Promoter 1. Promoter 2. Enhancer 2

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1 Essays 1. An animal normally has two copies of the Xan gene. The protein made by this gene helps the animal clear petroleum-based pollutants from its body. Tine Xan What we know about the Xan gene is shown in the cartoon above. We know that and work together to express the Xan gene. Also shown is a bit of information about the neighboring gene. It is called Tine (pronounced Tiny). We know that and work together to express Tine. You are going to analyze a collection of mutant animals and compare them to the wild type animals. Wild type diploid animals have two copies of both genes and will be represented like this---> Mutant 1 is an animal in which one copy of the genes have been deleted. It is represented like this ---> It is heterozygous for the deletion. The bottom chromosome has had both genes deleted. In Mutant 2, the deletion is homozygous. All copies of the genes have been removed. In Mutant 3, both genes have been duplicated. The top chromosome has 2 copies of the genes and the bottom chromosome has 2 copies of the genes. Therefore the animal has 4 copies of both genes. Mutant 3 will be represented like this--> In Mutant 4, one chromosome carries the duplication of the genes while the other chromosome carries only one copy of the gene. Therefore it carries 3 copies of Tine and 3 copies of Xan. Mutant 4 will be represented like this --> 1

2 In this experiment, you will be measuring the expression level (amount of mrna produced) from Xan. You never measure the amount produced by Tine. You will be shown a gel that shows the relative expression level from (Xan) from wild type and mutant animals. The gel will look somewhat like this --> This is the well in which the sample was loaded. wildtype mutant 1 mutant 2 I have also given you a histogram that displays the level of Xan expression in wild type and mutant animals. This is the mrna produced from (Xan) Here are the results of the study. Experiment 1 compares the expression level from (Xan) in wild type and mutant animals. Wild type wildtype Data showing expression from gene 2 mutant 1 mutant 2 Mutant 1 amount of mrna wildtype mutant 1 mutant 2 Mutant 2 Mutant 2 does not produce any detectable Xan mrna. This one is homozygous for the deletion. Experiment 2 compares the expression level from (Xan) in wild type and mutant animals. Wild type wildtype mutant 3 Data showing expression from gene 2 mutant 4 Mutant 3 amount of mrna Mutant 4 wildtype mutant 3 mutant 4 On the exam, I will ask you some questions. What might I ask? 2

3 2. You have developed an antibody that is specific for methylated cytosines. It does not recognize anything else. You are working with two cell lines called cell line A and cell line B. Cell line B was made from cell line A. Here is the process: cell line A is treated with a chemical that causes it to behave like a cancer cell (loss of growth control). Thus, cell line B is a "cancerous" form of cell line A. You would like to understand how cell line A and B differ. You grow cell lines A and B in separate petri dishes. You take 20,000 cells of each one and purify the genomic DNA from the cells. This is pure genomic DNA with no contaminants. You shear this DNA into 250 bp fragments. To tubes containing sheared genomic DNA A and sheared genomic DNA B you add the antimethylated cytosine antibody. You then add protein A to each tube. Protein A will bind the antibody and can be pelleted by centrifugation. You centrifuge these material and isolate the pellet. At this stage you have Pellet A and Pellet B (A is from genomic A and B is from genomic B). In the exam I will ask you to accomplish a specific goal with this material. You should try to anticipate how these pellets could be used to accomplish specific goals. What techniques will you need? You should consider multiple solutions to these questions. 3. You are studying a protein. To learn more about the protein, you need to have a clone of the gene that encodes the protein. You choose to clone this gene by PCR. To do so, you first determine the sequence of fragments of the protein. The entire protein is 1000 amino acids long. The technology available at the time of protein sequencing allows you to sequence only small peptides. So, the protein was digested into small pieces. For each piece, the sequence of a seven-amino-acid length was determined. Protease! digestion The original protein. You obtained 9 peptide sequences. They are: The protein has been digested into fragments. FLSYCWP PHQRIMT MNKVADE MCPCFIE HEDKFYC HEDWKFM DNKMYFH WPQHHRR RR Unfortunately, you do not have any more information about the sequence of the protein or the gene. 3

