斯氏狸殖吸虫线粒体Cytb基因在不同虫期的转录水平分析

目的 研究斯氏狸殖吸虫线粒体Cytb(cytochrome B)基因在不同虫期的转录水平以更深入了解该吸虫的物质代谢途径,探索药物作用的药靶。方法 分别提取斯氏狸殖吸虫5个虫期的总RNA,并反转录为cDNA。将目的基因质粒作为标准品制作标准曲线,以5个虫期的cDNA为模板,特异引物为实验组引物,卫氏并殖吸虫的18SrDNA基因引物作为内参引物做实时荧光定量PCR,分析斯氏狸殖吸虫线粒体Cytb基因分别在5个虫期的转录水平。结果 标准曲线线性关系良好,回归系数γ=0.994。产物熔解曲线分析结果均显示为单一波峰。定量分析结果显示,斯氏狸殖吸虫线粒体Cytb转录主要在囊蚴期、30 d幼虫期及60 d童虫期,并且是逐步升高趋势,但成虫期转录较少,虫卵期没有转录。结论 斯氏狸殖吸虫线粒体Cytb基因在不同虫期的转录水平有差别,在60 d童虫期高转录,提示Cytb基因在幼虫的发育和移行中起着重要作用。     

斯氏并殖吸虫线粒体Cytb基因的扩增、测序以及遗传进化分析

目的扩增出斯氏并殖吸虫线粒体Cytb（cytochromeB）基因片段并测序,分析该基因对吸虫遗传分类的意义。方法提取斯氏并殖吸虫成虫基因组DNA,PCR扩增线粒体Cytb基因,克隆质粒并测序。在NCBI的GeneBank中作相似性和同源性检索,用DNAsis等软件分析这些吸虫的线粒体Cytb基因,采用最大简约法（MP）和邻位相连法（NJ）构建系统进化树,用MEGA4软件对遗传距离进行计算分析。结果获得542bp的斯氏并殖吸虫线粒体cytb基因片段,通过在Gene—Bank数据库中Blast发现,斯氏并殖吸虫的Cytb序列与日本血吸虫（中国流行区虫种）、异盘并殖吸虫、哈氏并殖吸虫以及巨睾吸虫等7种吸虫有相似性,斯氏并殖吸虫与日本血吸虫的遗传距离最近。结论线粒体国tb基因可作为吸虫遗传进化分类的依据之一,也是斯氏并殖吸虫与血吸虫等易出现较高的交叉反应率的原因之一。     

日本血吸虫和斯氏狸殖吸虫的cox1遗传多态性研究

目的分析日本血吸虫和斯氏狸殖吸虫线粒体cox1基因的遗传多态性。方法检获日本血吸虫和三峡库区上下游流行区斯氏狸殖吸虫样本,提取其细胞DNA,PCR特异性扩增线粒体cox1基因并测序;分别用Clusterx和DNAsis软件对两虫种线粒体cox1基因片段进行序列的变异分析和转录因子基因序列分析。结果PCR产物经1%琼脂糖凝胶电泳,日本血吸虫与斯氏狸殖吸虫cox1基因片段大小分别为445 bp和418 bp。两虫种cox1基因序列有138位碱基相同,占斯氏狸殖吸虫cox1碱基的33.01%。两虫种cox1基因包含的转录因子基因序列全部相同,均为12种,18个。结论日本血吸虫和斯氏狸殖吸虫在基因水平上存在明显差异,但在遗传进化上具有明显的相似性,从基因水平上证明了两虫种存在交叉抗原的可能性。     

湖南省怀化地区山羊胰阔盘吸虫线粒体pcox1基因和核糖体18S rRNA基因序列遗传变异研究

目的　了解湖南省怀化地区山羊体内分离的胰阔盘吸虫遗传变异情况。方法　应用PCR技术对分离自湖南怀化地区山羊体内的18株胰阔盘吸虫分离株线粒体细胞色素c氧化酶亚基I基因部分序列（pcox1）和核糖体18S rRNA基因进行扩增,对扩增产物进行测序并进行遗传变异和系统发育分析。结果　分离自湖南怀化地区山羊体内的18株胰阔盘吸虫分离株pcox1和18S rRNA基因序列长度分别为430 bp和1 857 bp,分别存在14个和35个变异位点,种内遗传差异分别为0 ~ 1.4%和0 ~ 0.8%;与GenBank数据库中收录的胰阔盘吸虫中国株基因序列同源性最高,分别为99.0% ~ 99.8%和99.5% ~ 99.8%。基于两种基因构建的系统发育树分析结果一致,本研究获得的18株胰阔盘吸虫分离株与GenBank数据库中已知胰阔盘吸虫分离株聚为同一分支,与支睾阔盘吸虫等阔盘属吸虫相隔较近,与其他吸虫所属分支相隔较远。结论　湖南省怀化地区山羊源胰阔盘吸虫分离株遗传变异度较低,线粒体pcox1基因和核糖体18S rRNA基因均适合作为羊源胰阔盘吸虫遗传变异研究的分子标记。     

