PHYLOGENETIC RELATIONSHIPS AMONG GIANT PANDA AND RELATED SPECIES BASED ON RESTRICTION SITE VARIATIONS IN rDNA SPACERS

In this study, non radioactive Digoxigenin labeled ribosomal DNA(rDNA) probes were used for Southern blotting analysis to study the molecular phylogeny of the giant panda and related species. Restriction maps in the regions of rDNA spacers were compared between giant panda( Ailuropoda melanoleuca ), lesser panda( Ailurus fulgens ), Asiatic black bear( Selenarctos thibetanus ), sun bear( Helarctos malayanus ), raccoon( Procyon lotor ) and lynx( Felis lynx ). Phylogenetic trees for these species were constructed using maximum likelihood and parsimony method. The results show that in respect to rDNA RFLPs, the giant panda is more closely related to bear than to lesser panda; while the lesser panda is slightly related to the raccoon.

Plastid Inheritance in Sweet Potato as Revealed by DNA Restriction Fingerprinting

The inheritance of chloroplast DNA (cpDNA) in sweet potato (Ipomoea batatas Lain.) was analyzed using DNA restriction fingerprinting. The cpDNA fingerprints of hybrids from reciprocal crosses between Xushu18 and AB78-1 were found to be identical to those of their female parents, which reveals that cpDNA of sweet potato is maternally inherited in this intervarietal crossing. This maternal cpDNA transmission pattern does not accord with the putative one based on former cytological studies. The plastid inheritance in Convolvulaceae has been briefly reviewed in this study, and the utility of DNA restriction fingerprinting analysis in the study of plastid inheritance is also discussed.

Comparison of the Two Chromosome Forms of Paragonimus Westermani Using DNA Restriction Fragment Size and DNA Hybridization

Restriction enzyme digestion of total genomic DNA of two chro-mosome type Paragonimus westermani showed the presence of homologus highlyrepeated DNA in diploid and triploid forms. Southern blot analysis providedfurther evidence that the distribution of restriction enzyme sites (with 3enzymes) on repeated DNA of both was similar. But with Pst Ⅰ、Dde Ⅰ、Hae Ⅲand Hpa Ⅱ. They revealed polymorphism which also occured individually ineach form tested separatedly with hybridization technique. Mutation of recog-nize sites by enzymes, which is resulted from the alternation of gene sequance,has occured during evolution. Additionally, it is probably possible, based on PstⅠand Dde Ⅰdigest pattern to distinguish between diploid and triploid Parag-onimus westermani and group the Paragonimus simply and rapidly.

Discovery and Comparison of Restriction Patterns of Porphyra Total DNAs

Total DNA is isolated from Porphyra cells which have been prepared from sea snail enzyme digests of Porphyra thalli. The crude extracts of total DNA are rapidly purified with Wizard DNA Clean-up resin by using modified washing solution. DNA bands are clearly observed in the restriction endonuclease patterns of P. yezoensis and P. haitanensis total DNAs. Both P. yezoensis and P. haitanensis have specific DNA bands in the restriction patterns of EcoRI, PastI and HaeIII. According to the band pattern of total DNA, the discrimination between P. yezoensis and P. haitanensis is very easy especially in the restriction pattern of HaeIII. The comparative results indicate that the band pattern of total DNA is a new type and reliable DNA marker for identification and classification of Porphyra species.

RESTRICTION ENDONUCLEASE ANALYSIS OF MITOCHONDRIALDNA FROM HUMAN LUNG ADENOCARCINOMA CELLLINE SPC-A-1

Objective: To understand the role of mitochondrial DNA (mtDNA) in carcinogenesis. Methods: single-step method was used to isolate the mtDNA from human lung adenocarcinoma cell line SPC-A-1. The mtDNA was analyzed by restriction fragment length polymorphism (RFLP) with 11 kinds of restriction endonuclease, which were Pvu II, Xho I, Pst I, EcoR I, BstE II, Hind III, Hpa I, Bc1 I, EcoR V, Sca I and Xba I. Restriction map of mtDNA from SPC-A-1 cell was obtained by the single and double-digestion method. Results: It was found that no variation at 32 restriction-sites could be detected in the coding region of mtDNA from SPC-A-1 cell line. But a new site was found at nucleotide 16276 (EcoR V) within the noncoding region. Conclusion: These results indicate that the primary structure of gene coding region of mtDNA isolated from SPC-A-1 cell is highly stable. While the major variation of nucleotide is probably located in the noncoding region.

