CSB349H1_09FALL1_116 - Copy

CSB349H1_09FALL1_116 - Copy - 1. A gene mutation in mouse...

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Unformatted text preview: 1. A gene mutation in mouse embryonic stem cells is believed to affect a new sequence specific transcription factor that activates expression of the BLU gene. (a) You analyze the amino acid sequence of your putative transcription factor. Name two possible domains you would expect to find? (2 marks) - Activation domain (1) - DNA-binding domain (1) (subtypes are homeodomain, zinc containing motifs, basic leucine zipper, basic helix-loop-helix) OR – dimerization/protein interaction domain (subtypes are acidic type, glutamine rich, proline-rich) (1) (b) What are the two major rate-limiting steps that could be affected by your transcription factor to control the rate of transcription? (2 marks) (c) List the steps in a ChIP experiment and state two controls that are needed to interpret your results (3.5 marks). (d) You use an antibody to RNA pol II and mouse embryonic stem cells that contain a homozygous deletion of the gene encoding the transcription factor. What ChIP experiment would distinguish between the two mechanisms listed in (b)? Describe how the results of your ChIP experiment would differ, assuming that a mutation in your transcription factor affected each of the mechanisms listed in (b). (2 marks) - ChIP at BLU transcription start site (tss) for PolII in deletion and wildtype (wt) (1) - If deletion has no PolII, but wt does, then TF is likely affecting initiation (0.5) - If deletion and wt have same PolII at tss, then TF is likely affecting elongation; or, could ChIP downstream of tss to see that deletion has no PolII (0.5) OR will also accept: ChIP on CHIP - ChIP with RNAPII in deletion and wt (1) 1 ut or on - Crosslink cells (0.5) - Shear chromatin (0.5) - Enrich with antibody (0.5) - Reverse crosslink (0.5) - PCR using primers to specific region of interest (0.5) - Use un-enriched chromatin (input) as comparison (0.5) - Use IgG protein to control for non-specific antibody binding (0.5) to .s tu de nt bu dd y. c - Initiation (PIC assembly) (1) - Elongation (e.g., recruitment of PTEFb) (1) or RNAPII stalling (1) om - Hybridize resulting DNA to a tiling array, focusing on sequences at or around the TSS(1) - If TF affects elongation, then wildtype would have a signal at a sequence corresponding to the first 50 bp of the gene, but mutant would not(0.5) - If TF affects initiation, then wildtype would have a signal at the TSS but the mutant would not.(0.5) 5’ Based on this sequence, what could you hypothesize about the conformation your transcription factor adopts when binding DNA? (1 mark) Design an experiment to test this hypothesis (2 marks). 2. You are studying the NDD gene, which is mutated in a neurodevelopmental disorder. You decide to identify the regulatory module responsible for expression of the NDD gene in the cerebellum of the brain. (a) Briefly explain the principle behind the comparative genomics method to identify regulatory modules. (1 mark) - Regulatory sequences are conserved over evolution (because mutations in them are deleterious and removed from the population) (1) ut or on to - Express protein with interaction domain deleted (1) - DNAse I footprinting or gel-shift experiment. If hypothesis is right, DNA binding domain will be reduced. (1) .s tu - Hypothesis: binds DNA as a dimer because binding site appears palendromic (1) de nt bu dd y. c AACCCTAGGGTT 3’ 3’ TTGGGATCCCAA 5’ om (e) You identify the following 12 bp sequence in the promoter of the BLU gene that is bound directly by your transcription factor: 2 (b) What experiment is needed to test whether the regulatory module you had identified was an enhancer that drives NDD expression in the brain. What control experiment would you use? (2 marks) - Put conserved sequence upstream of a reporter and look at expression in a transgenic animal model system (vertebrate) (1) - Put in reporter with basal promoter alone (1); or construct with mutated enhancer (1) (c) What are the two reasons that the comparative genomics method might fail to identify the enhancer? (2 marks) (d) Let us assume the comparative genomics method failed to identify the enhancer in this case. If you knew that the enhancer