单个拟穴青蟹核内多拷贝线粒体COI假基因序列的研究(简报)

线粒体DNA(mtDNA)因它的高拷贝数、易扩增、高突变率、中性、低重组和母系遗传等特征,已广泛应用在系统进化和群体遗传研究方面[1,2].但是,由于mtDNA本身的特征,如异质性.     

Frequent assimilation of mitochondrial DNA by grasshopper nuclear genomes

Multiple copies of mitochondrial-like DNA were found in the brown mountain grasshopper, Podisma pedestris (Orthoptera: Acrididae), paralogous to COI and ND5 regions. The same was discovered using the ND5 regions of nine other grasshopper species from four separate subfamilies (Podisminae, Calliptaminae, Cyrtacanthacridinae, and Gomphocerinae). The extra ND5-like sequences were shown to be nuclear in the desert locust, Schistocerca gregaria (Cyrtacanthacridinae), and probably so in P. pedestris and an Italopodisma sp. (Podisminae). Eighty-seven different ND5-like nuclear mitochondrial pseudogenes (Numts) were sequenced from 12 grasshopper individuals. Different nuclear mitochondrial pseudogenes, if descended from the same mitochondrial immigrant, will have diverged from each other under no selective constraints because of their loss of functionality. Evidence of selective constraints in the differences between any two Numt sequences (e.g., if most differences are at third positions of codons) implies that they have separate mitochondrial origins. Through pairwise comparisons of pseudogene sequences, it was established that there have been at least 12 separate mtDNA integrations into P. pedestris nuclear genomes. This is the highest reported rate of horizontal transfer between organellar and nuclear genomes within a single animal species. The occurrence of numerous mitochondrial pseudogenes in nuclear genomes derived from separate integration events appears to be a common phenomenon among grasshoppers. More than one type of mechanism appears to have been involved in generating the observed grasshopper Numts.     

Pseudogenes and DNA-based diet analyses: a cautionary tale from a relatively well sampled predator-prey system

Mitochondrial ribosomal DNA is commonly used in DNA-based dietary analyses. In such studies, these sequences are generally assumed to be the only version present in DNA of the organism of interest. However, nuclear pseudogenes that display variable similarity to the mitochondrial versions are common in many taxa. The presence of nuclear pseudogenes that co-amplify with their mitochondrial paralogues can lead to several possible confounding interpretations when applied to estimating animal diet. Here, we investigate the occurrence of nuclear pseudogenes in fecal samples taken from bottlenose dolphins (Tursiops truncatus) that were assayed for prey DNA with a universal primer technique. We found pseudogenes in 13 of 15 samples and 1-5 pseudogene haplotypes per sample representing 5-100% of all amplicons produced. The proportion of amplicons that were pseudogenes and the diversity of prey DNA recovered per sample were highly variable and appear to be related to PCR cycling characteristics. This is a well-sampled system where we can reliably identify the putative pseudogenes and separate them from their mitochondrial paralogues using a number of recommended means. In many other cases, it would be virtually impossible to determine whether a putative prey sequence is actually a pseudogene derived from either the predator or prey DNA. The implications of this for DNA-based dietary studies, in general, are discussed.     

Multiple nuclear pseudogenes of mitochondrial DNA exist in the canine genome

Many copies of nuclear counterparts of mitochondrial DNA (mtDNA) were found in nuclear DNA from sperm heads of the domestic dog, Canis familiaris, by DNA-DNA hybridization and DNA sequencing. Nuclear counterparts homologous to the mtDNA D-loop region were cloned into lambda phage vectors (EMBL4 and lambda gt11), and nucleotide sequences of seven different mtDNA pseudogenes were then determined. The seven pseudogenes were E3 (474 bp; 82% homology with canine mtDNA), E13 (1867 bp; 67%), 8B (2375 bp; 78%), 12A (2650 bp; 79%), 33 (4131 bp; 86%), 47 (4251 bp; 86%), and E17 (5721 bp; 71%). These seven mtDNA pseudogenes corresponded to portions of cytoplasmic mtDNA containing the genes ile, ND1, leu, 16S rRNA, val, 12S rRNA, phe, D-loop, pro, thr, cytb, and glu. A neighbor-joining phylogenetic tree constructed from 12S rRNA sequences in mtDNA pseudogenes 8B, 33, 47, and E17 and in 10 mtDNA fragments from other species showed that these four pseudogenes form a monophyletic clade with canine mtDNA. A neighbor-joining phylogenetic tree based on the 318-bp cytb region showed that the canine pseudogenes existed before the divergence of 17 related canids, and their divergence dates were calculated at around 4.4 to 8.6 million years ago.     