Implications of oxidative damage to proteins and DNA in aging and its intervention by caloric restriction and exercise

In this short review we describe implications of age-related changes of protein and DNA oxidation as a public mechanism of biological aging. Oxidatively modified protein and DNA have been demonstrated to increase with advancing age in rodents. Half-life of proteins is extended and DNA repair activity declines in old animals. Dietary restriction initiated late in life can shorten the half-life of proteins to levels of young animals, thus contributing to reduce level of altered proteins in old animals by the regimen. Regular exercise reduced oxidatively modified proteins in the brain with improved cognitive functions. It attenuated oxidative stress in the liver, i.e., ameliorating activation of nuclear factor κB, increasing reduced glutathione, and decreasing oxidized guanine base in nuclear and mitochondrial DNA. These findings suggest that regular exercise has systemic effects in reducing oxidative stress. Thus, life-styles such as diet and exercise may extend health span, by up-regulating overall anti-oxidant capacities that include proteins involved in protein turnover and DNA repair, resulting in reduction of damaged proteins and DNA that potentially promote physiological and pathological aging.

The Effects of Dietary Restriction and Aging on in Vivo and in Vitro Binding of Aflatoxin B_1 to Cellular DNA

Laboratory animals maintained on a reduced calorie but nutritionally adequate diet have extended life spans and lowered incidences of spontaneous and chemically induced cancers compared to ad libitum- fed counterparts. Many of the effects of dietary restriction on laboratory animals have been suggested to be related to a deceleration of the aging process. The inhibition of age-related changes in xenobiotic metabolizing enzyme activities by dietary restriction has previously been reported. Alterations of these enzyme activities may cause changes in metabolic activation of carcinogens and, therefore, carcinogen-DNA binding. DNA-repair capability has also been reported to be enhanced in diet-restricted rats. Using AFB1 as a model carcinogen, we have studied in vivo and in vitro hepatic AFB1 -DNA binding, demonstrating that dietary restriction (60% of ad libitum consumption) may decrease the metabolic activation of AFB1, and subsequently reduce AFB 1-DNA binding. Our preliminary results obtained from the AFB 1-DNA binding experiments in isolated hepatocytes suggest that the observed age-dependent reduction in AFB 1-DNA binding which may be attributed to a loss of metabolic activating capability was delayed in the diet-restricted rats.

Polymorphism in Mitochondrial DNA of Six Fowl Breeds Revealed by Restriction Endonuclease

The patterns of cleavage of mtDNA by restriction endonucleases was analysed for six fowl breeds and of. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White, Abor Acres and mtDNA polymorphisms were detected in the restriction patterns with the following eight enzymes, Ava Ⅰ, Ava Ⅱ, EcoR Ⅰ, Hind Ⅲ, Bam HI, Pvu Ⅱ, Pst Ⅰ, Hinc Ⅱ. The restriction cleavage patterns were identical among these breeds. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White—A type. The patterns of Abor Acres were B type. Based on their mtDNA restriction types, all the breeds were classified into two groups. Genetic distances among these groups were calculated in order to define their phylogenetic relationships. The relationship among five egg line breeds is close, while Abor Acres (Broiler fowl) is relatively far from them. The results suggest that the difference of mtDNA could result from the different origins. The polymorphic sites in mtDNA of Hyline White has been located on a restriction map.

