D Ferroportin and inhibit the iron uptake E Ferroportin increases the secretion of iron Corrects answer: C 21. Trimeric G-proteins consist of , and subunits. The G subunit is a GTPase switch protein that cycle between active and inactive states. Gs isoform stimulates,
while Gi isoform inhibits adenylyl cyclase. Presume that you have isolated a mutant of Gs with decreased hydolysis of GTP.

What would be the physiological effect of this? A No activation of adenylyl cyclase B Increased association with the and subunits C Inhibition of and complex formation D Increased activation of adenylyl cyclase and increased cAMP-mediated signalling E Decreased activation of adenylyl cyclase and decreased cAMP-mediated signalling Corrects answer: D

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22. The protein Rb plays a crucial role in cell cycle regulation. The function of Rb is most precisely described as: A Inhibits transcription by phosphorylation of E2F B Inhibits transcription by binding to E2F C Inhibits transcription by activation of cyclin D D Is a protoonkogen which activates the transcription by binding to E2F E Binds E2F and P16 kinase and subsequently activates transcription Corrects answer: B 23. Individuals with mutations in BRCA1 and BRCA2 genes are predisposed to develop breast and ovarian cancer during lifetime. The BRCA1 protein is: A A growth factor B A tumour suppressor protein C Involved in inhibition of estrogen-independent intracellular signalling D Involved in DNA repair E A kinase crucial in estrogen receptor-mediated signalling Corrects answer: D 24. Cancers often involve mutation in several proteins participating in intracellular signalling and transcriptional regulation. Assume that a somatic mutation has arisen in the protein c-Ras. What could be the consequences of such a mutation? A Activation of tumour suppressor proteins B Constitutive activation of signalling pathways C Inhibition of tyrosine phosphorylation D Cell cycle arrest Corrects answer: B 25. Insulin is secreted from -cells in pancreas in response to increased blood glucose level, and calsium plays an important role in this process The molecular signalling mechanisms involved in this process involves the following process: A ATP-dependent activation of Ca2+ channels B IP3-gated release of Ca2+ from endoplasmatic reticulum (ER) C Voltage-independent Ca2+ channels D Ca2+-mediated endocytosis

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E Ca2+ influx from extracellular compartment Corrects answer: E 26. The main genetic risk in type 1 diabetes is tied to A Genes in the HLA - complex B Gene coding for insulin C Genes regulating glucose uptake D Genes regulating glucose metabolism Corrects answer: A 27. Two major forms of diabetes are named type 1 and type 2. Which of the following alternatives constitutes an important difference between these forms? A Blood glucose is higher in type1 than in type 2 diabetes B Antibodies against pancreatic islet tissue are found in type 1 but not in type 2 diabetes C Type 1 diabetes is always treated with insulin, type 2 diabetes never D Type 1 diabetes is more common in females, type 2 diabetes more common in men Corrects answer: B 28. Cells in the Islets of Langerhans in pancreas is responsible for secretion of insulin How is insulin released from the cells? A Insulin is mainly released from the cells by exocytosis after proinsulin is divided into insulin and C-peptid B Insulin is mainly released from the cells by diffusion after proinsulin is divided into insulin and C-peptid C Insulin is mainly released from the cells by exocytosis as proinsulin. Proinsulin is later divided into insulin and C-peptid D Insulin is mainly released from the cells by diffusion as proinsulin. Proinsulin is later divided into insulin and C-peptid Corrects answer: A 29. Insulin lowers the levels of blood glucose, while other hormones increase the blood glucose level. Which of the following hormones increases the blood glucose levels? A thyroxin B testosterone C glucagon 13

