7.012 Problem Set 6 Solutions

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1 Name Section Problem Set 6 Solutions Question 1 The viral family Orthomyxoviridae contains the influenza A, B and C viruses. These viruses have a (-)ss RNA genome surrounded by a capsid composed of lipids and proteins. Infection of your cells by an influenza virus occurs through the process of receptor-mediated endocytosis. Specific glycosylated proteins on the surface of your cells are co-opted by the virus as a receptor for viral entry. One of the viral capsid proteins, hemagglutinin (HA), acts as the ligand. a) For what purpose do cells normally use receptor-mediated endocytosis? To internalize specific macromolecules. b) Once influenza virus is inside your cell, it must first make viral mrna so that viral proteins can be produced. i) Which of the components required for transcription are from the virus. RNA-dependent RNA polymerase. The template is of viral origin. ii) Which of the components required for transcription are from the host. The nucleotides and the energy are from the host cell. c) After viral mrna has been transcribed, the influenza virus uses the cellular ribosomes to make viral proteins. Ribosomes can either be free in the cytosol or attached to the endoplasmic reticulum. When translation is initiated, where are the ribosomes found? All translation is initiated on ribosomes in the cytosol. d) For the viral capsid protein hemagglutinin, completion of translation must be performed by ribosomes attached to the endoplasmic reticulum. If free cytosolic ribosomes were used for translating the entire hemagglutinin protein, what effect would this have on the ability of new viral particles to infect other cells? Translated this way, the hemagglutinin protein would never be inserted into the host cell plasma membrane. When the new viral particles were packaged, they would lack surface hemagglutinin and thus would not have the ligand for the host cell receptor and would therefore not be infectious.

2 e) After the virus has replicated itself within the host cell, new viral particles bud off the cell s plasma membrane (as depicted below) to infect other cells. i) What is the origin of the lipids in the viral capsid? The lipids are from the host cell. Budding. ii) What is this process called? Question 2 a) Mammalian cells are often grown in tissue culture dishes to study their growth properties. i) If an asynchronous population of fibroblasts is put in a dish with medium, but no serum, what phase(s) of the cell cycle could they be in initially? All phases of the cell cycle could be represented. ii) After 48 hours, what phase(s) of the cell cycle would you predict these cells be in? The cells would all accumulate in Go. iii) After 48 hours, you replace the medium on the cells with medium containing serum. If the serum added has only epidermal growth factor (EGF) and the cells have only platelet-derived growth factor receptors on their surface, will they be stimulated to grow? Explain your answer. No. The PDGF receptor will not recognize EGF as a ligand. b) The retinoblastoma protein (prb) negatively regulates the transcription factor, E2F. When prb binds to E2F, E2F can no longer activate transcription of genes required to go from G1 to S phase. This helps ensure that DNA replication occurs at the correct time. Some individuals are prone to the familial disease retinoblastoma because they have inherited a mutant copy of the gene resulting a prb that can not bind to E2F. i) What consequence would inheriting a mutant copy of the RB gene have on cell cycle progression? As long as the cell had one normal RB gene and the resulting functional prb, the cell cycle would progress normally. 2

3 ii) What consequence would this defect have on the risk of developing of cancer? It would increase the risk of cancer. c) The prb protein is itself regulated by covalent modification. Phosphorylation of the protein prevents its association with E2F. i) During which phase of the cell cycle is prb first phosphorylated? prb is first phosphorylated at the end of G1. ii) What consequence does phosphorylation of prb have on cell cycle progression? Phosphorylation of prb releases the E2F transcription factor, which turns on the transcription of genes whose proteins are required for S phase. This allows the cell to progress past the restriction point and commits it to completing the cell cycle. d) After the completion of S phase, E2F function is no longer needed. prb must be dephosphorylated so that it can once again bind to E2F. Name the type of enzyme responsible for removing phosphate groups from proteins. Phosphatases remove phosphate groups. Question 3 There are a number of ways in which a virus can transform a normal cell into a cancer cell. In 1911, a New York farmer brought his prize chicken to a scientist named Peyton Rous because it had developed a tumor. Rous was able to determine that a virus was the cause of the tumor, and in his honor this virus is now known as the Rous sarcoma virus (RSV). RSV is identical to a non-tumorogenic retrovirus called the avian leukosis virus with the exception that RSV possesses one additional gene, viral src or v-src. RSV= Avian Leukosis Virus + v-src a) When a chicken cdna library was screened using v-src as a probe, it was discovered that the chicken genome normally carries a version of the src gene. i) We know that tumorogenisis is associated with the presence of v-src. Would you expect v-src to act as an oncogene or a tumor suppressor gene? Why? The tumor phenotype associated with the v-src gene is dominant, i.e. a single copy of the v-src gene is sufficient to induce tumors even though the chicken cells have their own src gene. This indicates that v-src is an oncogene. 3

