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Digestion by micrococcal nuclease of mouse submandibular-salivary-gland chromatin
Abstract:
Nucleic prepared from mouse submandibular salivary gland show marked fragility during isolation. Hwever, intact nuclei relatively free from cytoplasmic contamination were obtained by homogenization in buffers containing 0.88 M-sucrose, Ca2+, spermine, spermidine and the proteinase inhibitor aprotinin, followed by centrifugation through 2.2 M-sucrose. The kinetics of digestion by the micrococcal nuclease of chromatin in these nuclei are similar to those of chromatin from mouse liver nuclei. Base-pair size analysis of the solubilized DNA from both organs shows a stable high-molecular weight species of chromatin, which is further digested to mononucleosome and subnucleosome species. With extensive digestion the chromatin becomes insoluble. The mononucleosomes produced from salivary-gland chromatin after the inhibition of endogenous proteinase activity exhibit an s20,w value of 11S and contain histones H1, H2A, H2B, H3 and H4.
Insights
Researchers developed a method to isolate fragile mouse salivary gland nuclei, enabling chromatin analysis. This technique preserves high-molecular-weight DNA, revealing nucleosome structures similar to liver chromatin.
Area of Science:
- Molecular Biology
- Chromatin Structure
- Nucleic Acid Isolation
Background:
- Mouse submandibular salivary gland nuclei exhibit significant fragility during preparation.
- Cytoplasmic contamination is a common issue in isolating nuclear material.
- Understanding chromatin structure requires intact and pure nuclear samples.
Purpose of the Study:
- To establish a reliable method for isolating intact nuclei from mouse submandibular salivary glands.
- To analyze the chromatin structure and DNA digestion kinetics in these isolated nuclei.
- To compare salivary gland chromatin with that of mouse liver nuclei.
Main Methods:
- Homogenization of salivary gland tissue in a buffer containing 0.88 M sucrose, Ca2+, spermine, spermidine, and aprotinin.
- Centrifugation through a 2.2 M sucrose gradient to purify nuclei.
- Micrococcal nuclease digestion of chromatin followed by base-pair size analysis of solubilized DNA.
Main Results:
- Intact nuclei, free from cytoplasmic contamination, were successfully isolated using the developed buffer and centrifugation method.
- Chromatin digestion kinetics in salivary gland nuclei mirrored those observed in mouse liver nuclei.
- Stable high-molecular-weight chromatin species were identified, yielding mononucleosome and subnucleosome fragments upon digestion.
Conclusions:
- The optimized isolation protocol effectively yields pure, intact nuclei from mouse salivary glands, overcoming inherent fragility.
- Salivary gland chromatin exhibits structural characteristics and micrococcal nuclease digestion patterns comparable to liver chromatin.
- Mononucleosomes derived from salivary gland chromatin contain core histones (H1, H2A, H2B, H3, H4) and exhibit an 11S sedimentation coefficient.