Date of Award
Doctor of Philosophy (PhD)
John A. Duerre
The in vitro methylation of non-histone chromosomal proteins (NHCP) was investigated in nuclei isolated from the brain, liver, and thymus of 6-8-day-old rats. After incubating the nuclei in the presence of 20 μM S-adenosyl-L-[methyl-3H]methionine(l Ci/mmol), the NHCP were separated from histones on hydroxylapatite, and fractionated further on SDS acrylamide slab-gel electrophoresis. After the gels were dried, autoradiography was used to detect 3H-methyl groups associated with these proteins. Four NHCP from the liver and thymus were methylated, while six methylated proteins were detected from the brain. None of the methylated proteins in these tissues corresponded with those from other organs, except for the component with a molecular weight of 66,000. It was evident that the methyl groups were esterified to the free carboxyl groups of NHCP since they are heat labile, yielding methanol.
The carboxyl methylated NHCP from these organs were tightly bound to chromatin. Nucleoplasm and loosely associated NHCP were essentially devoid of methylated protein. However, nucleoplasm contained proteins with molecular weights similar to the methylated proteins.
The Carboxyl methyltransferase has a pH optimum of 6.9-7.0, was present in nucleoplasm, extractable from chromatin with 0.4 M NaCl, and non-specifically methylated nucleoplasmic protein when nucleoplasm was incubated with S-adenosyl-L-[methyl-3H]methionine.
The carboxyl methylation of NHCP proteins was verified, in vivo. Six-day-old rats were given L-[methyl-3H]methionine(7 mCi/mmol) by an intraperitoneal injection. The rats were killed at varying time periods and the NHCP isolated from gradient purified nuclei. Chromosomal proteins from the cerebellum, cerebrum, kidney, liver, and thymus contained significant amounts of 3H-methyl groups. Turnover of the 3H-methyl esters was extremely rapid when compared to the turnover rates of the NHCP. Interestingly, a methylesterase, was observed to be tightly associated with chromatin, and could account for the rapid turnover of methyl esters in the absence of protein synthesis.
It is possible that methylation of certain NHCP resulted in their association with chromatin. Methylated NHCP were tightly bound to chromatin while nucleoplasm contain similar molecular weight proteins that were not methylated. These observations give support to the idea that certain newly synthesized NHCP, present in nucleoplasm, were methylated and as a result of this reaction, tightly bind to chromatin.
It is attractive to suppose that the methylation and demethylation of specific proteins could activate or inactivate genes by the binging or release of these proteins from chromatin. The carboxyl methylated proteins appeared to be tissue specific, and the methyl esters associated with these proteins turnover at a high rate. These observations suggested that the methylation and demethylation of specific proteins could result in gene regulation.
Quick, Douglas P., "Carboxyl Methylation of Non-Histone Chromosomal Proteins" (1980). Theses and Dissertations. 621.