driving NDD expression in the cerebellum was silent in the rest of the brain because of repression by a PRC chromatin-remodeling complex. How could this help you locate the cerebellum enhancer? What experiment would you perform? (2 marks) 3. To study the function of the mediator complex in differentiation of white blood cells, you delete the med23 subunit and make a series of measurements of mRNA levels using twocolour mircoarrays. (a) List the major steps in a microarray experiment. (2 marks) - Extract (0.25) RNA (0.25) - Make labeled (0.25) cDNAs (0.25) - Hybridize(0.25) to array (0.25) 3 ut or OR will also accept -ChIP-seq (1) -For partial marks: Tiling array, Northern blot, DNAse I assay, DNAse Footprinting (0.5) on to .s tu - The enhancer might show specific chromatin marks (e.g. H3K27 methylation) or be bound by components of PRC (1) - Do a ChIP-chip assay for PRC components and/or chromatin marks in the whole mouse brain and look for peaks around the NDD gene (1) de nt bu dd y. c om - Could be a species-specific regulatory sequence (1) - Could be a conserved element that doesn't show sequence similarity (1) - OR Convergent evolution (1) - Scan for fluorescence (0.5) OR “look at colours 0.25” (b) How would you identify the group of genes whose transcripts increased over a differentiation time course? (0.5 marks) - Cluster analysis (0.5) (c) Write the corresponding letter on each of the components indicated in the diagram below. A = RNA PolII C-terminal Domain B = Mediator Complex C = TATA binding protein Indicate with an ‘X’ the interaction that would be disrupted at the promoter of a gene whose expression pattern depended on the med23 subunit (2 marks) 4. The fish Vtx1 gene belongs to a family of conserved vertebrate genes encoding transcription 4 ut or (d) Fill in the graph below showing the typical expression pattern you expect to observe in your microarray experiment for a gene you identified whose activation in differentiated cells depends on the med23 subunit. (1 mark) on X de nt bu dd C B to .s tu y. c A Line should go _down_ over time. Any shape is fine. NO marks for 2 separate lines, even if one is down om factors. Vtx1 is expressed exclusively in the developing fish brain. In a recent cDNA library screen you identify a novel gene that you believe is a duplicate of Vtx1, called Vtx2. Unlike the Vtx1 gene, Vtx2 is only expressed in the developing retina but not in the developing brain. (a) You compare the expression patterns of the fish, chick and mouse Vtx genes and find that in both chick and mouse Vtx is expressed exclusively in the developing brain. What model might explain the overall pattern of evolution of these genes in vertebrates and why? (2 marks) - Mutations in the promoter/enhancer sequences of Vtx2 to yield a novel expression pattern in the developing retina (1) (c) What do you predict the expression pattern of the ancestral vertebrate Vtx gene would look like? (1 mark) 5. During a Ty1/copia retrotransposition event, the RNA copy of the retroelement is converted to double-stranded DNA. (a) After tRNA priming of the RNA, briefly describe 2 specific processes that ensure that the entire RNA transcript is copied into a complete first strand of DNA? (2 marks) - 5' end degradation of RNA/DNA hybrid sequence (0.5) leaving a DNA overhang encoding the "R" motif in the 5' LTR of the first strand DNA (0.5) OR 5’ R motif, OR 5’LTR - Template switching (0.5) where the R motif in the 3' LTR of the RNA copy hybridizes to the complementary R motif in the 5' LTR of the newly generated single stranded DNA (0.5) 5 ut or on - Exclusive expression in the developing brain (1) to .s tu de nt bu dd (b) What change(s) to the components of the gene(s) could account for the different patterns of expression observed between Vtx1 and Vtx2 in the developing fish? (1 mark) y. c om - Neofunctionalization of one copy after the fish specific duplication (1); or, novel diversified function of one copy of the gene in fish (1) - The conserved pattern of expression in all vertebrates tested is in the developing brain (Vtx1 in fish, Vtx in other vertebrates) (0.5), but the retinal expression pattern of the duplicate Vtx2 gene in fish is novel (0.5) If order is incorrect, NO marks (b) What feature of the sequence surrounding a retroelement would