The heavy strand dilemma of vertebrate mitochondria on genome sequencing age: number of encoded genes or G + T content?

IF 1.1 4区 生物学 Q4 GENETICS & HEREDITY Mitochondrial Dna Part a Pub Date : 2018-02-17 DOI:10.1080/24701394.2016.1275603
Nicholas Costa Barroso Lima, F. Prosdocimi
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These studies have been classically conducted for many clades (Corneo et al. 1966; Sinclair et al. 1967; Brack et al. 1972; Beridze & Tabidze 1976; Garber & Yoder 1983) and they are still done in a smaller pace to isolate cell components for further analyses (Zhao et al. 2005). However, with the development of PCR and Sanger sequencing techniques, researchers did not need to separate the mitochondria from other cellular components to perform the sequencing of their DNA. Nowadays, more than 10 years after the development of next-generation sequencing (NGS) techniques, mitochondrial genomes can be obtained as a byproduct of whole genome sequencing. Although the amount of mitochondrial reads can differ because of the type of tissue, animal group and/or sample extraction protocols, we have found that one mitochondrial read can be found for each 200–1000 reads of the nuclear genome (Uliano-Silva et al. 2015; Perini Vda et al. 2016; Prosdocimi et al. 2016). In some cases the amount of reads from mitochondrial DNA is so high that assemblers masked them as repeats while performing whole genome assemblies. It was just after assembling and annotating many complete mitochondrial genomes of various organisms (Gomes de S a et al. 2015; Uliano-Silva et al. 2015; Perini Vda et al. 2016; Prosdocimi et al. 2016; Souto et al. 2016; Uliano-Silva et al. 2016) that we decided to take a closer look into the H and L strand assignments. We were astonish to realize that many vertebrate mitochondrial genomes published to date seem to be inaccurately annotated in terms of light and heavy strands (Arnason et al. 2006, 2007; Zhang et al. 2012; Yoon et al. 2013; Ren et al. 2014; Pan et al. 2015; Zhang et al. 2015; Zou et al. 2015; Meng et al. 2016; Sun et al. 2016). Classic works showed that mitochondrial L-strand could be defined as the one that has the lower content of guanines and thymines (Taanman 1999; Munn 1975). According to Vinograd et al. (1963), the titration of N–H protons of thymines and guanines would increase the buoyant density of denatured DNA, showing that Gþ T content is responsible for the buoyant density of mitochondrial strands. When sequencing and analyzing the mitogenome of the blue-fronted amazon parrot, Amazon aestiva (Lima et al. manuscript in preparation), we were confronted with observation that the majority of the genes in this molecule was encoded by the L-strand, i.e. the strand presenting the lower Gþ T content (in this case 38.06%). The first work describing a complete mitochondrial genome from a species from the Amazona genus suggested that most genes were present in the H-strand (Urantowka et al. 2013). We were also amazed to observe that the most significant avian mitochondrial genome classically described for the chicken (Desjardins & Morais 1990), have provided contradictory information about heavy and light strand assignments. While saying that the heavy strand presented most of genes in the first paragraph of their results, Desjardins and Morais (1990) have also shown that this strand presents the lower amount of Gþ T content (37.3%) and showed most of their genes being encoded on this strand, according to the legend of their Figure 2. On the other hand, in one of the most classical and cited work of mitochondrial genomes dated from 1981, Anderson and collaborators described for the first time the whole human mitochondrial genome. They state that most genes of the human mitochondrion were encoded in the light-strand (Anderson et al. 1981). However, contrarily to Anderson’s work (Anderson et al. 1981), Taanman (1999) states that the human mitochondrion H-strand is the one with higher amount of genes encoded. In order to bring light to this question, we downloaded all available vertebrate mitochondria from RefSeq (November 2016) and calculated their Gþ T content, associating this information with the number of genes described on each strand. We found out that nearly all vertebrate mitogenomes (4200 out of 4205) have their light strand presenting most genes and the lowest Gþ T (Supplementary Table 1). The single exceptions are from 5 fishes from different clades, with 2 from the same genus Johnius. Those exceptions present the majority of genes encoded in the heavey strand (Accession numbers: NC_005800, NC_013879, NC_021130, NC_022464 and NC_008222). 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引用次数: 17

Abstract

