菜豆花粉发育中质体和线粒体及其DNA存在的状况——着重阐明质体遗传的细胞学基础

应用电镜和DNA的DAPI荧光检测技术研究了菜豆(Phaseolus vulgaris L.)小孢子/花粉发育中质体和线粒体及其DNA存在的状况。观察表明:在小孢子分裂时质体全部分配到营养细胞中,初形成的生殖细胞已不含质体。线粒体和质体的DNA在花粉发育中也先后降解,生殖细胞从刚形成时发育至成熟花粉时期这两种细胞器DNA均不存在。研究结果为菜豆质体母系遗传提供了确切的细胞学证据。遗传分析的研究曾确定菜豆质体为双亲遗传,对与本研究结论不同的原因进行了讨论。     

不同生理状态下膜状急纤虫大核DNA和线粒体DNA的多态性比较

为探索纤毛虫在营养及休眠条件下两套遗传系统的作用关系,对膜状急纤虫(Tachysomapellionella)营养细胞和休眠包囊大核DNA、线粒体DNA进行了RAPD比较。结果显示,在所选用的34条随机引物中,大核DNA共扩增出203条片段,其中以休眠包囊大核DNA为模板扩增出45条特有片段,以营养细胞大核DNA为模板扩增出36条特有片段,两者存在40%的差异。在所选用的32条随机引物中,线粒体DNA共扩增出216条片段,其中以休眠包囊线粒体DNA为模板扩增出35条特有片段,以营养细胞线粒体DNA为模板扩增出47条特有片段,两者有38%的差异。结果表明,膜状急纤虫休眠包囊与营养期的大核DNA结构存在显著的差异;两者的线粒体DNA结构也存在较大差异。这表明,膜状急纤虫在包囊形成过程中,大核及线粒体DNA结构可能都发生了一定的变化,并且这些变化可能与包囊形成过程中的形态结构和代谢活动等剧烈变化以及休眠状态下的生理生化变化密切相关。     

玉竹雄配子的发育——着重阐明质体在生殖细胞和营养细胞中的分布和变化

玉竹(Polygonatum simizui Kitag)小孢子在分裂前,质体极性分布导致分裂后形成的生殖细胞不含质体,而营养细胞包含了小孢子中全部的质体。生殖细胞发育至成熟花粉时期,及在花粉管中分裂形成的两个精细胞中始终不含质体。虽然生殖细胞和精细胞中都存在线粒体,但细胞质中无DNA类核。玉竹雄性质体的遗传为单亲母本型。在雄配子体发育过程中,营养细胞中的质体发生明显的变化。在早期的营养细胞质中,造粉质体增殖和活跃地合成淀粉。后期,脂体增加而造粉质体消失。接近成熟时花粉富含油滴。对百合科的不同属植物质体被排除的机理及花粉中贮藏的淀粉与脂体的转变进行了讨论。     

家马核基因组中线粒体核内插入序列分析

在各种真核生物核基因组中,存在一些由线粒体基因组转移进入核基因组中的DNA片段,这些被认为是分子化石的片段叫做线粒体核内插入序列(Numt)。由于Numt与真实的线粒体序列高度相似,因此它的存在必然会成为PCR扩增线粒体DNA的不利因素。利用已经公布的家马(Equus caballus)基因组序列(2007年9月公布,GenBank登录号为NC_009144-NC_009175)对家马Numt进行了深入分析,共发现200个可能的Numt,长度范围为29到3727bp,其中有10个的长度大于800bp。分析结果显示由于不存在线粒体控制区域的疑似Numt,因此对基于此区域的群体遗传学研究不会产生影响。本研究还发现在家马进化过程中,第1号和27号染色体更倾向于接受线粒体序列的转移。以上结果将为今后马科动物的研究提供重要的参考信息,有助于避免在线粒体DNA研究中由于Numt污染的存在而得出错误的实验结果。     

豌豆生殖细胞的发育—着重论述质体母系遗传的细胞学基础

超微结构的研究证明,豌豆（Ｐｉｓｕｍ ｓａｔｉｖｕｍ Ｌ．）生殖细胞自形成直至成熟花粉时期,始终存在少量质体和较多的线粒体。ＤＮＡ 荧光的观察表明,在发育早期的生殖细胞中不含细胞质ＤＮＡ 类核,但在成熟花粉的生殖细胞中有许多的类核。在花粉离体萌发过程中,随着花粉管的生长,生殖细胞中的类核逐渐降解。在花粉培养２４ ｈ 后,生殖细胞的类核全部消失。研究结果确定了豌豆质体母系遗传的细胞学基础,支持遗传分析及ＲＦＬＰ研究的结论,阐明了过去在细胞学上认为是双亲遗传的判断不正确的原因     

