Study of the interaction of DNA and histones by spin-stretching and droplet evaporation

Molecular combing is a powerful and simple method for aligning DNA molecules onto a surface. Using this technique combined with fluorescence microscopy, DNA-histone complexes are stretched on a hydrophobic polymethyl methacrylate (PMMA) surface and observed directly. We have developed a new method to stretch single DNA-histone complexes, termed spin-stretching. The results show that the histones markedly enhance DNA binding to the PMMA surface. DNA winds around the histones and therefore decreases in length. The number of histones that bind to each DNA molecule is found to correlate with the histone concentration. The combed DNA-histone complexes are found to depend on two factors: the binding force on the surface and the centrifugal force at its local position. Na+ ions should compete with histones for binding to DNA; however, the observed competitive binding effect of Na+ ions at low concentrations was negligible.     

DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA

Mammals use DNA methylation for the heritable silencing of retrotransposons and imprinted genes and for the inactivation of the X chromosome in females. The establishment of patterns of DNA methylation during gametogenesis depends in part on DNMT3L, an enzymatically inactive regulatory factor that is related in sequence to the DNA methyltransferases DNMT3A and DNMT3B. The main proteins that interact in vivo with the product of an epitope-tagged allele of the endogenous Dnmt3L gene were identified by mass spectrometry as DNMT3A2, DNMT3B and the four core histones. Peptide interaction assays showed that DNMT3L specifically interacts with the extreme amino terminus of histone H3; this interaction was strongly inhibited by methylation at lysine 4 of histone H3 but was insensitive to modifications at other positions. Crystallographic studies of human DNMT3L showed that the protein has a carboxy-terminal methyltransferase-like domain and an N-terminal cysteine-rich domain. Cocrystallization of DNMT3L with the tail of histone H3 revealed that the tail bound to the cysteine-rich domain of DNMT3L, and substitution of key residues in the binding site eliminated the H3 tail-DNMT3L interaction. These data indicate that DNMT3L recognizes histone H3 tails that are unmethylated at lysine 4 and induces de novo DNA methylation by recruitment or activation of DNMT3A2.     

组氨酸对DNA电荷逆转和凝聚的影响

以组氨酸为研究对象，通过动态光散射观察到当组氨酸浓度为1 mg/mL时，电泳迁移率由负变为正，DNA发生电荷逆转。同时通过原子力显微镜可以观察到，随着组氨酸浓度的升高，DNA分子逐渐堆积折叠，直至在组氨酸浓度为1 mg/mL时凝聚成球，说明了组氨酸可以导致DNA凝聚和电荷逆转。由于组氨酸的等电位点为7.59，相信组氨酸侧链的质子化和去质子化对其带电状态起调节作用。作为生物体内的基本遗传物质，DNA影响着生物的生长发育，与细胞癌变、突变等异常活动也息息相关。在细胞中，组蛋白是DNA染色体的重要组成部分，在DNA的转录和复制等过程中调节DNA的凝聚状态。组氨酸是组蛋白的主要成分，研究组氨酸对DNA凝聚以及相关的电荷逆转过程的影响，对于理解组蛋白的调控作用有重要的基础意义。     

NaCl对果蝇生物学特性的影响

以三种不同果蝇品系为材料 ,观察不同浓度 Na Cl(0 m mol/L,10 mm ol/L,5 0 mm ol/L,10 0 mm ol/L,2 0 0 mmol/L)对果蝇生物学特性的影响。结果表明 :低浓度 Na Cl(10 mm ol/L)能加快果蝇幼虫生长发育 ,子代数量相应增加 ,高浓度 Na Cl(10 0 m mol/L,2 0 0 mm ol/L)延滞果蝇发育 ,子代数量减少 ;并且果蝇子代中雌蝇多于雄蝇。Na-Cl能缩短果蝇寿命 ,但低浓度组雄蝇寿命却延长。实验结果表明 ,一定量的 Na Cl能够影响果蝇生长发育、子代数量和成虫寿命 ,干扰群体的雌雄平衡。     

Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9

Specific modifications to histones are essential epigenetic markers---heritable changes in gene expression that do not affect the DNA sequence. Methylation of lysine 9 in histone H3 is recognized by heterochromatin protein 1 (HP1), which directs the binding of other proteins to control chromatin structure and gene expression. Here we show that HP1 uses an induced-fit mechanism for recognition of this modification, as revealed by the structure of its chromodomain bound to a histone H3 peptide dimethylated at Nzeta of lysine 9. The binding pocket for the N-methyl groups is provided by three aromatic side chains, Tyr21, Trp42 and Phe45, which reside in two regions that become ordered on binding of the peptide. The side chain of Lys9 is almost fully extended and surrounded by residues that are conserved in many other chromodomains. The QTAR peptide sequence preceding Lys9 makes most of the additional interactions with the chromodomain, with HP1 residues Val23, Leu40, Trp42, Leu58 and Cys60 appearing to be a major determinant of specificity by binding the key buried Ala7. These findings predict which other chromodomains will bind methylated proteins and suggest a motif that they recognize.     