Reliability of mitochondrial DNA in an acanthocephalan: the problem of pseudogenes

The utility of mitochondrial DNA as a molecular marker for evolutionary studies is well recognized. However, several problems can arise when using mitochondrial DNA, one of which is the presence of nuclear mitochondrial pseudogenes, or Numts. Pseudogenes of cytochrome oxidase I were preferentially amplified from Acanthocephalus lucii (Acanthocephala) using a universal PCR approach. To verify the presence and abundance of pseudogenes, length heterogeneity analysis of the PCR fragments was performed. PCR products obtained with universal primers often contained fragments of different sizes. Cloned sequences from universal PCR products nearly always contained sequence abnormalities such as indels and/or stop codons. Based on these sequences, new primers were developed to specifically target mitochondrial DNA. Sequences obtained with these specific primers lacked abnormalities. Phylogenetic analysis produced a single most parsimonious tree in which pseudogenes obtained with universal primers grouped together as did putative mitochondrial DNA sequences obtained with specific primers. The pattern of codon bias observed in the pseudogenes suggests a single nuclear integration event from the mitochondria. This is the first reported occurrence of pseudogenes in an acanthocephalan, and it demonstrates the potential dangers associated with the use of universal primers.     

Mitochondrial pseudogenes are pervasive and often insidious in the snapping shrimp genus Alpheus

Here we show that multiple DNA sequences, similar to the mitochondrial cytochrome oxidase I (COI) gene, occur within single individuals in at least 10 species of the snapping shrimp genus Alpheus. Cloning of amplified products revealed the presence of copies that differed in length and (more frequently) in base substitutions. Although multiple copies were amplified in individual shrimp from total genomic DNA (gDNA), only one sequence was amplified from cDNA. These results are best explained by the presence of nonfunctional duplications of a portion of the mtDNA, probably located in the nuclear genome, since transfer into the nuclear gene would render the COI gene nonfunctional due to differences in the nuclear and mitochondrial genetic codes. Analysis of codon variation suggests that there have been 21 independent transfer events in the 10 species examined. Within a single animal, differences between the sequences of these pseudogenes ranged from 0.2% to 20.6%, and those between the real mtDNA and pseudogene sequences ranged from 0.2% to 18.8% (uncorrected). The large number of integration events and the large range of divergences between pseudogenes and mtDNA sequences suggest that genetic material has been repeatedly transferred from the mtDNA to the nuclear genome of snapping shrimp. Unrecognized pseudogenes in phylogenetic or population studies may result in spurious results, although previous estimates of rates of molecular evolution based on Alpheus sister taxa separated by the Isthmus of Panama appear to remain valid. Especially worrisome for researchers are those pseudogenes that are not obviously recognizable as such. An effective solution may be to amplify transcribed copies of protein-coding mitochondrial genes from cDNA rather than using genomic DNA.     

Identification of the entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice

Summary The entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice (Oryza sativa cv. Nipponbare) was identified using clone banks that cover the chloroplast and mitochondrial genomes. The mitochondrial fragments that were homologous to chloroplast DNA were mapped and sequenced. The nucleotide sequences around the termini of integrated chloroplast sequences in the rice mtDNA revealed no common sequences or structures that might enhance the transfer of DNA. Sixteen chloroplast sequences, ranging from 32 bases to 6.8 kb in length, were found to be dispersed throughout the rice mitochondrial genome. The total length of these sequences is equal to approximately 6% (22 kb) of the rice mitochondrial genome and to 19% of the chloroplast genome. The transfer of segments of chloroplast DNA seems to have occurred at different times, both before and after the divergence of rice and maize. The mitochondrial genome appears to have been rearranged after the transfer of chloroplast sequences as a result of recombination at these sequences. The rice mitochondrial DNA contains nine intact tRNA genes and three tRNA pseudogenes derived from the chloroplast genome.     