镉对小鼠精母细胞凋亡及附睾内精子质量的影响

采用人工水质染毒的方法,利用透射电镜技术及流式细胞术（FCM）,探讨重金属镉对小鼠精巢内生殖细胞凋亡及附睾内成熟精子质量的影响.结果表明：各试验组小鼠生殖细胞处于凋亡时期的数量显著高于对照组,凋亡时期的生殖细胞超微结构呈现出线粒体空泡、核膜内陷、染色质周缘化及核固缩等形态特征,表明镉容易引起小鼠生殖细胞凋亡;各试验组精子早期凋亡的比例显著高于对照组,而活性精子的比例显著低于对照组（P<0.05）,其中高剂量组（0.10 mmol·L^-1）精子成活率（75.1%）显著低于对照组和其他试验组,而早期凋亡率（22.6%）则显著高于对照组;高剂量组睾丸生殖细胞DNA断裂率（18.2%）及附睾精子断裂率（26.5%）均显著高于对照组（3.3%、5.6 %）（P<0.05）.各试验组小鼠睾丸内DNA断裂的生殖细胞数量低于附睾内DNA断裂的精子数量.随着添加剂量的增加,小鼠睾丸内生殖细胞及附睾内精子凋亡率逐渐升高.表明小鼠生殖细胞凋亡及DNA损伤数量与镉剂量具有一定相关性.     

原发性扩张型心肌病的线粒体tRNA基因突变分析

为寻找原发性扩张型心肌病病例是否存在已知以及未知的线粒体tRNA致病性突变,以探讨扩张型心肌病可能的发病原因。收集2例原发性扩张型心肌病患者和10例正常对照尸检心肌组织石蜡标本,针对22种线粒体tRNA基因分别设计一对引物,PCR扩增后并测序分析线粒体tRNA基因突变情况。结果在对照样本中未检测到线粒体tRNA变异位点,在1例患者中检测到了tRNA~(Val)基因G1664A变异,Mitomap已有报道为多态性位点;于另1例患者中检测到tRNA~(Met)T4454C变异,有文章报道该位点与线粒体功能障碍有关,Mitomap报道为多态性位点。本研究中2例病例中未检测到线粒体tRNA致病性突变位点,可能与病例个体的心衰程度有关,有必要扩大样本量深入研究线粒体tRNA以及mt DNA其他基因突变与原发性扩张型心肌病之间的关系,以寻找可能的致病突变位点、易感的多态性位点或者单倍体群,为认识原发性扩张型心肌病的发病机制进一步提供理论基础和依据。     

杜鹃精细胞的超微结构及DNA荧光观察——着重阐明质体双亲遗传的细胞学基础

迎红杜鹃 ( Rhododendron mucronulatum Turcz.)的成熟花粉为二细胞型 ,精细胞在花粉管中形成。花粉管中的两个精细胞及与营养核之间互相联结 ,形成雄性生殖单位。两个精细胞的细胞质中均含有丰富的细胞器 ,包括质体、线粒体、小泡及微管 ,内质网和高尔基体稀少。具正常结构的精细胞质体在切面上多呈环形或哑铃形 ,内膜不发达 ,基质电子密度高。线粒体为球形或棒状 ,基质电子密度较低。 DNA特异性荧光染色显示 ,生殖细胞及精细胞中均含有大量类核 ( nucleoid) ,两个精细胞中的类核数量无明显差异。结果证明了杜鹃精细胞中存在大量具 DNA的可遗传细胞器 ,为杜鹃属植物的双亲细胞质遗传方式提供了细胞学证据。     

不同培养条件对鸡胚胎生殖细胞Oct4启动子DNA甲基化的影响

旨在在体外通过胚胎生殖细胞(EGC)和细胞外基质共同构建一个EGC的小生境(niche)模型,通过比较Oct4DNA甲基化的变化,研究niche中特有的表观遗传效应对胚胎生殖细胞自我更新的影响.构建EGC细胞标准培养方案(对照组)和改良培养方案(试验组),采用甲基化特异性PCR( MS-PCR)技术、RT-PCR技术,对比分析两种培养方案中Oct4启动子的甲基化状态,判断基因表达与其CpG岛甲基化的关系,分析DNA甲基化模式与EGC细胞自我更新的关系.结果显示,试验组可扩增出Oct4的非甲基化扩增产物,而对照组为其甲基化扩增产物,试验组Oct4表达水平明显高于对照组.试验组EGC生长状态明显好于对照组.试验表明,在改良培养条件下,Oct4基因启动子DNA甲基化程度较低,且与基因表达水平呈负相关,更有利于维持胚胎生殖细胞的自我更新状态.     

