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1 Genetics 3500 winter 2016 Test ii 10 marks The goal of the ENCODE project is to provide a detailed understanding of how genetic information is organized on our chromosomes. This insight have been made possible by the completion of the Human genome sequencing program in 2000 and the development of newer high throughput DNA sequencing technologies. Describe in detail what the ENCODE project has told us about how genes and how DNA encoding elements are organized on the human genome. See table in paper by Gerstein Expectation is that you enumeration several examples and include a few words of explanation. Should include: Much of the genome is transcribed as NcRNA some of which overlap protein sequences Exons can be shared by unrelated proteins. Introns can contain open reading frames of oother genes. RNA editing so proteins do not reflect DNA sequence Chromatin modification, Methylation of DNA and Histone modification affect gene regulation (information not embedded in DNA sequence Abundance of Transposable elements, some of which are still active CNV affect gene expression and an important form of genetic variation. Enhancer and silencers may be distant from genes that re regulated Abundance of pseudogene some of which are transcribed Why? Alternative splicing is very extensive. Proteins have an average of >5 splice forms. Transplicing of transcript on different chromosomes makes some novel proteins. Protein modification cannot be predicted from DNA sequence. Rna genes suchas MiRNA and SNrna’s 2 6marks Describe the application and significance of the Ames test. The Ames test is used to determine whether metabolic products of a particular substance are mutagenic Salmonella strains that are his – and deficient in one or more DNA repair enzymes are used. The compound is added to cells and liver extract and the mix is plated on his- media plates Comp’d are mixed with liver enzymes because many compounds are not themselves mutagens but become mutagens as a consequence of metabolism. The bacteria cells that grow are revertants. The number of revertants is an indication of how mutagenic the compound is and the number of mutants associated with a particular strain indicated the type of DNA damage done by a particular mutagen. 4 marks What are the potential consequences of chromosome breakage and rearrangement events due to the activation of DNA repair systems On rare occasions chromosome breakage will result in the inactivation of a specific gene . That’s because the amount of chromosome space occupied by genes is pretty small (1-2%). More frequently chromosome breakage and rearrangements are likely to move genes so that their expression is affected by other enhancer sequences. The example the text referred to is this one. https://en.wikipedia.org/wiki/Philadelphia_chromosome 3 6marks Describe the principle of PCR and indicate why this technology warranted the awarding of a Nobel Prize in 1994. Define. PCR is a technique that enable the amplification of specific Piece of DNA form a template DNA that is determined by ss DNA primers that flank the region that you wish to amplify. Define components. Primers, nucleotides, heat stable DNA polymerase, buffer and PCR machine aka thermocycler. Explain role of heating step, the n what happens when sample is cooled and primers bind specifically. Consequence of repeated cycles, …..logarithmic amplification of DNA. 4 4 marks For decades “Lamarkism” aka the theory that Giraffes acquired long necks because they need to reach foliage at the top of trees (inheritance of acquired characteristics ) was derided as junk science. We now now know that the effects of some environmental exposures can be heritably passed on to the progeny for at least one generation. This is called epigenetic inheritance. What is the molecular basis for this kind of inheritance? Methylation of DNA specifically the cytosine residues. Modification of histone proteins methylation (decreases access of TF to DNA) Deacetylation. Chromatin remodeling…..shifting the position of histones along the DNA to provide access of TF to promoter regions. 4 marks What are the common feature of a plasmid vector used in recombinant DNA research? See plasmid images on web. Restriction sites Promoter to control rate of transcription of the inserted gene product Termination sequence Selection marker Antibiotic resistance. 5 Answer one of the following two questions for 6marks (i) What role does Rb play in the regulation of the cell cycle and how the does p53 regulate the cell cycle when DNA damage is detected? (ii) What is the purpose of the RAS pathway? How does it work? More details are expected in your essay. You may find it helpful to include an image and describe all the point that the figure is intended to show the reader. Rb is synthesized and binds to the transcription factor E2F. Phosphorylation of RB by specific cyclinCDK complex (cyclin and cyclin-dependent kinase) results in Rb releasing E2F. This protein is then able to act as a TF to promote synthesis of key genes needed to replicate DNA. When DNA damage is detected, P53 acts as a TF to promote the synthesis of several genes, one being p21. P21 prevents the cyclin CDK’s from phosphorylating Rb. For addiitonal background, Google images of p53 and Rb , cell cycle RAs mediates a signal from outside the cell that results in the promotion of cell division. Binding of a growth factor to a growth factor receptor on the membrane triggers activation of RAS. In its inactive state RAS has GDP bound to it . RAS is activated when GTP is bound to RAS displacing the GDP. Activation ras then activated other proteins such as RAF leading to a signal cascades that leads to replication of DNA and cell division. 6 7 6 marks Multidrug resistance in cancer therapy results from the increased expression of specific membrane pumps that prevent the accumulation of a wide variety of chemotherapeutic agents in the cancer cells. Explain how the technique of real time PCR could be used to determine the levels of MDR-1 in a biopsy of tissue comparing gene expression in tumor cells and normal tissues, in order to assess a patient’s prognosis. ***Start with a short definition of PCR PCR is technique that results in the logarithmic amplification of a specific sequence of DNA that is determined by short primers which flank the region of DNA to be amplified. ***First mRNA is extracted from tumour and normal cell tissues and converted to cDNA using reverse transcriptase. *** In real time PCR the amplification of ds DNA is measured after each cycle using a fluorescent dye. (more DNa that is made results in more fluorescence) **** The amount of fluorescence generated using primers for MDR-1 in a sample is compared to the amount of fluorescence generated using primers for a gene that is assumed not to change its expression under most conditions. ***The relative expression of MDR, relative to the expression of the control gene in each sample is used to assess whether MDR-1 is up-regulated or down-regulated. 8 6 marks Describe how RE enzymes ligase plasmid vectors and E coli Bacteria can be used to clone DNA fragments isolated from a DNA sample from the Zika virus Define the components used: a. Restriction endonucleases. What do they do, what makes them specific cutters? b. Ligase What does it do? How is this enzyme used in conjunction with RE to make a recombinant molecule. See below: Process: 1. RE is used to cut the plasmid and the DNA source (zika virus) to create sticky ends. 2. Plasmid +DNA is mixed and ligase is added 3. Transformation of plasmid and selection of colonies of plasmids with inserts. 4. Only colonies with the transformed plasmid grow (why?) 9 6 marks You have a choice of either using the technique of microarrays or RNAseq to study gene expression in a ripening tomato fruit. Chose one of these techniques ,briefly explain what information it can give you and generally outline how it can be used to help understand this biological process. RNAseq *RNAseq is a technique in which the MRNA from a tissue is extracted and converted to cDNA with reverse transcriptase. Sequencing of cDNA enables estimation of relative expression of all mRNA reads. Can see expression of ncRNAs, protein coding genes and their alternative splice forms Upregulation of key genes can suggest a particular molecular mechanism that can be experimentally tested. Short version of the process (Not needed) cDNA is sheared into smaller pieces, sequencing primers are ligated onto the fragments sequencing primers enable the sequencing of fragments generating millions of reads reads are assembled back into mRNAs and aligned to the genome. Number of reads of each gene type is calculated. Microarrays. Microarray chip consists of a series of spots in which ss DNAs are attached that are capable of binding to a portions of the sequence of each gene from the organism. mRNA is isolated form tissue converted to cDNA and tagged with a fluorescent dye. cDNa is hybridized to the chip ad the amount of fluorescence at each spot is a measure of the number of copies of mRNA for that gene in the sample Upregulation of key genes genes can suggest a particular molecular mechanism that can be experimentally tested.