Primers for a PCR-based approach to mitochondrial genome sequencing in birds and other vertebrates.

A PCR-based approach to sequencing complete mitochondrial genomes is described along with a set of 86 primers designed primarily for avian mitochondrial DNA (mtDNA). This PCR-based approach allows an accurate determination of complete mtDNA sequences that is faster than sequencing cloned mtDNA. The primers are spaced at about 500-base intervals along both DNA strands. Many of the primers incorporate degenerate positions to accommodate variation in mtDNA sequence among avian taxa and to reduce the potential for preferential amplification of nuclear pseudogenes. Comparison with published vertebrate mtDNA sequences suggests that many of the primers will have broad taxonomic utility. In addition, these primers should make available a wider variety of mitochondrial genes for studies based on smaller data sets.     

Fugu genome does not contain mitochondrial pseudogenes

Contrary to previous observations that fish genomes are devoid of nuclear mitochondrial pseudogenes, a genome-wide survey identified a large number of "recent" and "ancient" nuclear mitochondrial DNA fragments (Numts) in the whole-genome sequences of the fugu (Takifugu rubripes), Tetraodon nigroviridis, and zebrafish (Danio rerio). We have analyzed the latest assembly (v4.0) of the fugu genome and show that, like the Anopheles genome, the fugu nuclear genome does not contain mitochondrial pseudogenes. Fugu assembly v4.0 contains a single scaffold representing the near complete sequence of the fugu mitochondria. The "recent" Numts identified by the previous study in fugu assembly v2.0 are in fact shotgun sequences of mitochondrial DNA that were misassembled with the nuclear sequences, whereas the "ancient" Numts appear to be the result of spurious matches. It is likely that the Numts identified in the genomes of Tetraodon and zebrafish are also similar artifacts. Shotgun sequences of whole genomes often include some mitochondrial sequences. Therefore, any Numts identified in shotgun-sequence assemblies should be verified by Southern hybridization or PCR amplification.     

Nuclear mitochondrial pseudogenes

As has been demonstrated recently, the transfer of genetic material from mitochondria to the nucleus and its integration into the nuclear genome is a continuous and dynamic process. Fragments of mitochondrial DNA (mtDNA) are incorporated in the nuclear genome as noncoding sequences, which are called nuclear mitochondrial pseudogenes (NUMT pseudogenes or NUMT inserts). In various eukaryotes, NUMT pseudogenes are distributed through different chromosomes to form a “library” of mtDNA fragments, providing important information on genome evolution. The escape of mtDNA from mitochondria is mostly associated with mitochondrial damage and mitophagy. Fragments of mtDNA may be integrated into nuclear DNA (nDNA) during repair of double-strand breaks (DSBs), which are caused by endogenous or exogenous agents. DSB repair of nDNA with a capture of mtDNA fragments may occur via nonhomologous end joining or a similar mechanism that involves microhomologous terminal sequences. An analysis of the available data makes it possible to suppose that the NUMT pseudogene formation rate depends on the DSB rate in nDNA, the activity of the repair systems, and the number of mtDNA fragments leaving organelles and migrating into the nucleus. Such situations are likely after exposure to damaging agents, first and foremost, ionizing radiation. Not only do new NUMT pseudogenes change the genome structure in the regions of their integration, but they may also have a significant impact on the actualization of genetic information. The de novo integration of NUMT pseudogenes in the nuclear genome may play a role in various pathologies and aging. NUMT pseudogenes may cause errors in PCR-based analyses of free mtDNA as a component of total cell DNA because of their coamplification.     