Intrinsic host restriction factors of human cytomegalovirus replication and mechanisms of viral escape

Before a pathogen even enters a cell, intrinsic immune defenses are active. This first-line defense is mediated by a variety of constitutively expressed cell proteins collectively termed "restriction factors"(RFs), and they form a vital element of the immune response to virus infections. Over time, however, viruses have evolved in a variety ways so that they are able to overcome these RF defenses via mechanisms that are specific for each virus. This review provides a summary of the universal characteristics of RFs, and goes on to focus on the strategies employed by some of the most important RFs in their attempt to control human cytomegalovirus(HCMV) infection. This is followed by a discussion of the counter-restriction mechanisms evolved by viruses to circumvent the host cell's intrinsic immune defenses. RFs include nuclear proteins IFN-γ inducible protein 16(IFI16)(a Pyrin/HIN domain protein), Sp100, promyelocytic leukemia, and h Daxx; the latter three being the keys elements of nuclear domain 10(ND10). IFI16 inhibits the synthesis of virus DNA by downregulating UL54 transcription- a gene encoding a CMV DNA polymerase; in response, the virus antagonizes IFI16 via a process involving viral proteins UL97 and pp65(p UL83), which results in the mislocalizing of IFI16 into the cytoplasm. In contrast, viral regulatory proteins, including pp71 and IE1, seek to modify or disrupt the ND10 proteins and thus block or reverse their inhibitory effects upon virus replication. All in all, detailed knowledge of these HCMV counter-restriction mechanisms will be fundamental for the future development of new strategies for combating HCMV infection and for identifying novel therapeutic agents.

Preparation of Total DNA from "Recalcitrant Plant Taxa

Contamination problems on DNA isolation from 'recalcitrant plant taxa' which is rich in polysaccharides have been commonly encountered in a wide range of research fields such as plant population biology, biodiversity, and molecular marker-assisted breeding. Here we present an improved protocol to extract DNA efficiently from dry or fresh leaves of a 'recalcitrant plant taxa', Betula alnoides Buch. Ham. ex D. Don in which three key steps are involved: 1) washing out most of polysaccharides and other secondary compounds with CTAB-free buffer from homogenate; 2) adoption of 3% CTAB rather than 2% CTAB in the exaction medium; and 3) using of high concentration of salt prior to DNA precipitation with isopropanol to remove residual polysaccharides. The isolated DNA has been proved suitable for RAPD-PCR amplification and restriction digestion. This modified procedure is simple, inexpensive and reliable, and is also applicable to many other plant taxa with high polysaccharides.

草鱼和鲤鱼线粒体 DNA 的分离纯化及其 COI 基因的分子克隆

本文报道配合使用差速离心和DNaseI核酸酶处理等步骤,从草鱼和鲤鱼新鲜肝组织分离线粒体DNA(mtDNA)的实验方法。这种方法经济简便,纯化的mtDNA产率多,纯度高,经限制性内切酶消化后进行琼脂糖凝胶电泳分离,可以得到清晰的DNA片段谱带,并可直接用于构建酶切图谱和线粒体基因的分子克隆。用这样的mtDNA,我们已克隆了草鱼和鲤鱼的细胞色素氧化酶亚基I基因(COI基因)。

猪线粒体DNA多态性与中国地方猪种起源分化的关系

用24种限制性内切酶分析了我国21个具代表性地方猪品种、1个引进品种和2个来自中国和越南的野生近缘种mtDNA的RFLP。结果表明:在74个个体中检出的32种限制性态型可归结成7种单倍型,其间的差异主要来源于少数几个限制性位点的点突变;地方猪种4种单倍型间的平均遗传距离为0.143％,遗传多态程度(π值)仅为0.007％,说明遗传多样性非常贫乏,提示中国地方猪种可能起源于一个野猪亚种。

中国大陆不同地域隔离群湖北钉螺基因组DNA的限制酶切长度差异

为证实中国大陆湖北钉螺不同地理株的存在,制备了各地钉螺的基因组DNA,经限制性内切酶Bam HI和Pst I酶解后,置0.8%琼脂糖凝胶电泳,比较分析不同地域隔离群钉螺基因组DNA重复序列DNA的限制酶切长度差异.安徽、四川、云南、湖南、湖北、江苏、上海、福建、江西等9地隔离群钉螺的DNA酶切带型.主带基本一致;次带差异明显.以四川普格、云南巍山、江苏东台之间差异较大.湖北省的汉川、洪湖、荆州、石首、咸宁的钉螺酶切图谱基本一致.提示不同隔离群钉螺存在一定的同源和亲缘关系.在基因水平上的差异程度与隔离群之间距离和自然环境关系密切.在一定区域内的钉螺DNA相对稳定.

