非特异性DNA的催化活性（1）——应用酚试剂及FeCl3检测DNA的酯酶活性

分别应用酚试剂与ＦｅＣｌ３来检测萘酚，表明ＤＮＡ能催化乙醛萘酯的水解，印证了以前关于ＤＮＡ具有酯酶活性的结论。ＦｅＣｌ３法可以作为检测ＤＮＡ酯酶活性的一种定量方法。     

特异性DNA倍增技术(PCR)及其应用

特异性DNA倍增技术(PCR)是近年来发展的一种新技术,具有快速、简便、灵敏、特异性高和重复性好等优点,尤其适合于临床分子生物学检测。PCR技术包括三个循环过程:(1)模板DNA的变性,(2)模板DNA-引物的复性,(3)DNA聚合酶作用下的引物链的延伸。本文对耐高温Taq DNA聚合酶、PCR的反应体系、PCR产物特异性的影响因素和PCR技术的应用等几个方面进行了综述。     

Folin-Ciocalteu分光光度法测定夏枯草总酚含量的研究(英文)

通过采用Folin-Ciocalteu试剂,以没食子酸为对照品,用紫外可见分光光度法,研究测定夏枯草中总酚含量。结果表明,在装有样品的10 mL容量瓶中依次加入Folin-Ciocalteu试剂0.5 mL,20%Na2CO31.7 mL,室温放置60 min后,在波长660 nm或760 nm测定吸光度。多酚质量浓度在0～10.3μg/mL范围内与吸光度有良好的线性关系,回归方程在660 nm为Y=94.542X-0.0067,R2=0.9999,加样回收率为101.7～105.0%;760 nm为Y=101.13X-0.0191,R2=0.9990,加样回收率为105.1～107.6%。本研究在稳定性、准确性和重复性方面都具有较好的实验结果,可为夏枯草多酚的定量分析提供方法参考。     

~(125)Ⅰ标记乙型肝炎病毒DNA探针的制备及应用

本文报道用国产~125I-碘化钠标记乙型肝炎病毒DNA制备探针,可得到较高比度的产物,一般稳定在10~7—10(?)cpm/μg DNA。用该探针对不标记的乙型肝炎病毒DNA进行点分子杂交。可检测到5pg左右DNA。对17例血液标本进行点分子杂交,结果与32p-标记乙型肝炎病毒DNA探针杂交结果基本一致。     

应用树状DNA杂交(DDH)对生殖道尖锐湿疣中HPV DNA的分型检测

从手术切除的50例生殖道尖锐湿疣新鲜标本中,以及15例正常人血清中,提取基因组DNA,同时用树状DNA杂交(dendrimer DNA hybridizalion,DDH)技术和PCR进行HPV DNA的分型检测.结果50例尖锐湿疣中,以DDH方法检测,感染HPV6型者20例,感染11型者24例,6/11型混合感染者3例,阴性3例,总检测率达94%;以PCR方法检测,HPV6型感染者21例,11型感染者24例,6/11型混合感染者3例,阴性2例,总检测率为96%.15例正常人血清中,以DDH方法检测,HPV感染的假阳性率为0%;以PCR检测,假阳性率为6.67%.还以HPV阳性标本对DDH方法做了敏感度的测定,结果阳性病例DNA检测最低浓度为97.28pg/ml.研究表明,DDH技术具有较高敏感性和高特异性,且成本较低,操作安全简便,可适用于基层中小医院较大样本量筛查.     

遗传作图的第三代工具──DNA芯片(DNA chip)

遗传病相关基因的定位工作是目前世界各国医学遗传学研究的焦点。90年代以来,随着人类基因组计划的发展;各种方法被相继创立和应用到这一领域中。DNA芯片充分结合并灵活运用了大规模集成电路制造技术、计算机、半导体、激光共聚焦扫描、寡核苷酸DNA合成、荧光标记探针杂交及分子生物学的其它技术,在这一领域的研究中有着巨大的潜力。相信在不久的将来,这一技术必将在基因定位研究中得到广泛的应用。     

新型含硅氨基甲酸酯衍生物的合成、杀虫活性及抗乙酰胆碱酯酶活性研究

合成了14个(1-甲硫基亚乙基)氨基甲基氨基甲酸酯(灭多威)1的新型含硅衍生物3。 测定了其杀虫活性和抗乙酰胆碱酯酶活性。结桌表明该类化合物具有很好的杀虫活性,在50μg/mL浓度下,对粘虫Mythimna separata Walker几乎全部具有100%杀灭效果。以美洲大蠊Periplaneta americana为试材,大部分化合物的抗乙酰胆碱酯酶活性与母体灭多威1相当     

DNA微阵列(或芯片)技术原理及应用

DNA微阵列或芯片(DNA microarray or chip)技术是近年发展起来的又一新的分子生物学研究工具.它是利用光导化学合成、照相平板印刷以及固相表面化学合成等技术,在固相表面合成成千上万个寡核苷酸探针,或将液相合成的探针由微阵列器或机器人点样于尼龙膜或硅片上,再与放射性同位素或荧光物标记的DNA或cDNA杂交,用于分析DNA突变及多态性、DNA测序、监测同一组织细胞在不同状态下或同一状态下多种组织细胞基因表达水平的差异、发现新的致病基因或疾病相关基因等多个研究领域.     