4 ii) Assuming that prior to 1900 RSV did not exist, speculate how it could have evolved from the avian leukosis virus. Perhaps at one time, the Avian Leukosis Virus integrated into the chicken genome adjacent to the normal chicken src gene. At one point when the viral DNA was excised from the host DNA to form progeny viral particles, the chicken src gene was also packaged. The src gene within the virus was mutated into an active form, and the active src gene caused excessive cell proliferation. Excessive cell proliferation of these cells infected the src carrying virus increases the survival of this virus. b) Simian virus 40 (SV40) is a member of the dsdna virus family Polyomavirinae. One of the viral proteins produced by SV40 is called large T antigen. This protein performs several essential functions for the virus. One of these functions is to interact with cellular proteins that regulate growth, such as prb and p53, and inactivate them. i) prb and p53 are. Tumor suppressors ii) What is the consequence of inactivating both prb and p53 in the risk of tumor development? This would increase the risk of tumor development. c) Retroviruses are unique in that their genomes exist as both RNA and DNA during different parts of their life cycle. Retrovirus particles possess (+)ssrna genomes, but once the retrovirus infects a cell, its genome is converted into DNA which can then integrate into one of the host cell's chromosomes. The location of this insertion is random, but it can have profound effects on the host cell. i) If the retrovirus integrates in the middle of the reading frame of a protooncogene, would you expect an increased or decreased risk of tumor development? You might expect a decreased risk of tumor development, because you have inactivated a growth-promoting gene. (In actuality there may neither an increased or decreased riski.e. no change in the likelihood of tumor development.) ii) If the retrovirus integrates in the middle of the reading frame of a tumor suppressor gene, would you expect an increased or decreased risk of tumor development? You would expect an increased risk of tumor development, because you have inactivated a tumor suppressor gene. 4

5 Question 3 c) continued The genes of a retrovirus are surrounded on both sides by long terminal repeat (LTR) sequences that each possess a strong promoter. The 5' LTR promoter is used to activate transcription of viral genes. Once integrated into the host chromosome, the promoter in the 3' LTR may activate transcription of neighboring host genes. iii) If the 3' LTR of the retrovirus integrates adjacent to a proto-oncogene gene, will it likely stimulate or inhibit tumor development? It could stimulate tumor development be increasing the transcription of growth promoting genes. iv) If the 3' LTR of the retrovirus integrates adjacent to a tumor suppressor gene, will it likely stimulate or inhibit tumor development? It could inhibit tumor development be increasing the transcription of growth suppressing genes. 5

6 Question 4 The diploid number of chromosomes for any organism is typically referred to as 2N. The gametes (egg and sperm in the case of humans) which are haploid are said to have a 1N chromosome content. Scientists can often determine what phase of the cell cycle a given cell is in based upon its chromosome content. a) In a somatic cell, for each phase of the cell cycle, give the chromosome content (or the range of possible chromosome content) you would expect. G2 4N Go 2N G1 2N M 4N S 2N 4N b) Scientists often use drugs to arrest cells at specific stages of the cell cycle. Two of these drugs are hydroxyurea and colcemide. At which phase of the cell cycle does each of these drugs arrest the cell cycle? How do they function to cause this arrest? Hydroxyurea arrests cells in S phase (or just prior to S phase) by interfering with DNA synthesis. Colcemide arrests cells in M phase by interfering with microtubules. b) Which drug would you use to arrest a cell in metaphase? colchecine Colcemide Draw what a cell (with 2N=4 chromosomes) arrested in metaphase will look like. 6

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