猴脑线粒体DNA提取及限制性内切酶分析

PREPARATION AND RESTRICTION-ENZYME ANALYSISOF MITOCHONDRIAL DNA FROM BRAINTISSUE OF MACAQUES

本文用冷碱法从2只恒河猴和1只食蟹猴的脑组织中提取mtDNA,最后的得率大约为0.7μgmtDNA/g脑组织,是肝脏组织得率的1/3左右。与肝脏组织相比较,从脑组织中提取mtDNA有以下优点:1.匀浆方便。2.样品中蛋白杂质少,容易彻底抽提去除蛋白质。3.大分子RNA杂质极少,不经Sepharose-4B柱或RNawe处理,就可得到较纯的样品。加之哺乳动物脑的体积较大,哺乳动物脑组织不失为提取mtDNA的一个有用的组织来源。经16种限制性内切酶分析,并与来自同一个体肝脏组织的mtDNA比较,结果进一步证实,mtDNA无组织特异性。对12岁以上老年猴脑mtDNA的分析表明,在衰老中,动物mtDNA的序列可能没有变化,甲基化程度也无显著增高。

Miiochondrial DXAs (mtDNA) were isolated from brain tissue of three monkeys, i. e., an old (over 12 years old) and a young (2-3 years old) rhesus monkeys, and a young (1-2 years old) crab-eating monkey. The method we used is alkaline lysis as described in our previous paper. Compared with marker DNA (λ/EcoRI/Hind III) on gel, the final yield was about 0.7μg mol)NA/g brain tissue, a little lower than that from liver ; issue. On the other hand, the homogenization of brain sissue is very easy; and the quality of the mtDNA is better than mtDNA from liver tisue i. e.. there was much less protein and RNA in rongh mtDNA from brain tissue, so further purification of the mtRNA by Sepharose-4B column or RNase digestion is not needed. This nethod is thought to be useful in the study of mammal genelics. The mtDNAs from brain were compared with those from livers of the same monkey. When 16 restriction enzymes of 6-bp recognition were used. 38-42 sites were observed in the samples. By combining the cleavage patterns for eachenzyme, we classified the three samples into three different restriction types. Meanwhile, the mtDAXs from brains and livers of the same animals showed no difference. The results demonstrated further that mtDNA from different organs of the same animal had no difference. Our results about the old monkey also suggested that there might be no change in mtDNA scquences in the process of senility.