Structure and chromosomal distribution of human mitochondrial pseudogenes

Nuclear mitochondrial pseudogenes (Numts) have been found in the genome of many eukaryote species, including humans. Using a BLAST approach, we found 1105 DNA sequences homologous to mitochondrial DNA (mtDNA) in the August 2001 Goldenpath human genome database. We assembled these sequences manually into 286 pseudogenes on the basis of single insertion events and constructed a chromosomal map of these Numts. Some pseudogenes appeared highly modified, containing inversions, deletions, duplications, and displaced sequences. In the case of four randomly selected Numts, we used PCR tests on cells lacking mtDNA to ensure that our technique was free from genome-sequencing artifacts. Furthermore, phylogenetic investigation suggested that one Numt, apparently inserted into the nuclear genome 25-30 million years ago, had been duplicated at least 10 times in various chromosomes during the course of evolution. Thus, these pseudogenes should be very useful in the study of ancient mtDNA and nuclear genome evolution.     

Amplification of multiple copies of mitochondrial Cytochrome b gene fragments in the Australian freshwater crayfish,Cherax destructor Clark (Parastacidae: Decapoda)

Non-coding copies of fragments of the mitochondrial genome translocated to the nucleus or pseudogenes are being found with increasing frequency in a diversity of organisms. As part of a study to evaluate the utility of a range of mitochondrial gene regions for population genetic and systematic studies of the Australian freshwater crayfish, Cherax destructor (the yabby), we report the first detection of Cytochrome b (Cyt b) pseudogenes in crustaceans. We amplified and sequenced fragments of the mitochondrial Cyt b gene from 14 individuals of C. destructor using polymerase chain reaction (PCR) with primers designed from conserved regions of Penaeus monodon and Drosophila melanogaster mitochondrial genomes. The phylogenetic tree produced from the amplified fragments using these primers showed a very different topology to the trees obtained from sequences from three other mitochondrial genes, suggesting one or more nuclear pseudogenes have been amplified. Supporting this conclusion, two highly divergent sequences were isolated from each of two single individuals, and a 2 base pair (bp) deletion in one sequence was observed. There was no evidence to support inadvertent amplification of parasite DNA or contamination of samples from other sources. These results add to other recent observations of pseudogenes suggesting the frequent transfer of mitochondrial DNA (mtDNA) genes to the nucleus and reinforces the necessity of great care in interpreting PCR-generated Cyt b sequences used in population or evolutionary studies in freshwater crayfish and crustaceans more generally.     

Mitochondrial COI gene transfers to the nuclear genome of Dendroctonus valens and its implications

Mitochondrial gene transfer to the nuclear genome could affect the accuracy of results in population genetics and evolutionary studies using mitochondrial gene markers. In a population genetics study of the red turpentine beetle (Dendroctonus valens), an invasive species in China, we found numerous ambiguous sites existing in the Cytochrome Oxidase I (COI) gene sequences obtained directly from polymerase chain reaction (PCR) products amplified from total genomic DNA using universal primers. By comparing the profiles of restriction endonuclease digestions and the sequences of PCR products amplified from mitochondrial DNA and nuclear DNA of the same individuals, we confirmed it was a phenomenon of mitochondrial gene transfer to the nuclear genome. Large numbers of COI pseudogenes were detected in this species. According to different levels of condon position bias and phylogenetic analysis, these should have originated from independent integration events. The impact of nuclear mitochondrial DNA sequences on population genetics analyses was discussed.     

DNA changes involving repeated sequences in senescing soybean (Glycine max) cotyledon nuclei

Age-related DNA changes in soybean ( Glycine max L. cv. Ransom) cotyledon nuclei were investigated by Feulgen cytophotometry and cloned DNA probes. The amount of nuclear DNA in 17-day-old senescing cotyledons was 23% lower than that of 5-day-old young cotyledons. In order to understand the nature of senescence-related DNA loss, fragments of repetitive DNA from young cotyledons were cloned into Escherichia coli HB101 cells by DNA recombination. The cloned DNA probed changes in specific repeated nucleotide sequences in senescing cotyledons. The colony hybridization between cloned DNA and [³²P]-labeled total DNA from 5- and 17-day cotyledons indicated loss of specific repeated sequences. The selected sequences of repeated DNA further showed a loss in their copy numbers. The study suggested that some repetitive DNA sequences were degraded selectively in the genome of senescing cotyledons.     