DNA序列分析在吸虫种株基因差异研究方面的应用

近年来 ,分子生物学技术在现代寄生虫学种、株基因差异的研究中得到了广泛的应用 ,本文综述了核糖体、线粒体DNA序列分析技术在吸虫种、株基因差异研究的进展     

人体寄生虫的分类阶元

生物学分类可反映过去和现在种群间的进化关系。传统的人体寄生虫分类鉴定主要基于某些重要形态学特征,故在形态学特征相似的近缘物种分类中具有较大局限性。近年来分子生物学技术的发展,尤其是核糖体、线粒体基因等分子标记的有效应用和测序技术的快速发展,极大促进了人体寄生虫分子分类学的发展。国际上有关人体寄生虫分类也在不断修订和完善。本文对人体寄生原虫、吸虫、绦虫和线虫的分类阶元进行了梳理,旨在为研究寄生虫分子系统学和遗传进化提供参考。     

人类蠕虫线粒体基因组及其在种群遗传学和种系发生学中作为标记的应用

目前己报道了 10 0多种寄生虫的全长线粒体基因组。本文主要总结了后生动物门的线粒体基因结构 ,并且回顾了人类线虫和扁虫线粒体基因组的研究状况。一些蠕虫的全长或近于全长的线粒体基因组的发现 ,为蠕虫的种系发生分析和遗传变异性研究提供了极为丰富的遗传标记。本文举例说明了线粒体基因组在蛔虫、盘尾丝虫、血吸虫、片形属吸虫、并殖吸虫、棘口吸虫、棘球绦虫和绦虫属的研究中的应用     

DNA序列分析在吸虫种株基因差异研究方面的应用

近年来，分子生物学技术在现代寄生虫学种、株基因差异的研究中得到了广泛的应用，本文综述了核糖体、线粒体DNA序列分析技术在吸虫种、株基因差异研究的进展。     

人类蠕虫线粒体基因组及其在种群遗传学和种系发生学中作为标记的应用

目前已报道了100多种寄生虫的全长线粒体基因组，本文主要总结了后生动物门的线粒体基因结构，并且回顾了人类线虫和扁虫线粒体基因组的研究状况，一些蠕虫的全长或近于全长的线粒体基因组的发现，为蠕虫的种系发生分析和遗传变异性研究提供了极为丰富的遗传标记。本文举例说明了线粒体基因组在蛔虫，盘尾丝虫，血吸虫，片形属吸虫，并殖吸虫，棘口吸虫，棘球绦虫和绦虫属的研究中的应用。     

牛裂体吸虫线粒体DNA序列和基因排序分析

目的为探讨牛裂体吸虫(Schistosoma bovis)在裂体属内的系统发生位置,测定牛裂体吸虫线粒体基因部分序列,并分析该编码区域的基因序列和基因排序。方法以GNT-K法抽提牛裂体吸虫成虫基因组DNA,用兼并和特异引物扩增目的基因。扩增产物经纯化后克隆于pGEM1T质粒载体,并转化大肠埃希菌。抽提和纯化阳性质粒DNA,并测序。以纯化后的阳性质粒DNA为模板,根据已获得的序列设计内部特异引物,采用引物步移法获得全长目的片段。在GenBank中查找曼氏血吸虫等相关血吸虫线粒体基因序列,作基因排序及比较分析后,以邻接法绘制系统发生树。结果测定了牛裂体吸虫线粒体烟酰胺腺嘌呤二核苷酸脱氢酶亚基Ⅳ～Ⅰ基因序列(nicotinamide adeninedinucleotide dehydrogenase subunit4-1gene,nad4-nad1),其长度为2214bp。分析该编码区基因排序为nad4-trnQ(Gln)-trnK(Lys)-nad3-trnD(Asp)-nad1。牛裂体吸虫在该区域的线粒体基因排序与非洲支系血吸虫相似,与亚洲支系血吸虫有很大的不同;根据牛裂体吸虫与其他8种吸虫部分nad4,nad3,部分nad1和部分nad4+nad3+nad1基因序列比对结果,分别构建系统发生树。结果表明,牛裂体吸虫与埃及血吸虫位于同一簇,归属于非洲血吸虫支系,这与由基因排序推测的牛裂体吸虫的系统发生位置结果相一致。结论牛裂体吸虫属于非洲支系而非亚洲支系血吸虫。     

Biochemical and electron microscopic evidence that cell nucleus negatively controls mitochondrial genomic activity in early sea urchin development.

