MCB 2050 EXAM QUESTIONS WITH ALL CORRECT ANSWERS

MCB 2050 EXAM QUESTIONS WITH ALL CORRECT ANSWERS What is position effect variegation? - Answer-Altered gene expression occuring when a gene is transferred from euchromatic region to heterochromatic region (or vice versa). Explain the role of HATs, HDACs, and SWI/SNF in chromatin remodelling. - Answer-HATs: acetylate histone lysine residues resulting in loosening of DNA-nucleosome interaction - more open chromatin HDACs: remove acetyl groups resulting in chromatin compaction. SWI/SNF: shifts nucleosomes along DNA to clear promoter regions for TF binding. Explain the phenomena of DNA methylation and gene imprinting. - Answer-DNA methyltransferases attach methyl groups to cytosine residues in CpG dinucleotides to create C5-methylcytosine. Methylation of gene promoters recruits chromatin modifying proteins resulting in chromatin compaction and inhibition of gene expression. Imprinted genes: methylation pattern of a gene is determined by its parental origin. Why is the alternate splicing of mRNA significant? - Answer-mRNA transcripts can be processed to include or exclude different exons such that the various mature mRNAs can be translated into proteins with different functions. Explain the cytoplasmic control of mRNA stability. - Answer-PABP1/eIF-4E mediated circularization prevents mRNA decay. Deadenylation allows binding of deadenylating enzyme to destabilize sequence AUUUA in the 3'UTR of short-lived mRNAs. Describe the process of miRNA biogenesis. - Answer-miRNAS synthesized as pri-miRNA with ds hairpin structure. Drosha removes ends of hairpin producing pre-miRNA that is exported to cytoplasm. Dicer removes hairpin loop to produce short 22 nucleotide dsRNA. dsRNA associates with AGO2 protein to form the RISC. mRNA strands are unwound, one is destroyed, the other (guide) strand base pairs with complementary sequence in specific mRNA 3'UTR resulting in silencing. Explain how proteostasis is regulated by molecular chaperone proteins. - Answer-Molecular chaperone proteins bind to exposed hydrophobic domains on unfolded proteins and prevent aggregation. Assist in protein folding and intercellular transport. Deliver damaged proteins to the ubiquitin-proteasome system (UPS). Damaged and short-lived proteins are ubiquitinated and delivered to the proteasome for destruction. What are oncogenes? How are they generated? - Answer-Promote cell proliferation or survival. Arise by point mutation, chromosomal translocation or gene amplification of proto- oncogenes resulting in the overexpression of or overproduction of overactive regulators of cell proliferation and survival. Explain how the cell cycle is regulated. - Answer-Cell cycle progression is controlled by specific cyclin dependent kinases. Abundance of specific cyclins change throughout the cell cycle. Active CDKs phosphorylate proteins controlling cell cycle progression. CDK activity regulated by: cyclin synthesis and degradation, CDK phosphorylation and dephosphorylation, CDKI (p21) binding. Cancer cells lose control of cell cycle checkpoints. G1/S checkpoint: growth factors -> cyclin D -| Rb -| E2F -> cyclin E. DNA damage checkpoint: p53 -> CDKI p21 -| CDK2- cyclin E. Explain apoptosis regulation. - Answer-Cell death occurs by caspase activation and controlled cellular destruction. Effector caspases are cleaved and activated by initiator caspases. BCL-2 family proteins: regulate cytochrome c release from mitochondria which leads to apoptosome formation and caspase-9 (inhibitor) activation. Activated BAX protein (pro-apoptotic BCL-2 family protein) forms channels in the mitochondrial outer membrane allowing cytochrome c release. BCL-2 (anti-apoptotic BCL-2 family protein) prevents BAX activation. Pro-apoptotic BCL-2 family members inhibit the anti-apoptotic members and directly activate BAX. What is Src? How is it related to cancer? - Answer-Intracellular kinase controlling cell proliferation. V-src gene is truncated version of c-src producing a constitutively active kinase. Conversion of human src proto-oncogene to an oncogene occurs by acquisition of a point mutation leading to constitutively active src protein. What is Ras? How is it related to cancer? - Answer-Monomeric G-protein (GTP bound: active, GDP bound: inactive) which transmits signals from receptor tyrosine kinases to intracellular kinases that control cell proliferation. Point mutation in RasD Glycine 12 to Valine which abolishes GTPase activity resulting in constitutive activity. Describe the genetic mutations responsible for chronic myelogenous leukemia and Burkitt's lymphoma. - Answer-Philadelphia chromosome: reciprocal translocation between chromosomes 9 and 22 creating fusion protein bcr-abl with constitutive intracellular tyrosine kinase activity resulting in chronic myelogenous leukemia. Reciprocal translocation between chromosomes 8 and 14 places c-myc gene under the control of a strong immunoglobulin gene enhancer resulting in overproduction of c-myc protein (regulates apoptosis and cell growth) in Burkitt's lymphoma. What are tumour suppressor genes? - Answer-Genes that inhibit cell proliferation or survival. Onco-gene forms arise by deletion, point mutation or promoter methylation causing a loss-of- function mutation. How does the Rb (retinoblastoma protein) affect the cell cycle? - Answer-Acts as a brake on the cell cycle by inhibiting the activity of the transcription factor E2F. Growth factors stimulate cyclin D synthesis, which binds to CDK4 activating it, resulting in Rb phosphorylation. Hyper-phosphorylated Rb cannot inhibit E2F resulting in E2F stimulated transcription of the cyclin E gene. CDK2/cyclin E phosphorylates proteins driving cells into S phase. Loss of function mutation reduces Rb protein levels resulting in G1/S transition in the absence of growth factor signaling. What is p53? What role does it play in cancer? - Answer-Transcription factor that is normally kept at very low levels by ubiquitination and proteasomal degradation. p53 protein levels accumulate in stressed cells leading to increased transcription of genes controlling DNA repair, cell cycle arrest (p21) and apoptosis (BAX). Loss of p53 function leads to genome instability as cells with damaged DNA escape apoptosis and continue to divide with potentially tumorigenic mutations. What is DNA restriction mapping? - Answer-Create physical maps of DNA based on size of restriction enzyme fragments. Mapping requires the comparison of digestion patterns from different RE used individually and in combination. Describe the process of Southern blotting. What is it used for? - Answer-Cut genomic DNA with RE, separate fragments on a gel, transfer DNA fragments to a membrane, detect specific fragment by hybridization with radioactive DNA probe. Used to determine the presence of a specific DNA sequence in a DNA sample - applications include detection of highly or partially homologous sequences, RFLP analysis or disease diagnostics