Identification of mitochondrial DNA species in interspecific cybrids and reconstituted cells using restriction endonuclease
Identification of mitochondrial DNA species in interspecific cybrids and reconstituted cells using restriction endonuclease
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使用限制性核酸内切酶鉴定种间杂种和重组细胞中的线粒体 DNA 种类
DOI：
10.1016/0014-5793(80)80913-6
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发表时间：
1980
期刊：
影响因子：
3.5
通讯作者：
T. Sekiguchi
中科院分区：
文献类型：
--
作者：
J. Hayashi;O. Gotoh;Y. Tagashira;M. Tosu;T. Sekiguchi
已知线粒体蛋白的基因在核 DNA 和线粒体 DNA (mtDNA) 中编码。因此，阐明细胞核和线粒体是否能够在细胞质杂种（cybrids）和细胞核和线粒体属于不同物种的重组细胞中协调发挥作用非常重要。将氯霉素抗性（CApr）细胞质转移到不同物种的全细胞中是极其困难的[11；这可能是由于不同物种的核DNA和mtDNA产物的不完全合作所致[11。然而，可以通过将人核质体与 CAP 小鼠细胞质体融合来分离可行的种间重组细胞 [21。通过亲代细胞线粒体 DNA 浮力密度的差异来鉴定细胞的线粒体 DNA 种类 [2]。然而，这种识别技术仅适用于具有足够不同的浮力密度以被清楚地区分的mtDNA物种。我们开发了另一种使用限制性内切酶鉴定培养细胞中 mtDNA 种类的程序。该程序具有许多优点：（i）它需要 < Ol ml 包装细胞（〜2 X 10'细胞）而无需标记；（ii）mtDNA 的切割模式是物种特异性的 [3]，甚至可以清楚地区分种内不同的 mtDNA [4-61；（iii）mtDNA 的序列鉴定是明确的。
Genes for mitochondrial proteins are known to be encoded in both nuclear DNA and mitochondrial DNAs (mtDNAs). So, it is important to elucidate whether the nucleus and mitochondria can function in harmony in cytoplasmic hybrids (cybrids) and reconstituted cells in which the nucleus and mitochondria are of different species. Transfer of chloramphenicol-resistant (CApr) cytoplasm to whole cells of different species was extremely difficult [11; this may be due to the incomplete cooperation of the products of nuclear DNA and mtDNAs of different species [11. However, viable interspecific reconstituted cells could be isolated by the fusion of human karyoplasts to CAP’mouse cytoplasts [21. mtDNA species of the cells were identified by a difference in buoyant densities of the mtDNAs of the parent cells [2]. However, this identification technique is applicable only to mtDNA species having buoyant densities sufficiently different to be clearly distinguished. We developed another procedure for identification of mtDNA species in culture cells using restriction endonuclease. This procedure has many advantages:(i) It requires< Ol ml packed cells (~ 2 X 10’cells) without labelling;(ii) The cleavage patterns of the mtDNAs are speciesspecific [3], and even intraspecifically different mtDNAs can be clearly distinguished [4-61;(iii) Sequence identification of mtDNAs is unambiguous.