Enucleated halves of sea urchin eggs obtained by centrifugation contain almost all the mitochondrial population of the egg. Removal of the nucleus followed by parthenogenetic activation stimulates the incorporation of [3H]thymidine into the mitochondrial DNA, whereas no such incorportion is observed in activated whole eggs. The block is not the result of a modification in the permeability of the mitochondrial membrane. Electron microscopic observations demonstrated duplication of mitochondrial DNA molecules in activated enucleated halves. No duplication was found in the mitochondrial DNA from activated whole eggs or from nonactivated enucleated halves. We conclude that the cell nucleus exerts a negative control on the activity of the mitochondrial genome through some short-lived nuclear substance(s).     

Expression of petite mitochondrial DNA in vivo: zygotic gene rescue.

A protocol is introduced for probing the organization and regulation of expression of the yeast mitochondrial genome, termed "zygotic gene rescue." The procedure is based on the notion that genes retained on mitochondrial DNA of on the notion that genes retained on mitochondrial DNA of petites can be expressed in zygotes of a cross between petite and wild type. To test the validity of this notion, we have taken advantage of our ability to discriminate, by mobility differences on sodium dodecyl sulfate/polyacrylamide gels, different forms of the product of alleles of the mitochondrial gene, varI. In petite strains that have retained the varI gene, its characteristic product appears in zygotes 4-5 hr after mating; no product is observed in petite strains deleted in the varI locus. Our studies indicate that (i) expression in the zygote of the varI gene in the petite genome is not exclusively the result of recombination with mitochondrial DNA of the wild-type tester, and (ii) the varI gene is probably reiterated in the petite mitochondrial genome. The strength of the technique of zygotic gene rescue in the analysis of the mitochondrial genome is discussed.     

Mitochondrial DNA and nuclear DNA from normal rat liver have a common sequence.

Although Pst I does not cut the circular mitochondrial genome of the rat, BamHI generates from this genome two unequal fragments of DNA. Each of these fragments was cloned in pBR322. Nuclear DNA was digested from rat liver singly or doubly with Pst I and BamHI, and it was demonstrated that nuclear DNA shared a common sequence with the larger mitochondrial DNA BamHI fragment. The cloned larger mitochondrial DNA fragment was further subdivided with HindIII into four pieces that were labeled and then used to probe the double-digested nuclear DNA. The hybridization data showed that the common sequence is less than 3 kilobase pairs long and lies within the part of the mitochondrial genome containing the D-loop and a portion of the rRNA genes. It therefore appears that, as in lower eukaryotes, there are shared sequences between the nuclear and mitochondrial genomes in mammals.     

Properties of a Saccharomyces cerevisiae mtDNA segment conferring high-frequency yeast transformation.

The bakers' yeast Saccharomyces cerevisiae is a facultative anaerobe, tolerant to mutations in its mitochondrial genome. Individual cytoplasmic petite mutants retain genetic information derived from any portion of the parenteral mtDNA, prompting questions concerning distribution of the DNA replication origin(s) on the yeast mitochondrial genome. The experiments described in this paper were designated to test the possibility of using high-frequency yeast transformation as a selection for yeast mtDNA sequences conferring autonomously replicating function. A complete petite mitochondrial genome was inserted into the yeast vector YIp5, and the hybrid plasmid (YRMp1) was used to transform yeast. YRMp1 promoted high-frequency transformation of both wild-type yeast cells and petite mutant hosts lacking mtDNA and was maintained in each of these strains as a high-copy-number extrachromosomal element. The stability and copy-number properties of YRMp1 are similar to those of YRp12, a recombinant plasmid containing a yeast chromosomal autonomously replicating sequence.     