indicate a possible retrotransposition event? (0.5 marks) Why does this occur? (2 marks) - Direct repeat sequences on either side of the LTRs (0.5) OR “same sequence” flanking (0.5) - Because staggered cuts of a target sequence (0.5) are generated followed by insertion of blunted retroelement dsDNA (by ligation) (0.5) and the gaps are repaired by the host DNA repair mechanisms yielding a direct repeat sequence on either side of insertion (0.5) (c) What component of the retroelement is responsible for its transcription? How does this differ from the transcription of a transposon? (2 marks) 6. Your PhD project involves examining the function of AM1 in a juvenile vision disorder in mice. This gene is expressed in both extraembryonic and embryonic tissues, and the first knockout made in the lab was embryonic lethal due to a placental defect. Therefore, your supervisor instructs you to knockout this gene only in embryonic tissues using standard gene targeting techniques in ES cells. (a) State two independent strategies that you might use to accomplish this goal (1 mark). - Conditional gene targeting (or conditional knockout) (0.5) - Tetraploid complementation (0.5) 6 ut or If only have “retroelements have LTRs, transposons have ITRs” (0.5) on - 5'LTR (0.5) contains promoter (0.5) sequences for transcription (e.g. TATA box) - Transposons only have ITRs that do not contain promoter sequences (0.5) therefore they must rely on the fortuitous activity of a nearby active promoter that will transcribe the coding sequence of transposase into RNA (0.5) to .s tu de nt bu dd y. c om (b) Briefly state the steps involved for one of these strategies (2 marks) - Conditional gene targeting, or, conditional knockout (0.5) o Generate floxed allele of AM1 using homologous recombination in ES cells and breed to homozygosity (0.5) o Generate a Cre transgenic mouse by pronuclear injection using an a promoter that is only expressed in embryonic tissues (0.5) o Cross the Cre transgenic onto an AM1 heterozygous background (0.5) o Cross separate lines to generate conditional knockout only in embryonic tissues; placenta unaffected; genotype and analyze (0.5) Tetraploid complementation with null (-/-) ES cells (0.5) o Electrofuse host embryos (black strain) to generate 4N zygote and grow to blastocyst (or morula) stage in vitro (0.5) o Generate AM1-/- ES cell line by deriving de novo from ICM of AM1-/embryos using standard ES cell cultures (0.5) o Inject ES cells into blastocoel cavity of 4N embryo (or aggregate ES cells with 4N morula) (0.5) o Transfer to foster mother; offspring are all AM1-/- (placenta was normal); analyze phenotype directly (0.5) OR - 7. A genomic region situated in close proximity to the coding region of a gene is suspected to act as a promoter. (b) Indicate the expected distance of the features listed in (a) with respect to the transcription start site. (1 mark) - Initiator Element – can look for consensus sequence to be positioned around 10bp of TSS (0.5) - Downstream Promoter Element – can be within 50bp downstream of the TSS (0.5) (c) What specific feature of the primary transcripts would help you support the conclusion that this gene uses a broad type promoter? (1 mark) - Multiple tss identified resulting in transcripts with different lengths of the 5’UTR (1) 7 ut or on - Initiator Element (0.5) - Downstream Promoter Element (0.5) (Also histone variants, loose chromatin, protein binding sites) to .s tu (a) The sequence of the potential promoter does not seem to contain a TATA box. What two other features can suggest that it is a promoter? (1 mark) de nt bu dd y. c om 8. Briefly state why the excision of a single P element inserted near a gene could lead to different mutant alleles of that gene in different Drosophila embryos. (2 marks) Excision leaves behind overhangs of DNA that might be differentially degraded before the host DNA repair machinery (NHEJ) could repair the break (1) Different embryos could have different degrees of degradation (0.5) during the excision of their P element leading to different alleles of the affected gene (0.5) ----------------------------------------------END---------------------------------------------------- ut or on to .s tu de nt bu dd y. c om 8 ...
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