提取线粒体DNA方法的比较及较纯线粒体基因获取方法的确立

目的:对2种常用的线粒体DNA提取方法进行比较,分析提取物中核DNA的存在情况,同时建立新检测方法降低线粒体假基因干扰。方法:以仅在核DNA中存在的基因β-actin作为核DNA存在的标定基因,通过PCR方法扩增线粒体DNA上的一段基因MTND5-2,并以β-actin做参比,比较常用的2种提取线粒体DNA方法的优劣,即碱法Ⅰ(先提取完整线粒体后从中获得线粒体DNA)和碱法Ⅱ(根据线粒体DNA与核DNA的结构差异,从中获得双链环状的线粒体DNA)。结果:2种线粒体DNA提取方法并不能获得仅含线粒体DNA的纯提取物,碱法Ⅰ获得的线粒体DNA纯度相对较高;以碱法Ⅰ提取物为模板进行PCR,可获得更多较纯的线粒体目的基因。结论:碱法Ⅰ较碱法Ⅱ可获得更纯的线粒体来源的目的基因;新建方法可获得较纯的线粒体基因,且是一种简单、方便、经济的方法。     

Inheritance of chloroplast and mitochondrial DNA in alloplasmic forms of the genus Daucus

The inheritance of mitochondrial (mt) and chloroplast (ct) DNA in the progeny from interspecific crosses between the cultivated carrot (Daucus carota sativus) and wild forms of the genus Daucus was investigated by analysis of mt and ct RFLPs in single plants of the parental and filial generations. We observed a strict maternal inheritance of the organellar DNAs in all interspecific crosses examined. Previous studies on putative F₂ plants from a cross between Daucus muricatus x D. carota sativus suggested paternal inheritance of ctDNA. Our reinvestigation of this material revealed that the mtDNA of the putative F₂ plants differed from the mtDNA of both putative parents. Therefore, our data suggest that the investigated material originated from other, not yet identified, parents. Consequently, the analysis of this material cannot provide evidence for a paternal inheritance of ctDNA.     

Cloning and characterization of the Chlamydomonas moewusii mitochondrial genome.

Summary We report that the mitochondrial genome of Chlamydomonas moewusii has a 22 kb circular map and thus contrasts with the mitochondrial genome of Chlamydomonas reinhardtii, which is linear and about 6 kb shorter. Overlapping restriction fragments spanning over 90% of the C. moewusii mitochondrial DNA (mtDNA) were identified in a clone bank constructed using a Sau3AI partial digest of a C. moewusii DNA fraction enriched for mtDNA by preparative CsCI density gradient centrifugation. Overlapping Sau3AI clones were identified by a chromosome walk initiated with a clone of C. moewusii mtDNA. The mtDNA map was completed by Southern blot analysis of the C. moewusii mtDNA fraction using isolated mtDNA clones. Regions that hybridized to C. reinhardtii or wheat mitochondrial gene probes for subunit I of cytochrome oxidase (cox1), apocytochrome b (cob), three subunits of NADH dehydrogenase (nadl, nad2 and nad5) and the small and the large ribosomal RNAs (rrnS and rrnL, respectively) were localized on the C. moewusii mtDNA map by Southern blot analysis. The results show that the order of genes in the mitochondrial genome of C. moewusii is completely rearranged relative to that of C. reinhardtii.     

Mitochondrial DNA of the blowflyPhormia regina: restriction analysis and gene localization

A study of an invertebrate mitochondrial genome, that of the blowflyPhormia regina, has been initiated to compare its structural and functional relatedness to other metazoan mitochondrial genomes. A restriction map of mitochondrial DNA (mtDNA) isolated from sucrose gradient-purified mitochondria has been established using a combination of single and double restriction endonuclease digestions and hybridizations with isolated mtDNA fragments, revealing a genome size of 17.5 kilobases (kb). A number of mitochondrial genes including those encoding the 12 S and 16 S ribosomal RNA, the cytochromec oxidase I subunit (COI) and an unidentified open reading frame (URF2) have been located on thePhormia mtDNA by Southern blot analysis using as probes both isolated mtDNA fragments and oligonucleotides derived from the sequences of previously characterized genes from rat andDrosophila yakuba mtDNAs. These data indicate that for those regions examined, the mitochondrial genome organization of blowfly mtDNA is the same as that ofDrosophila yakuba, the order being COI-URF2-12 S-16 S. These data also report the presence of an A + T-rich region, located as a 2.5-kb region between the URF2 and the 12 S rRNA genes, and its amplification by the polymerase chain reaction is described.     