土壤Cd胁迫对三七生长和根系DNA损伤及抗氧化酶活性的影响

采用盆栽实验法,研究了土壤中添加0.0(对照)、0.1、0.3、0.6、1.0、3.0、6.0、10.0和30.0 mg·kg-1Cd对三七〔Panax notoginseng(Burk.)F.H.Chen〕生长和抗氧化酶活性的影响,并采用彗星实验对Cd胁迫条件下三七根尖细胞的DNA损伤进行了分析。结果显示:随土壤Cd添加量增加,三七的成活率、株高、单株复叶数和单株叶面积以及单株根、茎和叶片的干质量及鲜质量总体上呈先升高后降低的趋势;其中,各处理组三七植株的成活率和单株复叶数均高于对照,而株高和单株叶面积则在Cd添加量较低的条件下高于对照、在Cd添加量较高的条件下低于对照;单株根、茎和叶的鲜质量及干质量在Cd添加量30 mg·kg-1条件下均小于对照但差异不显著。随Cd添加量的提高,三七根系中SOD和CAT活性呈先上升后下降、再上升再下降的变化趋势,而POD活性总体上逐渐升高;其中,Cd添加量0.6、1.0、3.0和30.0 mg·kg-1处理组的SOD活性极显著或显著低于对照,而各处理组的POD和CAT活性总体上均高于对照且在Cd添加量1.0~30.0 mg·kg-1条件下与对照有极显著差异。彗星实验结果表明:各处理组三七根尖细胞的彗尾长、尾部DNA相对含量和Olive尾矩均有差异,其中,在Cd添加量较高的条件下各指标均高于对照但差异均不显著。研究结果显示:在较低水平的土壤Cd胁迫条件下,三七的生长、抗氧化酶活性和根系DNA均没有受到明显伤害,而较高水平的Cd胁迫则对其生长和抗氧化酶活性有抑制作用,且根尖细胞DNA损伤也较严重。     

DNA序列全对称群和多义密码子的对称性(Ⅰ)

为了完善DNA序列对称理论,本文将12阶DNA群(D群)推广为24阶DNA全对称群(Dd群).DNA全对称群被定义为特殊的交换群(S4),其交换元素是DNA序列的4个碱基.DNA群与四面体群(T群)同构,DNA全对称群与正四面体全对称群(Td群)同构,D群是Dd群的一个子群.本文还推导出了Dd群12个新元素的矩阵表,Dd群的乘法表,得到了在Dd群操作下四碱基A,C,G和T的变换表等.     

NMR Determination of the Absolute Configuration of a Macrophomate Synthase Inhibitor by Using an Axial Chiral Reagent

Macrophomate synthase catalyzes an extraordinary four-step transformation from oxalacetate and 2-pyrone to macrophomic acid by an intermolecular DielsAlder reaction. The absolute configuration of the most potent macrophomate synthase inhibitor; (?)-2-carboxylmethyl-1-methoxybicyclo[2.2.2]oct-5-ene-2-carboxylic acid, was determined to be (1S,2R,4R) by using an axial chiral reagent.     

外源酚对凤眼莲体内酚含量和与酚代谢有关酶的影响

凤眼莲能够吸收和在体内聚集外源苯酚,体内的酸含量随着环境中酚浓度的上升而上升。从生长于合酚培养液中的凤眼莲体内能够检测到酚糖苷,说明凤眼莲体内有酚精苷转移酶的存在。浓度小于５０ｍｇ／Ｌ的外源酚能提高凤眼莲体内的多酚氧化酶和过氧化物酶的活性。多酚氯化酶与过氧化物酶在线粒体和微粒体中均有不同程度的分布,而酚糖苷转移酶则不存在于这些细胞器中。     

Cooperative DNA Binding and Protein/DNA Fiber Formation Increases the Activity of the Dnmt3a DNA Methyltransferase

The Dnmt3a DNA methyltransferase has been shown to bind cooperatively to DNA and to form large multimeric protein/DNA fibers. However, it has also been reported to methylate DNA in a processive manner, a property that is incompatible with protein/DNA fiber formation. We show here that the DNA methylation rate of Dnmt3a increases more than linearly with increasing enzyme concentration on a long DNA substrate, but not on a short 30-mer oligonucleotide substrate. We also show that addition of a catalytically inactive Dnmt3a mutant, which carries an amino acid exchange in the catalytic center, increases the DNA methylation rate by wild type Dnmt3a on the long substrate but not on the short one. In agreement with this finding, preincubation experiments indicate that stable protein/DNA fibers are formed on the long, but not on the short substrate. In addition, methylation experiments with substrates containing one or two CpG sites did not provide evidence for a processive mechanism over a wide range of enzyme concentrations. These data clearly indicate that Dnmt3a binds to DNA in a cooperative reaction and that the formation of stable protein/DNA fibers increases the DNA methylation rate. Fiber formation occurs at low μm concentrations of Dnmt3a, which are in the range of Dnmt3a concentrations in the nucleus of embryonic stem cells. Understanding the mechanism of Dnmt3a is of vital importance because Dnmt3a is a hotspot of somatic cancer mutations one of which has been implicated in changing Dnmt3a processivity.     