4 4. This is an in vitro splicing reaction. Samples are taken from the reaction and loaded on a polyacrylamide gel. The precursor mrna (Band 3) used in the reaction is radiolabeled. Darker means that the RNA producing this band is more abundant. The precursor produces an extremely broad band because it is very abundant. T=0 T=5 T=10 T=15 T=20 T=25 Band 2 Band 1 Band 3 Band 4 Band 5 What will I ask you about this gel? What will I ask you about the reaction? 4

5 Multiple Choice tidbits 1. Can you descibe the chromatin immunoprecipitation assay step-by-step? Specifically how is it done? Can you tell me all of classes of "things" that ChromIP can be used to assay. 2. How can enhancers act at a distance? pg With respect to enhancers what do we mean by combinatorial code. pg What is exon definition? pg Could you choose a good set of degenerate primers for PCR? What criteria are important? What are the uses of degenerate primers? Do you understand that the down stream primer must "point" the opposite direction to the upstream primer? Can you do that in practice? Do you understand how to select the best region for your primers? Do you understand why degenerate PCR is useful? 6. I will probably ask you one or more multiple choice questions over the histone code pg The role of RNA polymerase II CTD domain in various processes (pg 429 and lecture). 8. Architectural transcription factors. LEF-1 is lymphoid enhancer-binding factor. TF is a HMG (high mobility group) protein. Causes 130 degree bending. Brings Ets-1 site closer to the promoter. The 2nd example in your book is of one that unbends DNA and thereby makes the DNA ready to bind proteins. 9. Insulators. What do insulators do? Read in your book. How can the use of an insulator activate a promoter? 10. Structure of nucleosomes and their affect on chromatin structure and on transcription. How many bp. what proteins etc. 11. How histones are involved in the process of chromatin remodelling. 12. What is the first thing that probably attracts an activating transcription factor to a transcriptional control region? What are:bromodomains, chromodomains? 13. DNase I sensitivity and how it relates to gene transcription DNase cuts up DNA. A sample of chromatin is very resistant to digestion. Can you describe all of the ways that this could occur? 14. What is a HAT? How were they purified? 15. What is a HDAC? 16. Do you understand degenerate PCR? Could you apply it to a problem? 17. Don't forget massively parallel sequencing as taught to you by Josh. 18. Could you diagram out RNA splicing and name all of the sequences and snrnps involved? 19. In what sense does the branch point "select" the acceptor site in an intron? 20. Could you draw a 5'-CAP? 21. Do you know the polya site motif? 22. If a mrna looses the 5'-CAP or polya tail - what happens? 23. What is H3K4methylation associated with? H3K27me? H3K9acetylation? H3S28phosphorylation? 24. Do you know how HATs were first purified? 25. What is 5'-RACE? 26. What histone modifications attract SWI/SNF? What does SWI/SNF do? 27. What histone modifications attract TFIID? 5