Unparalleled GC content in the plastid DNA of Selaginella

One of the more conspicuous features of plastid DNA (ptDNA) is its low guanine and cytosine (GC) content. As of February 2009, all completely-sequenced plastid genomes have a GC content below 43% except for the ptDNA of the lycophyte Selaginella uncinata, which is 55% GC. The forces driving the S. uncinata ptDNA towards G and C are undetermined, and it is unknown if other Selaginella species have GC-biased plastid genomes. This study presents the complete ptDNA sequence of Selaginella moellendorffii and compares it with the previously reported S. uncinata plastid genome. Partial ptDNA sequences from 103 different Selaginella species are also described as well as a significant proportion of the S. moellendorffii mitochondrial genome. Moreover, S. moellendorffii express sequence tags are data-mined to estimate levels of plastid and mitochondrial RNA editing. Overall, these data are used to show that: (1) there is a genus-wide GC bias in Selaginella ptDNA, which is most pronounced in South American articulate species; (2) within the Lycopsida class (and among plants in general), GC-biased ptDNA is restricted to the Selaginella genus; (3) the cause of this GC bias is arguably a combination of reduced AT-mutation pressure relative to other plastid genomes and a large number of C-to-U RNA editing sites; and (4) the mitochondrial DNA (mtDNA) of S. moellendorffii is also GC biased (even more so than the ptDNA) and is arguably the most GC-rich organelle genome observed to date—the high GC content of the mtDNA also appears to be influenced by RNA editing. Ultimately, these findings provide convincing support for the earlier proposed theory that the GC content of land-plant organelle DNA is positively correlated and directly connected to levels of organelle RNA editing.     

Nuclear and mitochondrial revertants of a yeast mitochondrial tRNA mutant

Summary We isolated revertants capable of respiration from the respiratory deficient yeast mutant, FF1210-6C/ 170, which displays greatly decreased mitochondrial protein synthesis due to a single base substitution at the penultimate base of the tRNA^Asp gene on mitochondrial (mt) DNA. Three classical types of revertant were identified: (1) same-site revertants; (2) intragenic revertants which restore the base pairing in the acceptor stem of the mitochondrial tRNA^Asp; and (3) extragenic suppressors located in nuclear DNA. In addition a fourth type of revertant was identified in which the mutant tRNA^Asp is amplified due to the maintenance of both the original mutant mtDNA and a modified form of the mutant mtDNA in which only a small region around the tRNA^Asp gene is retained and amplified. The latter form resembles the mtDNA in vegetative petite (rho ^-) strains which normally segregates rapidly from the wild-type mtDNA. Each revertant type was characterized genetically and by both DNA sequence analysis of the mitochondrial tRNA^Asp gene and analysis of the quantity and size of RNA containing the tRNA^Asp sequence. These results indicate that the mitochondrial tRNA^Asp of the mutant retains a low level of activity and that the presence of the terminal base pair in tRNA^Asp is a determinant of both tRNA^Asp function and the maintenance of wild-type levels of tRNA^Asp.     

RAPD Analysis in Crocus sativus L. Accessions and Related Crocus Species

In the present paper a Random Amplified Polymorphic DNA (RAPD) investigation was carried out on DNAs from five Crocus sativus L. (saffron) accessions cultivated in different countries and on six closely related Crocus species. Aims of the study are to check whether cultivated saffron has maintained a constant genomic organisation and to clarify its relationships with possible ancestor species. For the fifteen primers, which produced positive results, DNAs of saffron corms from different accessions present the same amplification pattern, in accordance with the similar DNA content and base composition pointed out in previous studies. The amplification of the seven Crocus species DNAs with twenty-one primers provided 217 repeatable and interpretable fragments, which were scored for presence/absence and employed for a cluster analysis. Results indicated that C. sativus is very closely related to C. cartwrightianus and also similar to C. thomasii. This result, concurring with part of the previous evidence, would rule out the hypothesis of close relationships between C. sativus and C. pallasii.     

