Collura R V, Auerbach M R, Stewart C B
Department of Biological Sciences, University at Albany, State University of New York 12222, USA.
Curr Biol. 1996 Oct 1;6(10):1337-9. doi: 10.1016/s0960-9822(02)70720-3.
Direct sequencing of mitochondrial DNA (mtDNA) following amplification using the polymerase chain reaction (PCR) has found widespread use in population genetic and phylogenetic research over the past few years. Recently, nuclear copies of mitochondrial genes have been reported in diverse eukaryotic species, often confounding such research (reviewed in [2,3]). Under certain circumstances, nuclear pseudogenes can be amplified more efficiently than the intended mtDNA target, even when using as template mtDNA that has been purified by gradient centrifugation. If the transfer of the gene copy to the nucleus happened recently, it can be difficult-if not impossible-to identify the legitimate mitochondrial sequence. Here, we present a simple method that can identify expressed mitochondrial genes, using the cytochrome b gene of the particularly problematical proboscis monkey as an example. Because mtDNA is transcribed and processed into polyadenylated mRNAs reverse transcription coupled to PCR can be used to amplify the expressed mitochondrial version. This method produced an unambiguous sequence for the proboscis monkey mitochondrial cytochrome b gene; in contrast, traditional DNA-based PCR methods produced ambiguous sequence, because many nuclear pseudogenes were present. Phylogenetic analysis of the cytochrome b gene suggests that the proboscis monkey groups with the Asian langurs, rather than forming a sister taxon to all Asian and African colobines as was previously suggested. Reverse transcriptase-coupled PCR should be applicable to many other cases of nuclear transfer of mtDNA, including those involving ribosomal genes.
在过去几年中，利用聚合酶链反应（PCR）扩增后对线粒体DNA（mtDNA）进行直接测序，已在群体遗传学和系统发育研究中得到广泛应用。最近，在多种真核生物物种中都报道了线粒体基因的核拷贝，这常常使此类研究变得复杂（见[2,3]综述）。在某些情况下，即使使用通过梯度离心纯化的mtDNA作为模板，核假基因也可能比预期的mtDNA靶标更有效地被扩增。如果基因拷贝向细胞核的转移是最近发生的，那么识别真正的线粒体序列可能会很困难——甚至是不可能的。在此，我们以特别成问题的长鼻猴的细胞色素b基因为例，介绍一种能够识别表达的线粒体基因的简单方法。由于mtDNA会被转录并加工成多聚腺苷酸化的mRNA，因此逆转录偶联PCR可用于扩增表达的线粒体版本。该方法为长鼻猴线粒体细胞色素b基因产生了明确的序列；相比之下，传统的基于DNA的PCR方法产生的序列不明确，因为存在许多核假基因。对细胞色素b基因的系统发育分析表明，长鼻猴与亚洲叶猴归为一类，而不是像之前所认为的那样与所有亚洲和非洲疣猴形成姐妹分类群。逆转录偶联PCR应该适用于mtDNA核转移的许多其他情况，包括涉及核糖体基因的情况。
