Waterborg J H
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri, Kansas City 64110-2499.
J Biol Chem. 1993 Mar 5;268(7):4912-7.
Histone synthesis in alfalfa tissue culture cells was studied by labeling with tritiated lysine, purification of histone proteins by reversed-phase high pressure liquid chromatography, and fluorography of acid/urea/Triton X-100 polyacrylamide gels. Minor histone variant H3.2 was synthesized twice as fast as major variant H3.1. The predicted difference in histone H3 variant turnover was examined during continued growth. More than 50% of newly synthesized histone H3.2 and 20% of new H3.1 were lost from chromatin over a period of 100 h. This produced a ratio between the stable remaining portions of each new histone H3 variant protein identical to that of the steady-state histone H3 variants. The labile portion of new histone H3.2 (half-life of 20 h) was rapidly lost specifically from transcriptionally active chromatin as judged by the acetylation level of nearly 1.5 acetylated lysines/histone molecule, a level 50% higher than the acetylation in histone H3.2 overall and three times that of histone H3.1. These results and the constitutive level of H3.2 gene expression identify histone H3.2 of alfalfa as a functional replacement histone variant. The extent of its preferential assembly into active chromatin nucleosomes and the rapid rate of its subsequent loss indicate significant dissolution of plant nucleosomes during gene transcription.
通过用氚标记的赖氨酸进行标记、利用反相高压液相色谱法纯化组蛋白以及对酸/尿素/ Triton X - 100聚丙烯酰胺凝胶进行荧光自显影，研究了苜蓿组织培养细胞中的组蛋白合成。次要组蛋白变体H3.2的合成速度是主要变体H3.1的两倍。在持续生长过程中，对组蛋白H3变体周转的预测差异进行了研究。在100小时的时间内，超过50%新合成的组蛋白H3.2和20%新合成的H3.1从染色质中丢失。这使得每种新的组蛋白H3变体蛋白稳定剩余部分的比例与稳态组蛋白H3变体的比例相同。根据每分子组蛋白近1.5个乙酰化赖氨酸的乙酰化水平判断，新组蛋白H3.2的不稳定部分（半衰期为20小时）从转录活跃的染色质中迅速特异性丢失，该水平比组蛋白H3.2整体的乙酰化水平高50%，是组蛋白H3.1乙酰化水平的三倍。这些结果以及H3.2基因表达的组成水平将苜蓿的组蛋白H3.2鉴定为一种功能性替代组蛋白变体。其优先组装到活性染色质核小体中的程度以及随后快速丢失的速率表明在基因转录过程中植物核小体有显著溶解。