Differential ultracentrifugation is a classic technique of cell biology and microbiology used to separate cell components, such as nuclei, mitochondria, chloroplasts, lysosomes, peroxisomes, vesicles, ribosomes, and cytoplasm (Garber & Yoder 1983). Regarding the study of the mitochondria, a cesium chloride (CsCl) solution has been used in a technique named Buoyant density ultracentrifugation to separate nuclear DNA from mitochondrial DNA (Welter et al. 1988; Zimmerman et al. 1988). Therefore, the two distinct bands obtained in the CsCl ultracentrifugation were used to define a heavy (H) and a light (L) DNA strand for a given mitochondrion. These studies have been classically conducted for many clades (Corneo et al. 1966; Sinclair et al. 1967; Brack et al. 1972; Beridze & Tabidze 1976; Garber & Yoder 1983) and they are still done in a smaller pace to isolate cell components for further analyses (Zhao et al. 2005). However, with the development of PCR and Sanger sequencing techniques, researchers did not need to separate the mitochondria from other cellular components to perform the sequencing of their DNA. Nowadays, more than 10 years after the development of next-generation sequencing (NGS) techniques, mitochondrial genomes can be obtained as a byproduct of whole genome sequencing. Although the amount of mitochondrial reads can differ because of the type of tissue, animal group and/or sample extraction protocols, we have found that one mitochondrial read can be found for each 200–1000 reads of the nuclear genome (Uliano-Silva et al. 2015; Perini Vda et al. 2016; Prosdocimi et al. 2016). In some cases the amount of reads from mitochondrial DNA is so high that assemblers masked them as repeats while performing whole genome assemblies. It was just after assembling and annotating many complete mitochondrial genomes of various organisms (Gomes de S a et al. 2015; Uliano-Silva et al. 2015; Perini Vda et al. 2016; Prosdocimi et al. 2016; Souto et al. 2016; Uliano-Silva et al. 2016) that we decided to take a closer look into the H and L strand assignments. We were astonish to realize that many vertebrate mitochondrial genomes published to date seem to be inaccurately annotated in terms of light and heavy strands (Arnason et al. 2006, 2007; Zhang et al. 2012; Yoon et al. 2013; Ren et al. 2014; Pan et al. 2015; Zhang et al. 2015; Zou et al. 2015; Meng et al. 2016; Sun et al. 2016). Classic works showed that mitochondrial L-strand could be defined as the one that has the lower content of guanines and thymines (Taanman 1999; Munn 1975). According to Vinograd et al. (1963), the titration of N–H protons of thymines and guanines would increase the buoyant density of denatured DNA, showing that Gþ T content is responsible for the buoyant density of mitochondrial strands. When sequencing and analyzing the mitogenome of the blue-fronted amazon parrot, Amazon aestiva (Lima et al. manuscript in preparation), we were confronted with observation that the majority of the genes in this molecule was encoded by the L-strand, i.e. the strand presenting the lower Gþ T content (in this case 38.06%). The first work describing a complete mitochondrial genome from a species from the Amazona genus suggested that most genes were present in the H-strand (Urantowka et al. 2013). We were also amazed to observe that the most significant avian mitochondrial genome classically described for the chicken (Desjardins & Morais 1990), have provided contradictory information about heavy and light strand assignments. While saying that the heavy strand presented most of genes in the first paragraph of their results, Desjardins and Morais (1990) have also shown that this strand presents the lower amount of Gþ T content (37.3%) and showed most of their genes being encoded on this strand, according to the legend of their Figure 2. On the other hand, in one of the most classical and cited work of mitochondrial genomes dated from 1981, Anderson and collaborators described for the first time the whole human mitochondrial genome. They state that most genes of the human mitochondrion were encoded in the light-strand (Anderson et al. 1981). However, contrarily to Anderson’s work (Anderson et al. 1981), Taanman (1999) states that the human mitochondrion H-strand is the one with higher amount of genes encoded. In order to bring light to this question, we downloaded all available vertebrate mitochondria from RefSeq (November 2016) and calculated their Gþ T content, associating this information with the number of genes described on each strand. We found out that nearly all vertebrate mitogenomes (4200 out of 4205) have their light strand presenting most genes and the lowest Gþ T (Supplementary Table 1). The single exceptions are from 5 fishes from different clades, with 2 from the same genus Johnius. Those exceptions present the majority of genes encoded in the heavey strand (Accession numbers: NC_005800, NC_013879, NC_021130, NC_022464 and NC_008222). We suspect that the statement proposed by Taanman (1999) that most vertebrate mitochondrial genes were on the H-strand was replicated downstream until most
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脊椎动物线粒体重链困境对基因组测序的影响:编码基因数还是G + T含量?
