Upon gentle lysis of exponentially growing mouse carcinoma cells FM3A by sodium dodecyl sulfate, DNA was released as a "DNA-protein complex" in a folded conformation. No histones could be detected in the DNA-protein complex. The proteins bound to DNA were found to be composed of several kinds of nonhistone proteins with a molecular weight range of 50,000 to 60,000; they appear to play a key role in stabilizing and maintaining the compact and folded structure of the complex. Removal of the proteins by Pronase or 2-mercaptoethanol produced a more relaxed structure sedimenting about half as fast as the original complex in a neutral sucrose gradient. DNA in the folded complex is supercoiled, as indicated by the characteristic biphasic response of its sedimentation rate to increasing concentration of various intercalating agents, actinomycin D, ethidium bromide and acriflavine, with which the cells were treated before lysis. Pronase- or 2-mercaptoethanol-treated relaxed DNA still possessed the characteristic of closed-circular structure as judged from its response to intercalating agents. Nicking with gamma-ray or 4NQO broke these superhelical turns and relaxed the folded complex to slower sedimenting forms equivalent to the relaxed DNA obtained on treatment with Pronase or 2-mercaptoethanol. Viscometric observations of DNA-protein complex were consistent with the above results. A tentative model for the structure of this DNA-protein complex is proposed in which supercoiled DNA is folded into loops by several kinds of nonhistone proteins. Autoradiographic examination of the complex appeared to support this model.
Abstract Background : Our previous study showed that L ‐cysteine (Cys) and methylmethionine sulfonium chloride (MMSC) inhibited ethanol‐induced gastric mucosal damage and increased the amount of surface mucin in rats. This study examined whether Cys and MMSC augmented mucin secretion and changed distribution of mucin vesicles ultrastructurally in mucous cells by using primary cultured mucous cells from rabbit glandular stomach. Changes in intracellular cyclic adenosine 3′,5′‐monophosphate (cAMP) and in levels of cytosolic free Ca 2+ were investigated by treatment with Cys and MMSC. Methods : Mucin content was measured by an enzyme‐linked lectin assay. Transmission electron micrography was used to examine ultrastructural distribution of mucin granules. The amount of cAMP or levels of free Ca 2+ were measured by enzyme immunoassay or by fura‐2. 16,16‐Dimethyl prostaglandin E 2 (dmPGE 2 ) or ATP was used as the positive control. Results : L ‐Cysteine and MMSC increased mucin secretion and decreased cellular mucin content. The same was noted for dmPGE 2 . Accelerated mucin granule movements toward the plasma membrane were shown by these agents. Intracellular cAMP increased with exposure to dmPGE 2 for 20 min, while neither Cys nor MMSC increased cAMP. No increase in cytosolic free Ca 2+ levels occurred after treatment with Cys or MMSC, but an increase was induced 10 s after the addition of ATP. Conclusions : The present findings indicate that the increase in mucin secretion by Cys and MMSC was not mediated through the cAMP or Ca 2+ signal transduction pathway, but might occur through non‐receptor‐mediated mechanisms.
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