Practice Exam 20071. A special JAK-STAT signaling system (JAK5-STAT5) was recently identified inwhich a gene called TS5 becomes selectively transcribed and expressed in theliver upon induction by a novel cytokine IL-25. TS5 encodes an important tumorsuppressor protein that keeps us from developing liver cancer. Patients withcertain liver tumors are found to express no or very little TS5. However,treatment with IL-25 can prevent liver cancer in these “responder” patients but notin other liver cancer patients.A. Briefly describe how IL-25 treatment might be able to help this“responder” subgroup of liver cancer patients.B. Why aren’t others or all liver cancer patients helped by IL-25 treatment?Describe 2 kinds of mutations that would lead to down regulated TS5.Briefly describe how specific small molecule drugs could reverse theeffects of this mutationC. The promoter and cis-regulatory sequence of the TS5 gene were clonedand sequenced. Which transcription factor binding sites would you expectto find? Explain your answer.D. (What in vitro assay would you use to determine the precise bindingsequence of your candidate transcription factor? Explain why you chosethis technique, and how it works.E. It was observed that treating your candidate transcription factor with aprotein phosphatase (an enzyme that removes phosphate groups fromproteins) obliterated the ability of your transcription to bind DNA.Explain why you would have expected this to happen?2. The ability to generate gene knockout and transgenic organisms have had a profoundimpact both on basic research capabilities as well as drug discovery and developmentin the pharmaceutical industry.A. (Briefly describe 4 of the technical or reagent “advances” that made itpossible to routinely generate KO mouse lines carrying mutation specificgenes.1.2.3.4.B. Today, one of the most important uses of KO mice involves drugdevelopment. Which 2 aspects of drug discovery and development havebeen most influenced by our ability to KO specific genes in mice andpropagate such KO mouse lines?C. If you compare the procedures for generating KO versus transgenicanimals, which of these do you think is more difficulty to successfullycarryout? Explain why.D. In constructing a specific vector for the tissue specific expression of acloned gene in a transgenic animal – what cis-control elements must youbuild into such a vector? For example, let’s say we want to over expresshuman insulin in red blood cells of sheep.E. There is a great deal of debate as well as suspicion and mis-informationabout genetically modified organisms (GMO’s) – especially agriculturalproducts such as rice, corn, wheat, etc. Describe 2 cases in which GMO’swould be or have already been generated that might make a big impact inthe 3rd World where food supply is limited relative to the population.Given this potential for tremendous “good” of GMO’s, why are certaingovernments and even some scientists reluctant to embrace GMO’s?F. Although we have successfully generated KO mice, has this technologybeen easily translated to work for many other organisms? Explain why?How about transgenics? Have they been applied to many species?3. One of the key steps in regulating differential gene expression in all organismsis the control of transcription. In prokaryotes, the transcriptional machinery is relativelysimple consisting of RNA pol and Sigma factors plus a few activators and repressors.However, in eukaryotes and especially metazoans, the transcriptional machinery hasevolved into a much more elaborate system of protein complexes that carry out multiplefunctions.A. Name and describe three of the critical and distinct stages of the transcriptionprocess for the synthesis of primary RNA products destined to become mRNA.B. What are 3 different classes of large multi-subunit protein assemblies thatparticipate in the formation of an activated pre-initiation complex (PIC) and thatpromotes gene transcription in animal cells?C. The TATA binding protein (TBP) is part of a large transcription complex – whatis it called and what are 3 functions attributed to the TBP-containing complex?D. Several of the different large multi-subunit complexes of the PIC also containpolypeptides that carry out various enzymatic activities. Name and brieflydescribe 3 such enzymes and the nature of their catalytic function.E. In addition to the integral components of the transcriptional pre-initiation complexthat must be assembled at eukaryotic promoters, what other class of co-regulatorsplay an important function in activating gene transcription? (HINT: recall whatthe nature of the DNA template is for transcription in eukaryotes). How does thisclass of co-regulators work to help activate specific genes?4. The DNA of Eukaryotic organisms is wrapped up into chromatin which is arepeating unit of protein and nucleic acids also called nucleosomes.A. Briefly describe an experiment that would allow you to determine thelength of DNA that is typically wrapped around and protected by amononucleosome. Do you expect the length of DNA that is wrappedaround single nucleosomes to be different from the inter-nucleosomeDNA length? Explain why.B. Once you have a test tube full of purified mononucleosomes, whatexperiments could you do to analyze the size and charge of the proteincomponents of mononucleosomes?C. If all or most of the DNA in the eurkaryotic nucleus is wound up intonucleosomes, how can sequence specific transcription factors like SP1bind to cis-regulatory DNA? Describe at least two different mechanismsthat would allow specific binding of transcription factors to chromatintemplates.D. Since cis-regulatory elements of metazoan genes can be anywhere from–50 to –50,000 bp upstream of the start site of transcription, whatexperiment could you perform to roughly map potential recognition sitesfor a given transcription factor (let’s say the well characterized EstrogenReceptor) at a gene when you only have the start site but not the cis-regulatory sequences identified. To refine this mapping whatcomplementary assay could you use?E. Once you have roughly mapped a putative ER control region (let’s say it’sbetween position –3250 and –3000) what assay would you use to showthat ER actually can occupy (ie be bound to) this putative ERE in a livingcell (ie in vivo). Briefly describe how your assay works.F. Given that ER is a hormone