6 Here are some multiple choice questions that I think might be helpful. They are from a previous year's exam. I think that these will be helpful. However, I guarantee that this list is incomplete. You will have questions that do not have a representative here. These Multiple choice are choose ALL of the correct answers. Your exam will be choose the correct answer. 5. Histone modifications A) All histone modifications are inherited in the germ line. B) Acetylation of histones neutralizes positive charge reducing their "grip" on the DNA and also reduces the amount of nucleosome cross-linking. C) Histones in the Tetrahymena macronucleus have a very low level of acetylation. D) More than 70 different modifications to histones have been identified. E) Unacetylated histones are associated with genes that are transcribed at a low level. 6. Here is an amino acid sequence. Please generate a degenerate primer that could recognize the DNA that encodes this amino acid sequence ---> This genetic codon table might be useful in this endeavor. Codon Usage by amino acid. F L S Y C W P H Q R TTT TTA TCT TAT TGT TGG CCT CAT CAA CGT TTC TTG TCC TAC TGC CCC CAC CAG CGC CTT TCA CCA CGA CTC TCG CCG CGG CTA AGT AGA CTG AGC AGG I M T N K V A D E G ATT ATG ACT AAT AAA GTT GCT GAT GAA GGT ATC ACC AAC AAG GTC GCC GAC GAG GGC ATA ACA GTA GCA GGA ACG GTG GCG GGG This degenerate primer has fold degeneracy. A) 15 B) 24 C) 48 D) 64 E) 128 MWCWHFS 7. One is monitoring a splicing reaction by gel electrophoresis of the RNA molecules. The precursor mrna is 1000 nucleotides and is radiolabeled. After 1 minute, you note the appearance of a radiolabeled band that migrates at 1200 nucleotides. A) This band is probably the final splice product. B) This band is probably a lariat containing the intron and exon 2. C) This molecule will probably disappear later in the reaction. D) This band is probably the spliceosome itself. 8. Insulator A. A mutation that eliminated insulator activity would cause genes to be turned on/off/up & down in inappropriate places or times. B. In order to work it is thought that 2 of them must team up C. Provides a barrier through which some forms of activation or repression cannot pass. D. These also act as transcription termination signals that prevent polymerase from transcribing from one gene to another in the genome. 9. Degenerate PCR A) Can be used to clone a fragment of a cdna that encodes a protein B) Primers with high degeneracy work better than primers with low degeneracy. C) These primers "degrade or degenerate" over time and therefore provide a way to 6

7 determine when a nucleic acid appears and when it dissapears. D) The word "degenerate" is derived from the idea of a degenerate genetic code. In this sense, degenerate means that the code employs many synonyms. 10. True or False. Choose all of the correct statements. A) Histones have a higher percentage of lysine and arginine residues than most proteins. B) In eukaryotes, histones are poorly conserved over evolutionary time. C) There are five different classes of histones. D) The nucleosome core contains histone H1. E) The 30nm fiber form of chromatin is more compact than the beads on a string and has 6 nucleosomes per turn of the solenoid. 11. A histone acetyl transferase modifies some chromatin A) This chromatin will now be more attractive to proteins that contain a bromodomain. B) This chromatin will now be less attractive to Restriction Enzymes. C) This chromatin will now be more attractive to TAF250 D) The DNA in this region will probably be more accessible. 12. Dimitris Thanos studies the regulation of the human interferon gene (IFN-beta) in response to viral infection. The follow are some of the interpretations of his work: A) Phosphorylation of serine #10 of histone H3 is required fro the acetylation of lysine #14 of histone H3. B) Phosphorylation of serine #10 of histone H3 occurs after TFIID has been bound. C) The pattern of histone modifications are random. D) TFIID binds after lysine #14 of histone H3 is acetylated. E) The SWI/SNF complex modifies the nucleosome over the promoter and changes it so that it can be physically moved or repositioned. 13. In Dimitris Thano's study GCN5 can acetlyate certain lysines on a histone only after the phosphorylation of a serine group on a histone. A) True B) False 14. The following is evidence that a histone code is used in regulated gene expression. A) Nucleosomes methylated at K9 of histone H3 can be bound by a repressor while nucleosomes methylated at K4 & K9 of histone 3 AND K20 of histone H4 are bound by an activator and are not bound by the repressor. B) Transcription factors can assemble into heteromeric multimers. These bind different sequences than the homomultimers. In addition, the effect of an enhanceosome is dependent on the unique combinations of transcription factors that it contains. 15. Polyadenylation A) occurs in the nucleus but not the cytoplasm B) mrnas encoding histones are not polyadenylated C) occurs on transcripts made from RNA polymerase I D) RNA polymerase II participates in the process E) Involves something called CTD 16. Lef-1 A) A transcription factor that stimulates transcription by bending the DNA and bringing other transcription factor binding sites closer to the core promoter. B) A transcription factor that stimulates transcription by dimerizing with the CREB transcription factor. The transcription of certain genes can only be simulated by the 7