A high frequency of intergenomic mitochondrial recombination and an overall biased segregation of B. campestris or recombined B. campestris mitochondria were found in somatic hybrids made within Brassicaceae

Mitochondrial segregation and rearrangements were studied in regenerated somatic hybrids from seven different species combinations produced using reproducible and uniform methods. The interspecific hybridizations were made between closely or more distantly related species within the Brassicaceae and were exemplified by three intrageneric, two intergeneric and two intertribal species combinations. The intrageneric combinations were represented by Brassica campestris (+) B. oleracea, B. napus (+) B. nigra and B. napus (+) B. juncea (tournefortii) hybrids, the intergeneric combinations by B. napus (+) Raphanus sativus and B. napus (+) Eruca sativa hybrids, and the intertribal combinations by B. napus (+) Thlaspi perfoliatum and B. napus (+) Arabidopsis thaliana hybrids. In each species combination, one of the two mitochondrial genotypes was B. campestris since the B. napus cultivar used in the fusions contained this cytoplasm. Mitochondrial DNA (mtDNA) analyses were performed using DNA hybridization with nine different mitochondrial genes as probes. Among the various species combinations, 43–95% of the hybrids demonstrated mtDNA rearrangements. All examined B. campestris mtDNA regions could undergo intergenomic recombination since hybrid-specific fragments were found for all of the mtDNA probes analysed. Furthermore, hybrids with identical hybrid-specific fragments were found for all probes except cox II and rrn18/rrn5, supporting the suggestion that intergenomic recombination can involve specific sequences. A strong bias of hybrids having new atp A-or atp9-associated fragments observed in the intra- and intergeneric combinations could imply that these regions contain sequences that have a high reiteration number, which gives them a higher probability of recombining. A biased segregation of B. campestris-or B. campestris-like mitochondria was found in all combinations. A different degree of phylogenetic relatedness between the fusion partners did not have a significant influence on mitochondrial segregation in the hybrids in this study.     

Optical isolation of individual mitochondria ofPhysarum polycephalum for PCR analysis

Summary We attempted to amplify a specific region of mitochondrial DNA (mtDNA) using the polymerase chain reaction (PCR) from fewer than ten mitochondria isolated individually by microdissection or use of an optical tweezer. We selected preliminarily isolated mitochondria fromPhysarum polycephalum as the model materials and tried to amplify the mtDNA region corresponding to the specific mitochondrial plasmid of this true slime mould. For separation of a few mitochondria from the mitochondrial population, we initially used a destruction method in which excluded mitochondria were disrupted by a UV laser. However, mtDNA was still amplified, although weakly, from mitochondria that had been destroyed by the UV laser. Therefore, we used an optical tweezer to trap individual mitochondria and separate them from the others. The required number of mitochondria were separated from the mitochondrial suspension through a narrow canal of isolation buffer and used directly for PCR amplification. The results showed that the mtDNA could be amplified from at least 9 mitochondria trapped by the optical tweezer.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EDTA ethylenediaminetetraacetic acid - mtDNA mitochondrial DNA - PCR polymerase chain reaction     

Defects in mitochondrial DNA replication and human disease

Mitochondrial DNA (mtDNA) is replicated by the DNA polymerase g in concert with accessory proteins such as the mtDNA helicase, single stranded DNA binding protein, topoisomerase, and initiating factors. Nucleotide precursors for mtDNA replication arise from the mitochondrial salvage pathway originating from transport of nucleosides, or alternatively from cytoplasmic reduction of ribonucleotides. Defects in mtDNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mtDNA deletions, point mutations, or depletion which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mtDNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders, such as progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). This review focuses on our current knowledge of genetic defects of mtDNA replication (POLG, POLG2, C10orf2) and nucleotide metabolism (TYMP, TK2, DGOUK, and RRM2B) that cause instability of mtDNA and mitochondrial disease.     

Rapid chloroplast segregation and recombination of mitochondrial DNA in Brassica cybrids

Summary Brassica cybrids were obtained after fusing protoplasts of fertile and cytoplasmic male sterile (CMS) B. napus lines carrying the original b. napus, and the Ogura Raphanus sativus cytoplasms, respectively. Iodoacetate treatment of the fertile line and X-irradiation of the CMS line prevented colony formation from the parental protoplasts. Colony formation, however, was obtained after protoplast fusion. Hybrid cytoplasm formation was studied in 0.5 g to 5.0 g calli grown from a fused protoplast after an estimated 19 to 22 cell divisions. Chloroplasts and mitochondria were identified in the calli by hybridizing appropriate DNA probes to total cellular DNA. Out of the 42 clones studied 37 were confirmed as cybrids. Chloroplast segregation was complete at the time of the study. Chloroplasts in all of the cybrid clones were found to derive from the fertile parent. Mitochondrial DNA (mtDNA) segregation was complete in some but not all of the clones. In the cybrids, mtDNA was different from the parental plants. Physical mapping revealed recombination in a region which is not normally involved in the formation of subgenomic mtDNA circles. The role of treatments used to facilitate the recovery of cybrids, and of organelle compatibility in hybrid cytoplasm formation is discussed.