差异超离心是细胞生物学和微生物学的经典技术,用于分离细胞成分,如细胞核、线粒体、叶绿体、溶酶体、过氧化物酶体、囊泡、核糖体和细胞质(Garber & Yoder 1983)。关于线粒体的研究,在一种名为浮力密度超离心的技术中,氯化铯(CsCl)溶液被用于分离核DNA和线粒体DNA (Welter et al. 1988;Zimmerman et al. 1988)。因此,在CsCl超离心中获得的两个不同的条带用于定义给定线粒体的重(H)和轻(L) DNA链。这些研究通常是针对许多进化支进行的(Corneo et al. 1966;Sinclair et al. 1967;Brack et al. 1972;Beridze & Tabidze 1976;Garber & Yoder 1983),他们仍然以较小的速度分离细胞成分以进行进一步分析(Zhao et al. 2005)。然而,随着PCR和Sanger测序技术的发展,研究人员不需要将线粒体从其他细胞成分中分离出来进行DNA测序。在新一代测序技术发展10多年后的今天,线粒体基因组作为全基因组测序的副产品已经可以获得。尽管线粒体读取量可能因组织类型、动物组和/或样品提取方案而有所不同,但我们发现,每200-1000个核基因组读取可以找到一个线粒体读取(Uliano-Silva et al. 2015;Perini Vda et al. 2016;prodocimi et al. 2016)。在某些情况下,线粒体DNA的读取量如此之高,以至于组装者在进行全基因组组装时将其掩盖为重复序列。正是在组装和注释了各种生物体的许多完整线粒体基因组之后(Gomes de S a et al. 2015;Uliano-Silva et al. 2015;Perini Vda et al. 2016;prodocimi et al. 2016;Souto et al. 2016;Uliano-Silva et al. 2016),我们决定更仔细地研究H和L链的分配。我们惊讶地发现,迄今为止发表的许多脊椎动物线粒体基因组似乎在轻链和重链方面的注释不准确(Arnason et al. 2006,2007;Zhang et al. 2012;Yoon et al. 2013;Ren et al. 2014;Pan et al. 2015;Zhang et al. 2015;Zou et al. 2015;bbb10等人2016;Sun et al. 2016)。经典研究表明,线粒体l -链可以定义为鸟嘌呤和胸腺嘧啶含量较低的那条链(Taanman 1999;穆恩1975)。根据Vinograd等人(1963)的研究,滴定胸腺嘧啶和鸟嘌呤的N-H质子会增加变性DNA的浮力密度,表明Gþ T含量是线粒体链浮力密度的原因。在对蓝额亚马逊鹦鹉(amazon aestiva)的有丝分裂基因组进行测序和分析时(Lima等人的手稿正在准备中),我们发现该分子中的大多数基因都是由l链编码的,即Gþ T含量较低的那条链(在本例中为38.06%)。第一个描述亚马逊属物种完整线粒体基因组的工作表明,大多数基因存在于h链中(Urantowka et al. 2013)。我们还惊讶地发现,鸡的最重要的鸟类线粒体基因组(Desjardins & Morais 1990)提供了关于重链和轻链分配的相互矛盾的信息。Desjardins和Morais(1990)在他们的结果的第一段中说重链呈现了大部分基因,但他们也表明,这条链的Gþ T含量较低(37.3%),并且他们的大部分基因都编码在这条链上,如图2所示。另一方面,在1981年最经典和被引用的线粒体基因组研究中,安德森和合作者首次描述了整个人类线粒体基因组。他们指出,人类线粒体的大多数基因都编码于光链(Anderson et al. 1981)。然而,与Anderson的研究相反(Anderson et al. 1981), Taanman(1999)认为人类线粒体h链是编码基因数量较多的一段。为了阐明这个问题,我们从RefSeq(2016年11月)下载了所有可用的脊椎动物线粒体,并计算了它们的Gþ T含量,并将这些信息与每条链上描述的基因数量相关联。我们发现几乎所有的脊椎动物有丝分裂基因组(4205个中有4200个)都有包含最多基因和最低Gþ T的轻链(补充表1)。唯一的例外是来自不同分支的5种鱼类,其中2种来自同一属的Johnius。这些例外存在于重链编码的大部分基因(登录号:NC_005800, NC_013879, NC_021130, NC_022464和NC_008222)。
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来源期刊
Mitochondrial Dna Part a
Mitochondrial Dna Part a Biochemistry, Genetics and Molecular Biology-Genetics
CiteScore
3.00
自引率
0.00%
发文量
6
期刊介绍: Mitochondrial DNA Part A publishes original high-quality manuscripts on physical, chemical, and biochemical aspects of mtDNA and proteins involved in mtDNA metabolism, and/or interactions. Manuscripts on cytosolic and extracellular mtDNA, and on dysfunction caused by alterations in mtDNA integrity as well as methodological papers detailing novel approaches for mtDNA manipulation in vitro and in vivo are welcome. Descriptive papers on DNA sequences from mitochondrial genomes, and also analytical papers in the areas of population genetics, phylogenetics and human evolution that use mitochondrial DNA as a source of evidence for studies will be considered for publication. The Journal also considers manuscripts that examine population genetic and systematic theory that specifically address the use of mitochondrial DNA sequences, as well as papers that discuss the utility of mitochondrial DNA information in medical studies and in human evolutionary biology.
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