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Unformatted text preview: Name ___________KEY_______________ MCB121 W12 Midterm 1 1 FIRST MIDTERM January 31, 2012 Question Points Score 1 32 2 42 3 24 4 12 5 40 Total 150 v This examination is closed book. v There are 8 pages to the exam. Please count them before you start to make sure all are present . v Please write your name on each page of the exam . v Answer each question in the space provided. If additional space is required use the back of the page and indicate clearly that you continued your answer on the back. Do not attach additional pages. v GOOD LUCK! Name ___________KEY_______________ MCB121 W12 Midterm 1 2 1. (32 points) A. Outline one strategy you might take to identify yeast centromere sequences. You should be able to explain your method in general terms in 4-5 steps using a clearly labeled diagram. (8 points) There are multiple ways you could do this. One way would be to create a yeast genomic library, shear or cut with restriction enzymes and clone into a yeast vector that contains a marker and an ARS sequence. Transform yeast with the library and select for the marker. Since centromeres are required for accurate chromosome segregation, those cells transformed with a plasmid carrying a centromere will grow better than those without. Plasmids can be isolated from these cells and sequenced. Another way you might do this is to do chromatin immunoprecipitation using and antibody to CENPA. To do this you would isolate genomic DNA from yeast and shear to small fragments using ultrasonic fragmentation or micrococcal nuclease. Next you would add an antibody to CENP that is attached to a bead. After centrifugation the antibody-bound chromatin will be separated from the rest of the chromatin. You can then use next generation sequencing (e.g. 454, Illumina or PacBio etc) or a microarray to identify the sequence. B. (8) You identify a 125 bp DNA sequence that can function as a centromere in yeast cells. One conserved feature of centromeres is the incorporation of the histone H3 variant CENP- A. You next want to see if this sequence can form a nucleosome incorporating CENP-A in place of H3. You add purified nucleosome subunits to a solution containing a 1000 bp fragment of DNA. Name all of the proteins you would include with your DNA fragment to create this specialized nucleosome. H2A, H2B, H4, CENPA C. (8)You use electron microscopy (EM) to visualize the DNA after the addition of the “CENP-A nucleosomes” you created. You notice that there are about 7 CENP-A nucleosomes assembled. In a control experiment using “H3 nucleosomes” you see only 5. You also notice that the diameter of the protein core of the CENPA nucleosome is about ½ the diameter of a H3 nucleosome. Which of the following hypotheses listed below are supported by these data (circle all that apply) i. The CENPA nucleosome core could be a tetramer....
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This note was uploaded on 03/16/2012 for the course MCB MCB 121 taught by Professor Gasser,burgess during the Winter '11 term at UC Davis.
- Winter '11