A two-step hypothesis on the mechanisms of in vitro cell aging: cell differentiation followed by intrinsic mitochondrial mutagenesis

Despite vigorous research, there is yet no agreement on the biochemical mechanisms responsible for the loss of replicative potential of diploid cultured cells. In contrast to the program theories of in vitro cell aging, we propose that, as already suggested by Minot in 1907, senescence is the result of cell differentiation. We further maintain that the fundamental cause of cell aging is an instability of the mitochondrial genome because of a lack of balance between mitochondrial repair and the disorganizing effects of oxygen radicals which arise in the respiring mitochondria of terminally differentiated cells. This probably results in intrinsic mitochondrial mutagenesis which may be followed by endonuclease degradation of the altered mitochondrial DNA. Since the mitochondrial genome controls the synthesis of several hydrophobic proteins of the inner mitochondrial membrane, the postulated denaturation or loss of mtDNA will prevent the replication of the organelles. Thus, deprived of the ability to regenerate their mitochondrial populations, the cells will sustain an irreversible decline in their ability to synthesize ATP, with concomitant senescent degradation of physiological performance and eventual death.     

Cleavage of Replicating Forms of Mitochondrial DNA by EcoRI Endonuclease

Digestion of mouse L cell mitochondrial DNA with EcoRI restriction endonuclease produces two linear duplex fragments comprising 86.3 +/- 2.0% and 14.2 +/- 1.0% of the circular genome length (16,000 +/- 470 nucleotide pairs). Digestion of human HeLa cell mitochondrial DNA with EcoRI produces three linear duplex fragments comprising 49.2 +/- 1.0%, 44.4 +/- 0.9%, and 6.4 +/- 0.4% of the circular genome length (16,590 +/- 710 nucleotide pairs). These fragments are shown to be generated by cleavage in unique regions of the mouse and human mitochondrial DNAs. An electron microscopic analysis of partially replicated molecules cleaved by EcoRI establishes a unidirectional mode of DNA replication for L cell mitochondrial DNA. The origin for DNA replication is located on the larger EcoRI fragment at a position that is 1,890 +/- 250 nucleotide pairs (11.8 +/- 1.2% of the circular genome length) from the proximal restriction site. Replication proceeds unidirectionally away from this restriction site throughout the length of the larger EcoRI fragment. Analysis of L cell, D-loop mitochondrial DNA cleaved by EcoRI indicates that a unique sequence is synthesized in formation of the D-loop in these nonreplicating molecules. The origin of D-loop synthesis is located on the larger EcoRI fragment at a position 1,760 +/- 180 nucleotide pairs (11.0 +/- 1.1% of the circular genome length) from the proximal restriction site and is, therefore, the origin for unidirectional displacement replication.     

Mitochondria and the heart

Since the identification of the first pathogenic mutations of mitochondrial DNA in 1988, a plethora of information about human mitochondrial diseases has been brought to light. Not surprisingly, many of these disorders affect the myocardium, because this tissue relies heavily upon oxidative metabolism. This review focuses on disorders of the respiratory chain, the only area of mammalian cellular metabolism under the control of two genomes, nuclear and mitochondrial. Consequently, defects of aerobic synthesis of adenosine triphosphate (ATP) can be due to mutations of either genome. We describe genetic mitochondrial cardiomyopathies and briefly review mouse models and the mitochondrial theory of presbycardia.     

Yeast mitochondrial genomes consisting of only A.T base pairs replicate and exhibit suppressiveness.

Mutants, called p-, that result from extensive deletions of the 75-kilobase Saccharomyces cerevisiae mitochondrial genome arise at high frequency. The remaining mitochondrial DNA is amplified in the p- cells, often as head-to-tail multimers, producing a cell with the normal amount of mitochondrial DNA. In matings, some of these p- mutants exhibit zygotic hypersuppressiveness, excluding the wild-type mitochondrial genome (p+) from all the diploids that are produced. From a hypersuppressive p- strain, we isolated two mutants with reduced suppressiveness. These mutants, one moderately suppressive and one nonsuppressive, retain only 89 and 70 base pairs, respectively, of the wild-type mitochondrial genome. Their sequences consist of 100% A . T base pairs. Replication of DNA in the mitochondrion, formation and amplification of new deletion genomes, and exhibition of suppressiveness do not require a single G . C base pair.     

Biogenesis of mitochondria: molecular mapping of the mitochondrial genome of yeast.

We have developed a new procedure for the detailed molecular mapping of any allele of the yeast (Saccharomyces cerevisiae) mitochondrial genome. The procedure employs a collection of different genetically characterized petite strains whose genomes have been physically defined by molecular hybridization. The map position of an allele is within the DNA segment common to all defined petites that can be shown by marker rescue to retain the locus. The same collection of petites can be used to locate the positions of mitochondrial rRNA and tRNA cistrons and DNA fragments produced by restriction endonucleases.