紫果猕猴桃嫩枝多酚氧化酶活性与总酚含量变化

采用紫果猕猴桃嫩枝和叶柄为试材,分析全年中植株多酚氧化酶（PPO）活性和总酚含量的变化规律。结果表明,紫果猕猴桃植株PPO活性的最适pH为7.0;最佳底物为邻苯二酚,且浓度为0.16 mol/L;最佳温度为25 ℃。紫果猕猴桃植株生长期的PPO活性明显高于休眠期,植株总酚含量在4月、5月、9 月达最低。     

SEC-HPLC法测定重组人生长激素注射液中苯酚含量

采用SEC-HPLC法测定重组人生长激素注射液中苯酚的含量。色谱柱为TSK G2000SWxL（7.8×300mm,5μ）,流动相为PB（pH值7.0）-异丙醇（97∶3）,流速0.6 mL/min,检测波长214nm,柱温25℃。苯酚在0.10～1.00 mg/mL浓度范围内线性关系良好,r=0.99915;最低检测限为0.5 ng/mL;平均回收率101.48%,RSD=0.58%（n=9）;3批重组人生长激素注射液中苯酚的含量分别为2.98、3.02和2.96 mg/mL,分别为标示量的99.33%、100.67%和98.67%。SEC-HPLC法环保、准确、快速、可靠,可用于重组人生长激素注射液中苯酚含量的测定。     

Cloning efficiency following ES cell nuclear transfer is influenced by the methylation state of the donor nucleus altered by mutation of DNA methyltransferase 3a and 3b

The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodeling of cloned embryos. In this study we investigated the effect of DNA methylation state of donor cells on the development of mouse embryos reconstructed with embryonic stem (ES) cell nuclei. Our results confirmed that deletion of the DNA methyltransferase 3a (Dnmt3a) and DNA methyltransferase 3b (Dnmt3b) distinctly decreases the level of DNA methylation in ES cells. In contrast to wild type ES cells (J1), Dnmt3a − / − 3b − / − (DKO) and Dnmt3b − / − (3bKO) donor cells significantly elevated the percentage of embryonic stem cell nuclear transfer (ECNT) morula, blastocysts and postimplantation embryos (P < 0.05). However, the efficiency of establishment of NT-ES cell lines derived from DKO reconstructed blastocysts was not improved, and the expression pattern of OCT4 and CDX2 in cloned blastocysts and postimplantation embryos was not altered either. Our results suggest that the DNA methylation state of the donor nucleus is an important factor in regulation of the donor nuclear reprogramming.     

螺旋藻多糖对核酸内切酶活性和DNA修复合成的增强作用

本文用核酸内切酶实验和放射自显影术研究了螺旋藻水溶性多糖对DNA切除修复的效应。结果表明,该多糖能显著增强辐射引起DNA损伤的切除修复活性和程序外DNA合成(UDS)。考察切除修复的时程,发现螺旋藻多糖的存在不但能加快损伤DNA切除反应和UDS的初时速度,而且能延缓以上两个重要修复反应的饱和。     

Replication of single-stranded porcine circovirus DNA by DNA polymerases α and δ

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64–66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39–57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase α and δ as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase α and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase α holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789–4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase α is blocked with the DNA polymerase α specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase δ can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.     

Further Study on the Esterase Patterns of Sibling Species in the Drosophila saltans Subgroup (saltans Group): Intraspecific and Interspecific Variations in the Development

Twenty of the 32 esterase bands previously detected in the adults of D. prosaltans, D. saltans and D.␣austrosaltans were found in larvae and pupae studied in this work. The results showed that, in addition to expressing the highest number of esterase bands, the adult stage of the three species exhibited the highest degree of expression (amount of synthesis) for most of the bands. Differences between larval and pupal stages were detected in the degree of expression (amount of synthesis) of the bands and in the frequency of samples expressing them. The frequencies of expression of the bands corresponding to genes in loci 1–3 were greater in pupae than in larvae while the frequencies of expression of the bands corresponding to genes in loci 4–9 were predominantly expressed in larvae or were equal in both developmental stages. Like the adults, larvae, pupae and empty pupal cases (which were also studied in this work) showed specific esterases. Taken together, the observations showed that, in the species studied, every developmental stage is characterized by specific bands and by specific frequency and degree of expression of the bands shared with other stages.