8 heterodimer. C) By itself Lef-1 can stimulate transcription from all core promoters. D) Regulates gene expression by cutting the DNA. This is a eukaryote restriction enzyme. E) When bound to DNA it can cause the DNA:protein complex to migrate aberrantly in a gel. 17. True or false. Please choose all of the true answers A) Histone acetyltransferases were first purified from a protozoan called Tetrahymena. B) Histone acetyltransferases were first purified from a yeast called Saccharomyces. C) Histone acetyltransferases have not yet been purified. 18. True or false. Please choose all of the true answers. A) During transcription, all of the histones must be removed from a gene before RNA polymerase can transcribe it. B) Architectural transcript factors can change the shape of the DNA to help transcription factors that have bound elsewhere to interact with the promoter. C) Activity staining was used to identify histone acetyltransferase in a gel. 19. Purposes of chromatin immunoprecipitation are: A) To determine if anti-repression has occurred. B) To determine if the DNA is in the 30nm fiber form or beads on a string form C) Determine the relative abundance of acetyl groups in a specific region of the genome D) Determine the relative rate of expression of a specific gene in the chromatin. E) Determine the relative abundance of a specific protein bound to a specific region of the genome 20. True or false. Please choose all of the true statements. A) In molecular biology "expression" means the level of production of a particular RNA or protein encoded by a particular gene. B) In molecular biology "expression" means the look on Dr. Atkinson's face when he realizes that he has forgotten what day and time it is and has just repeated a lecture! C) In molecular biology "expression" means the quantity of positively acting transcription factors bound to the transcriptional control region of a gene. 21. True of False. Choose the true statements. A) When one shears DNA the breaks in the DNA are introduced in a more random manner than when breaks are introduced by enzyme digestion. B) Shearing of DNA means to use physical stress to tear the DNA into fragments. 22. What does relative expression mean? A) It refers to the expression level from a gene in terms of the actual number of transcripts produced by the gene. B) It can mean the amount of expression from one gene in comparison to another gene (perhaps expressed as a ratio). C) It can mean the amount of expression from a gene in comparison to something like total cellular RNA. 23. Which of the following two outlines are correct for the chromatin immunoprecipitation assay? A) To do this one would cross link the chromatin, purify it, shear it into small fragments, perform immunoprecepitiation, reverse the cross links in the pelleted material, destroy the protein using a protease, purify the DNA, perform quantitative PCR against the parts of the genome under study. B) To do this one would cross link the chromatin, purify it, perform immunoprecepitiation, reverse the cross links in the pelleted material, destroy the protein using a protease, purify the DNA, shear the DNA into small fragments, perform quantitative PCR against the parts of the genome under study. 8

9 24. Here is a map of a plasmid. It is in a mammalian cell and it has been wrapped up in nucleosomes. It contains one promoter. The promoter is turned on and then the DNA with it's nucleosomes is isolated from the cell. This chromatin is digested with DNase I. The concentration of DNaseI is low and therefore hypersensitive sites are cut first. You stop this digestion at this point and remove all of nucleosomes from the DNA. Then the DNA is divided into three aliquots. One aliquot is digested with the restriction enzyme PstI, one with SphI and one with ClaI. The digested DNA is then run on an agarose gel. To produce the pattern shown ---->. Merely based on the pattern of bands you should be able to answer the following question. Where is the most likely location of the promoter? Choose one. A. At position 3. B. At position 6. C. At position 9. D. At position In the following list which best describes the sensitivity of the DNA to DNaseI digestion. A. Naked DNA > a 30 nm fiber form of chromatin > beads on a string form of chromatin > heterochromatin > actively transcribed chromatin B. Naked DNA > beads on a string form of chromatin > actively transcribed chromatin > a 30 nm fiber form of chromatin > heterochromatin C. Naked DNA > beads on a string form of chromatin == actively transcribed chromatin > a 30 nm fiber form of chromatin > heterochromatin D. heterochromatin > Naked DNA > beads on a string form of chromatin == actively transcribed chromatin > a 30 nm fiber form of chromatin 9