青蟹线粒体COI假基因的分离和特征分析

线粒体DNA标记在遗传结构和系统进化研究中得到广泛应用,然而核假基因的存在对此有很大威胁。本文以中国东南沿海的青蟹(Scylla paramamosain)为研究对象,利用线粒体COI基因的通用引物和特异性引物进行扩增,分别得到34个假基因（nuclear mitochondrial pseudogenes, Numts）和5个线粒体COI基因序列。在所获得的34个假基因中共定义了29种单倍型,根据序列的相似度,这些假基因可以分为2类,每类假基因都有各自保守的核苷酸序列。第Ⅰ类假基因存在2处插入序列和1处8 bp的缺失序列,这些位点导致了整个阅读框的移位;在第Ⅱ类假基因和5个线粒体COI序列中只有碱基替换,未发现插入和缺失序列。实验结果分析表明,这两类假基因分别代表了2次核整合事件,即核转移事件的最低值。研究结果提示了     

Hyperactive recombination in the mitochondrial DNA of the natural death nuclear mutant of Neurospora crassa.

In Neurospora crassa, a recessive mutant allele of a nuclear gene, nd (natural death), causes rapid degeneration of the mitochondrial DNA, a process that is manifested phenotypically as an accelerated form of senescence in growing and stationary mycelia. To examine the mechanisms that are involved in the degradation of the mitochondrial chromosome, several mitochondrial DNA restriction fragments unique to the natural-death mutant were cloned and characterized through restriction, hybridization, and nucleotide sequence analyses. All of the cloned DNA pieces contained one to four rearrangements that were generated by unequal crossing-over between direct repeats of several different nucleotide sequences that occur in pairs and are dispersed throughout the mitochondrial chromosome of wild-type Neurospora strains. The most abundant repeats, a family of GC-rich sequences that includes the so-called PstI palindromes, were not involved in the generation of deletions in the nd mutant. The implication of these results is that the nd allele hyperactivates a general system for homologous recombination in the mitochondria of N. crassa. Therefore, the nd+ allele either codes for a component of the complex of proteins that catalyzes recombination, and possibly repair and replication, of the mitochondrial chromosome or specifies a regulatory factor that controls the synthesis or activity of at least one enzyme or ancillary factor that is affiliated with mitochondrial DNA metabolism.     

香港红山茶个体内ITS多态性及物种鉴定的应用

核糖体DNA的内转录间隔区(ITS)一直被作为一种重要的分子标记,却很难用于山茶物种中。通过对1个疑似香港红山茶(Camellia hongkongensis)的样本进行ITS区域的扩增、克隆和测序,从中获得74种不同序列。研究结果表明,其ITS区域具有高度的多态性,其中76%的序列为假基因。系统发育分析显示,超过半数的假基因源自同一祖先。这些假基因在经历多次基因重复后分化成至少5个谱系,且每个谱系中的序列非常相似,这表明一些假基因不但未被剔除,反而通过快速复制事件幸存下来。由于山茶物种个体内ITS的高度多态,使用这个区域区分山茶物种可能导致错误。然而,通过比较香港红山茶中的1个种间特异性r DNA假基因,确定该样本属于香港红山茶。     

Nuclear insertions and heteroplasmy of mitochondrial DNA as two sources of intra‐individual genomic variation in grasshoppers

We examined the level of intra‐individual variation in a region of the mitochondrial genome coding for cytochrome oxydase 1 (COI) in two grasshopper species using a clone‐and‐sequence analysis of hundreds of sequences. In both Locusta migratoria and Chortoicetes terminifera, we found that 60–65% of the clones were unique COI‐like sequences. Among these COI‐like sequences, 70–75% diverged by less than 1% from the real mitochondrial haplotypes, and were likely to represent microheteroplasmic molecules. About 20% of the COI‐like sequences diverged by more than 9% from the mitochondrial haplotypes, and generally included stop codons, suggesting that these sequences were nuclear mitochondrial pseudogenes (NUMTs). Only six sequences, diverging by 2–6% from the mitochondrial haplotypes, were identified as potentially misleading in phylogenetic studies. In addition, we found that five sequences from C. terminifera were associated with mobile elements or repetitive DNA families.