1. The first paper to demonstrate different chromatin structures in active and i
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1. The first paper to demonstrate different chromatin structures in active and inactive genes used nucleases to probe the globin loci in chicken red blood cells, which express globin mRNA, and in chicken fibroblasts, which do not. Isolated nuclei from these cells were treated with either micrococcal nuclease or DNase I, and then DNA was prepared and hybridized in vast excess to a 3H-thymidine-labeled globin cDNA. If the nuclear DNA has not been degraded, it will hybridize to the globin cDNA and protect it from digestion by S1 nuclease, which is specific for single strands of DNA.
Digestion of red-cell nuclei or fibroblast nuclei with micrococcal nuclease (so that about 50% of the DNA was degraded) yielded DNA samples that still protected greater than 90% of the cDNA from subsequent digestion with S1 nuclease. Similarly, digestion of fibroblast nuclei with DNase I (so that less than 20% was degraded) yielded DNA that protected greater than 90% of the cDNA. An identical digestion of red-cell nuclei with DNase I, however, yielded DNA that protected only about 25% of the cDNA. These results are summarized in Table 1.
When nucleosome monomers were isolated from red blood cells by digestion with micrococcal nuclease, their DNA protected more than 90% of globin cDNA. If the monomers were first treated with DNase I, the isolated DNA protected only 25% of globin cDNA. If the monomers were briefly treated with trypsin to remove 20–30 amino acids from the N-terminus of each histone, digestion of the modified nucleosomes with micrococcal nuclease yielded DNA that protected only 25% of globin cDNA (Table 1).
A. Which nuclease—micrococcal nuclease or DNase I—digests chromatin that is being expressed (active chromatin)? How can you tell?
B. Does trypsin treatment of nucleosome monomers appear to render a random population or a specific population of nucleosomes sensitive to micrococcal nuclease? How can you tell?
1. The first paper to demonstrate different chromatin structures in active and inactive genes used nucleases to probe the globin loci in chicken red blood cells, which express globin mRNA, and in chicken fibroblasts, which do not. Isolated nuclei from these cells were treated with either micrococcal nuclease or DNase I, and then DNA was prepared and hybridized in vast excess to a 3H-thymidine- labeled globin cDNA. If the nuclear DNA has not been degraded, it will hybridize to the globin c and protect it from digestion by S1 nuclease, which is specific for single strands of DNA Digestion of red-cell nuclei or fibroblast nuclei with micrococcal nuclease (so that about 50% of the DNA was degraded yielded DNA samples that still protected greater than 90% of the cDNA from subsequent digestion with S1 nuclease. Similarly, digestion of fibroblast nuclei with DNase I (so that less than 20% was degraded yielded DNA that protected greater than 90% of the cDNA. An identical digestion of red-cell nuclei with DNase I, however, yielded DNA that protected only about 25% of the cDNA. These results are summarized in Table 1 When nucleosome monomers were isolated from red blood cells by digestion with micrococcal nuclease, their DNA protected more than 90% of globin cDNA. If the monomers were first treated with DNase I, the isolated DNA protected only 25% of globin cDNA. If the monomers were briefly treated with trypsin to remove 20-30 amino acids from the N-terminus of each histone, digestion of the modified nucleosomes with micrococcal nuclease yielded DNA that protected only 25% of globin cDNA (Table 1) A. Which nuclease micrococcal nuclease or DNase I digests chromatin that is being expressed (active chromatin? How can you tell? B. Does trypsin treatment of nucleosome monomers appear to render a random population or a specific population of nucleosomes sensitive to micrococcal nuclease? How can you tell? C. Is the alteration that distinguishes active chromatin from bulk chromatin a property of individual nucleosomes, or is it related to the way nucleosome monomers are packaged into higher-order structures within the cell nucleus? Protected globin cDNA a treatment Source of DNA DNase I micrococcal 91% 91% Fibroblast nucle 93% 95% 92% 25% Red-cell nuclei Red-cell nucleosomes 91% 25% 25% ed-cell nucleosomes (trypsin)Explanation / Answer
Ans-A- the micrococcal is more accurately used as the table shows that the globin protected was higher when micrococcal nuclease was used hence it is responsible for digestion of chromatin.
Ans-B- according to article the trypsin treatment reduce the amount of protected globin DNA and hence we can say that it renders the population sensitive to micrococcal action.
Ans-C-it is related to the packaging of monomers in the cell nucleus
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