PRACTICE EXAM ANSWERS 20071.A. Essentially, we want the tumor suppressor TS5 to be expressed at normal level.You should know that the Jak Stat pathway regulates this tumor suppressor, and thatTS5 induction is good. IL25 is a cytokine that will cause the upregulation of TS5.IL25 binds to the receptor on the outside of the cell activating a signal transductionpathway that ultimately leads to expression of TS5 which is needed for normal liverfunction. Since treatment of these patients with IL25 can prevent liver cancer thenwe can surmise that the receptor and corresponding pathway is intact otherwise noamount of IL25 would mitigate the disease. Therefore, this responder sub-group hasliver cancer because of a decreased amount of IL25 production for some reason.B. This question is divided into two parts—the first part would include potentialmutations in the Stat proteins, or mutations in the Jak proteins. All liver cancerpatients are not helped by IL25 treatment because the problem lies somewhere in thepathway that IL25 induces, not in the amount of IL25 present in the body. Possible mutations and how a small molecule drug could help:1. in the receptor – use a small molecule that binds and activates (causesdimerization) the receptor even though it does not recognize IL25.2. a mutation at one of the residues that is phosphorylated in the pathway –you could use a small molecule that binds to the protein that should bephosphorylated that mimics phosphorylation and allows the pathway to beactivated3. a mutation in the phosphorylation recognition module of JAK or STAT –use a small molecule that activates the protein anyway, even though it doesnot recognize its normal phosphorylated partnerC. The simplest answer is that these are Stat binding sites. STAT binding sites aretypically inverted repeats of a specific DNA sequence – this is because STAT’sare obligate dimers.D. The best in vitro assay would be footprinting but you could, with some effort, alsouse gel shift. Additionally, a type of SELEX experiment could be used. DNasefootprinting is best because it most accurately maps the sequence of the STAT5recognition element. You could confirm this by using point mutations of themapped binding site to be absolutely sure.E. In the STAT pathway, there must be a Jak mediated Phosphorylation of thetranscription factor STAT5. This happens because phosphorylation is requiredfor STAT dimerization, which in turn is required for DNA binding and activationof trxn. Treating STAT5 with a phosphatase would prevent dimerization and thesubsequent formation of an active STAT5.2.A. The following represent major advances that allowed KO technology1. ES cell discovery, isolation and cell culture2. Targeted homologous recombination in mammals3. Development of blastocyst injection techniques4. Mammalian cell culture transfection and vector developments5. “Plus-minus” dominant selection of transfected ES cellsB. 1. Creating animal models of human diseases2. Testing the consequences of inhibiting a specific target gene / Proofof Concept for potential drugs3. Drug testing / looking for side effects and/or drug specificityC. KO animals are more technically demanding. The procedure is morecomplicated, and success relies on achieving and detecting a very rare event –targeted homologous recombination as well as germ line transmission.D. The overexpression vector needs to contain regulatory sequence that will berecognized by factors in the tissue you would like to express your gene in. In thiscase, you require a sheep (or mammalian or at least eukaryotic) basal promoterand enhancer elements from sheep red blood cells—probably also useful to have agood ribosome binding site, splicing donor/acceptor sites, poly-A signal etc.E. Several possible examples although we specifically talked about in 2 class :sheep that produce clotting factor and golden rice. Hopefully some people willdescribe other examples and were able to list two genetically modified foodorganisms, since that is what the question is about. Social resistance to change,potential metabolic side effects of the transgene, and potential danger to theenvironment all issues that would be accepted in the second part. Since thequestion asks for reasons why both governments AND scientists sometimes rebuffaccepting GMOs, only listing social issues will get only partial credit. Also, theycan’t just say “because people think GMOs might be bad.F. While transgenics have been used across different species, knockout’s have notbeen as successful. The problem is mostly with homologous recombinationfrequencies although establishing good ES cells can also be problematic.3A. Pre-initiation / initiation; promoter clearance/elongation; termination/ processing.Pre-initiation: involves the formation of stable protein/DNA complexes betweenpromoter DNA, the RNA pol II complex and various auxiliary factors (TFIIA, B,D,E,F,H).Promoter clearance/ elongation: involves the ATP (energy) dependent disengagementof the RNA pol II complex from the core promoter, the opening up or strand separationfor laying down the 1st few RNA products; the elongation step requires the processivemovement of RNA pol II aided by various elongation factors to synthesize long primarytranscripts.Termination/processing: involves the release of the elongating RNA pol II complexaccompanied by 3’ cleavage and poly A addition of the RNA transcript.B.• Activator/enhancer complexes (enhancesomes)• RNA polymerase II and the basal transcription complex including TFIIA, B,D, E,F, H.• Co-activator or co-repressor (co-regulatory) complexes (TBP/TAFs; SAGA;CRSP/mediator; CBP etc.)• Chromatin remodeling and modification complexes: BAF, SWI/SNF/ Brahma,HDAC, HATC. TBP is part of the TBP/TAF complex also called TFIID. This multi-subunitcomplex has the following documented functions:• Binding to specific seq elements of the core promoter (ie TATA, INR, DPE)• Targeted interaction with activation domains of enhancer binding activators (ie.the TAFs can serve as co-activators linking activators to the pre-initiationcomplex.)• Some of the TAF’s have the ability to catalyze various enzymatic reactionsincluding kinase, acetyl transferase, UB-ligase.• At least one TAF subunit is also involved in a chromatin recognition function viabromodomains that bind to acetylated lysines on the N-terminal tails of H3 andH4.D. Catalytic Activities:RNA pol II