3400Su11Quiz2answers[1]

3400Su11Quiz2answers[1] - 1a How is DNA organized in...

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1a) How is DNA organized in 100A (10nm) and 300A (30nm) filaments? The 100A fil is ~147bp of double stranded DNA wrapped ~2x around a nucleosome. The 300A fil is a coil of the 100A fil (solenoid) that is held together by histone H1. b) DNA and nucleosomes have the ability to shift or move with respect to each other (phasing). How is this possible? The CRC and nucleosome chaperones are able to loosen the winding of DNA around the nucleosome and move it relative to the DNA which is associated with the nucleosome. This shifting of nucleosomes along the DNA is known as phasing. c) What is the role of Chromatin Remodeling Complexes? The role of the CRC is to loosen the chromatin structure so that transcription factors can gain access to the DNA strand. This is one of the first steps in making the 300A fil available for transcription. d & e) Design TWO experiments that would demonstrate that a specific gene was being expressed in a cell. The first might be one that we described in class. Isolate DNA from a cell thought to be expressing the gene, cut the DNA with a restriction enzyme and fractionate the fragments by gel electrophoresis, blot or transfer the DNA from the gel to a nitrocellulose filter and then probe the DNA on the blot with a cDNA or synthetic nucleotide specific for the mRNA encoded in the gene i.e. a cDNA. A second might be to isolate mRNA from a cell, fractionate it by gel elect., blot to a nylon membrane and then probe with cDNA. A third might be to use an IgG (antibody) to determine if the cell is making the protein in question. This would not be as good an experiment as it would not really show that the gene was being transcribed; the mRNA from the protein may have been made earlier. But we will accept this experment. 2a) Are histone proteins conserved among plants and animals? Why? Histones are extremely well conserved ; histone H3 from a garden pea and man differs by only one a.a. The conservation is due to the fact that the proteins are small and each a.a. is involved in an interaction with DNA or another histone. A single mutation would most likely negates (negative affects) its functionality. b) What histones form a nucleosome and how are nucleosomes put together? Nucleosomes contain two each of H2a, H2b, H3 and H4 – i.e. an octomer. They are assembled by histone chaperones and initially form a H2a/H2b dimers and H3/H4 tetramers that associate with a nucleosome chaperone when they enter the nucleus. Two dimers and
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a tetramer are then assembled into a nucleosome with the aid of the chaperone. c) What property of histone proteins allow them to play a role in gene expression? What enzymes make this regulatory role dynamic and what do these enzymes do? The folding of histones exposes a long amino terminal tail that sticks out from the rather globular proteins.
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