D estradiol Corrects answer: C 30. The term oncogenes is used on genes that when mutated promote oncogenesis. Which property of these genes is responsible for effect? A Oncogenes regulate cell growth B Oncogenes regulate cell death C Oncogenes encode tyrosine kinases D Oncogenes encode tyrosine phosphatases E Oncogenes regulate antibody production Corrects answer: A 31. We use the term tumor suppressor genes on genes that prevent tumor development. Why do one ore more tumor suppressor genes have to be inactivated in the development of a tumor? A Because tumor suppressor genes bind oncogenes in a cell B Because tumor suppressor genes are inherited in only one copy C Because tumor suppressor genes induce cell cycle halt and/or apoptosis D Because tumor suppressor genes lead to less expression of IgH (immunoglobulin Heavy Chain) in cells E Because tumor suppressor genes inhibit cell adhesion to basal membranes Corrects answer: C 32. Inheritance of one deficient allele for a tumor suppressor gene may predispose for cancer Why is this? A The tumor suppressor genes are present in only one copy B Having one deficient allel destroys the expression of other genes C Having only one healthy allel increases the risk for loss of gene function D The mutated allele suppresses the healthy allele Corrects answer: C 33. A set of related protein sequences can be represented for example by a consensus sequence (representative single sequence) or by a profile. What is the main advantage from using a profile compared to a consensus sequence? A Searching with profiles will only return related (homologous) sequences B It is not possible to search with a consensus sequence 14

Profiles make it possible to distinguish between conserved and non-conserved positions in the sequences D Profiles are easier to implement in iterative methods E There are no specific advantages; profiles and consensus sequences perform equally well Corrects answer: C 34. When using for example Blast or PSI-Blast to search with an individual sequence in a library of sequences we often evaluate the result based on an E-value. What does an E-value of 10 in a PSI-Blast search mean? A The score value is among the 10% best values measured over the full sequence library B We can expect the on average 10 sequences from the data base get equally good or better score value if we search with a randomized sequence C Only the 10 first hits in the PSI-Blast result are significant D We can expect a minimum of 10 significant hits per data base E We have to use 10 PSI-Blast iterations to get a statistically significant result Corrects answer: B 35. When Blast or PSI-Blast are used for comparing sequences we have to take sequence properties into account, in particular regarding level of complexity. Why may a low-complexity protein sequence give problems in Blast? A It will give too few hits in the score matrix B Only high-complexity sequences may cause problems C The sequence is often short, and will therefore return few significant hits D It will give a complex sequence alignment that is difficult to interpret E The sequence may have a very atypical distribution of amino acids Corrects answer: E

36. Endocytosis of signalling receptors is a way of limiting signalling. Following endocytosis ubiquitinated receptors are recognized at the early endosome (EE), and via the endosomal sorting complex required for transport (ESCRT), transferred onto intra luminal vesicles of the EE, where signalling can no longer occur. What is the name of the key protein for the recognition of ubiquitinated receptors on the limiting membrane of the EE, and what component is vital for the binding of this protein to the limiting membrane of the EE? A Hepatocyte growth factor-regulated substrate (HRS), and clathrin B Clathrin, and phosphatidylinositol 3-phosphate (PI(3)P)

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C HRS, and PI(3)P D Early endosomal antigen-1 (EEA1), and PI3P Corrects answer: C 37. A major component of the gram-negative bacterial wall is lipopolysaccharide (LPS). What toll-like receptor (TLR) is vital for the initiation of LPS signalling? A TLR3 B TLR5 C TLR4 D TLR9 Corrects answer: C. 38. The Toll-like receptors (TLRs) utilize leucin-rich repeat motifs in the extracellular domain for ligand binding; and a shared intracellular domain called the Toll/interleukin-1 receptor domain (TIR)-domain for the recruitment of TIR-adapters for the initiation of intracellular signaling pathways. What is the name of the TIR-adapter used for signaling by most of the TLRs? A TRAM (TRIF-related adaptor molecule) B TRIF (TIR-domain-containing adapter-inducing interferon-) C MYD88 (Myeloid differentiation primary response gene (88)) D TIRAP (TIR- domain-containing adaptor protein) Corrects answer:C. 39. All receptors are proteins that upon binding trigger a biological response. What is the name of a compound that binds a receptor and triggers its biological response? A Competitive agonist B Agonist C Competitive antagonist D Antagonist Corrects answer: B 40. Three enzymes participate in ubiquitin attachment to a protein substrate. The E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitinprotein ligase (E3-ligase). It is the E3-ligase that shows the substrate specificity. How many E3ligase genes are there in the human genome? A More than 500

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B 40 C 1 D 2 Corrects answer: A

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