浙江地方猪种线粒体DNA多态及遗传多样性研究

本实验用ＡｐａⅠ、ＡｖａⅠ、ＢａｍＨⅠ、ＢｃｌⅠ、ＢｇｌⅠ、ＢｇｌⅡ、ＣｌａⅠ、ＤｒａⅠ、ＥｃｏＲＩ、ＥｃｏＲＶ、ＨａｅⅡ、ＨｉｎｃⅡ、ＨｉｎｄⅢ、ＨｐａⅠ、ＫｐｎⅠ、ＰｓｔⅠ、ＰｖｕⅠ、ＰｖｕⅡ、ＳａｃⅠ、ＳａｌⅠ、ＳｃａⅠ、ＳｍａⅠ、ＳｔｕⅠ、ＸｂａⅠ和ＸｈｏⅠ等２５种识别６个碱基对的限制性内切酶分析了浙江地方品种猪、浙江野猪等３０个个体的线粒体ＤＮＡ，共检出３０种限制性态型（ｍｏｒｐｈ），可归结成３种单倍型，其中ＢａｍＨⅠ－Ｂ、ＢｃｌⅠ－Ｂ、ＤｒａⅠ－Ｂ和ＸｂａⅠ－Ｃ为首次发现；各单倍型间平均遗传距离（ｐ）为０．００５，群体的平均多态度（π）为０．０００８，均处于遗传多样性贫乏范围，提示浙江地区猪可能起源于一个共同的祖先。结合前人资料，对金华Ⅲ系猪进行分析，分析结果提示金华Ⅲ系猪可能有两个母系起源。

糖尿病肾病大鼠胰基因组DNA甲基化状态的变化

分别从正常大鼠、糖尿病肾病大鼠、中药糖微康治疗的糖尿病肾病大鼠、西药开博通治疗的糖尿病肾病大鼠的胰组织中提取其基因组DNA ,用对DNA甲基化作用敏感的限制性核酸内切酶HpaⅡ和MspⅠ (为一组同裂酶 )进行限制性酶切 ;并进行染色质DNaseⅠ敏感性分析 .实验发现中药糖微康治疗后能向正常方向逆转胰组织DNA中CCGG位点甲基化状态 ,胰组织中处于转录活性状态的染色质明显增加 .

海水养殖沉积环境微生物总DNA的提取方法研究

传统的微生物分离与培养技术：无法揭示微生物群落的动态变化．为了阐释虾池沉积环境微生态格局的原始组成情况，本研究先以TENP缓冲液去除沉积物中的腐殖酸。继之以溶菌酶-SDS温和裂解，其总DNA的提取效率达90％以上．所获DNA产量达2～20μg／g沉积物（湿），片段大小均在23kb左右，不经纯化即可直接进行PCR扩增和限制性酶切．以该DNA为模板进行PCR—DGGE分析，揭示了虾池沉积物丰富的微生物多样性，该方法是一种适用于虾池沉积物总DNA提取的简便、可靠方法．

糖尿病大鼠肾、胰基因组DNA甲基化状态的变化

从正常大鼠、糖尿病大鼠、中药糖微康治疗的糖尿病大鼠、西药开博通治疗的糖尿病大鼠的肾、胰等组织中提取出其基因组DNA ,应用对DNA甲基化作用敏感的限制性核酸内切酶HpaⅡ和MspⅠ (为一组同裂酶 )进行了限制性酶切图谱分析 ,结果表明 ,DNA甲基化作用的主要位点在CpG二核苷酸处 ,且“糖微康”在关闭患糖尿病后肾组织中被异常活化的基因的同时 ,还能活化糖尿病动物胰组织中处于静息状态的基因 .由此可见糖尿病的发病和治疗都与DNA甲基化作用密切相关 .

Inheritance of Chloroplast and Mitochondrial DNA in Chinese Fir (Cunninghamia lanceolata)

The inheritance of mitochondrial (mt) DNA and chloroplast (cp) DNA was investigated in intergeneric hybrids from crossing between Cunninghamia lanceolata (Lamb.) Hook. and Cryptomeria fortunei Hooibrenk. The chloroplast trnL trnF region and one intra genic segment of the mitochondrial gene, Cox Ⅲ, were amplified from those of the parents and hybrids by PCR using gene specific primers. Cp and mtDNA polymorphisms of the amplified regions were detected between the parents after restriction digestions. Restriction fragment length polymorphism (RFLP) analysis revealed that all the F 1 individuals possessed Cox Ⅲ restriction fragment patterns (characteristic of the paternal parent Cryptomeria fortunei ) and the trnL trnF region (identical to the maternal parent Cunninghamia lanceolata ) showing that a different mode of inheritance for organelle DNA has occurred in the hybrids. Furthermore, the maternal inheritance of chloroplast DNA is reported here for the first time in coniferophyta.

土壤微生物分子生态学研究中总DNA的提取

建立了一种土壤DNA提取方法。根据DNA产量和纯度2个评价指标,从3种手提土壤DNA方法中优选出方法B为手提DNA方法(Labmethod),它包括样品预处理,细胞裂解,粗DNA纯化,其中细胞裂解组合了玻璃珠击打,SDS裂解,溶菌酶裂解。进一步应用PCR-限制性片段长度多态性(Restrictionfragmentlengthpolymorphism,RFLP)技术及PCR-温度梯度凝胶电泳(Temperaturegradientgelelectrophoresis,TGGE)技术,结合DNA产量、纯度、片段大小以及所反映的微生物群落结构特性等指标评价了手提方法(Labmethod)得到的总DNA质量,并将这些结果与2种应用较广的商业试剂盒(MoBioUltraCleanSoilDNAKit和Bio101FastDNASPINKit(ForSoil))所得DNA的各种指标进行了比较。结果表明,手提方法(Labmethod)的粗DNA产量低于Bio101Kit的,但高于MoBioKit的。这些方法所得DNA的长度都在21kb左右,Labmethod提取的DNA没有严重被剪切现象,而2种试剂盒提取的DNA都有不同程度的剪切。手提方法得到的DNA经纯化后,应用细菌及真菌特异引物进行PCR扩增,均能获得目的片段,表明该方法能从土壤中同时有效提取细菌和真菌总基因组DNA。并且,手提方法所得DNA的细菌16SrDNA和真菌18SrDNA的PCR-RFLP图谱与两种商业试剂盒的图谱基本相似,但3种方法提取的DNA在细菌16SrDNAV3区和真菌28SrDNA片段的PCR-TGGE图谱上都存在一定差异,主要表现在一些弱势条带的有无或强弱上。这说明手提方法提取的总DNA与2种商业试剂盒一样,在一定程度上能反映微生物群落的多样性和组成。总之,手提DNA方法(Labmethod)所用试剂普通,价格便宜,能在4h以内获得够质够量的土壤DNA用于微生物分子生态学研究,较适合于广大普通实验室进行土壤DNA提取工作。

紫菜基因组DNA提取及酶切

利用两种市售的DNA提取试剂盒对几种紫菜进行基因组DNA提取。该方法需要的组织量少 ,需时短 ,获得的基因组DNA纯度高 ,可被AFLP内切酶 ,特别是EcoRⅠ完全酶切 ,完全满足AFLP研究的需要。不同的DNA提取方法及提取出的基因组DNA的质量对酶切效果及以后的实验结果有影响。