Structural determinants for generating centromeric chromatin

Mammalian centromeres are not defined by a consensus DNA sequence. In all eukaryotes a hallmark of functional centromeres--both normal ones and those formed aberrantly at atypical loci--is the accumulation of centromere protein A (CENP-A), a histone variant that replaces H3 in centromeric nucleosomes. Here we show using deuterium exchange/mass spectrometry coupled with hydrodynamic measures that CENP-A and histone H4 form sub-nucleosomal tetramers that are more compact and conformationally more rigid than the corresponding tetramers of histones H3 and H4. Substitution into histone H3 of the domain of CENP-A responsible for compaction is sufficient to direct it to centromeres. Thus, the centromere-targeting domain of CENP-A confers a unique structural rigidity to the nucleosomes into which it assembles, and is likely to have a role in maintaining centromere identity.     

超富集植物垂序商陆的锰吸收动态研究

以锰超富集植物垂序商陆为实验材料,通过温室实验模拟锰污染环境,研究垂序商陆生长在不同锰浓度生长介质的锰吸收动态.研究结果表明:垂序商陆对锰的吸收和积累模式依赖于生长介质中锰的浓度和处理时间.垂序商陆根、茎和叶的锰含量基本随着生长介质中锰浓度的升高而增加;在生长介质不同锰浓度(0.2,0.5和5.0 mmol/L)条件下,垂序商陆根、茎和叶锰含量均随着生长时间呈现波动变化,在植物处理5d时出现一个峰值,其后有所降低;垂序商陆在锰处理开始的48 h内存在一个快速的吸收过程,但是,生长介质高锰浓度(5.0mmol/L)变化幅度要大于低锰浓度(0.2和0.5 mmol/L);超富集植物垂序商陆对锰的吸收和转运受生长介质中锰浓度和植物体内锰含量状况双重控制.     

油茶SRAP-PCR反应体系的建立

以油茶品种大别山2号和赣兴46为试验材料,利用Me5Em8正反向引物组合进行了SRAP-PCR反应体系的L9(34)正交试验,采用正交设计直观分析及方差分析对影响SRAP反应的Taq聚合酶、Mg2+、dNTP和引物浓度进行分析,并对模板DNA浓度进行了单因素试验分析。结果表明,油茶SRAP-PCR 20 μL反应体系的最佳组合为: Taq聚合酶1.20 μmol/min、Mg2+浓度125 mmol/L、dNTP浓度0.15 mmol/L、Primer浓度0.60 μmol/L、模板DNA含量60 ng,并含有2 μL 10×buffer(Mg2+ free)。各因素对油茶SRAP-PCR反应的影响大小依次为:dNTP、 Mg2+ 、Taq聚合酶、Primer。     

ITX/EDAB光引发丙烯酸溶液聚合动力学

以异丙基硫杂蒽酮(ITX)/对-(二甲基氨基)苯甲酸乙酯(EDAB)为复配引发剂,研究了光引发丙烯酸溶液聚合动力学。结果表明,R_P与单体浓度呈一次关系,引发剂反应级数在低浓度(<0.2mmol/L)下为0.2057,高浓度(>0.8mmol/L)下为-0.1;反应速率存在最大值,此时引发剂浓度为0.8mmol/L;聚合反应表观活化能为9.53kJ/mol。     

亚硫酸钠、亚硫酸氢钠对小麦幼苗生长及细胞分裂的影响

研究二氧化硫体内衍生物——亚硫酸钠、亚硫酸氢钠混合液对小麦幼苗生长和细胞分裂的影响.结果表明:低浓度(0.1mol/L)的亚硫酸钠、亚硫酸氢钠的混合液对小麦幼苗生长及细胞分裂有促进作用;高浓度(0.2mol/L)的亚硫酸钠、亚硫酸氢钠的混合液抑制小麦幼苗生长及细胞分裂.     

亮氨酸拉链结构蛋白在大肠杆菌重组子中的表达

酵母转录因子GCN4是通过亮氨酸拉链(bZIP)结构结合DNA的蛋白质之一，当GCN4二聚体与DNA结合时，亮氨酸拉链区的2个单体结合为平行的卷曲螺旋结构，而其基区由无规线团结构变为α螺旋．为探讨亮氨酸拉链蛋白与DNA的结合机理，设计了含有GCN4亮氨酸拉链蛋白基区结合DNA的必需氨基酸的折叠片段，并将其克隆到Escherichia coli BL21，讨论了此亮氨酸拉链蛋白的表达条件．在蛋白质的小量表达试验中，重组子Escherichia coli BL21于5mL含有50μg／mL氨节青霉素和34μg／mL氯霉素的LB液体培养基中培养至对数期，加入不同浓度的IPTG，继续培养以诱导蛋白质的表达，在不同的时间(如：诱导前，诱导2，4，6，8h)取样100μL到1．5mL离心管中、离心收集沉淀，将沉淀悬浮于样品缓冲液中，用10％SDS-PAGE检测；在10L含氨苄青霉素和氯霉素的LB液体培养基中进行了大量表达，根据小量表达的试验结果确定了IPTG的浓度和诱导时间．结果表明：含有这种拉链蛋白质的重组子Escherichia coli BL21在37℃下小量培养时，0．1-0．8mmol的IPTG均可在2-10h内诱导该蛋白质表达；而大量培养时，0．2mmol和0．4mmol的IPTG在37℃均不可能诱导表达，只在28℃时才表达；小量培养和大量培养的最佳诱导时间为4-6h，诱导剂IPTG的浓度为0．2mmol，大量表达的温度为28℃而不是37℃．     

一种新型钌(Ⅱ)多吡啶配合物的制备及其与ctDNA的作用研究

以1,10-邻菲啰啉、对硝基苯肼和三氯化钌等为原料合成一种新型钌(Ⅱ)多吡啶配合物-高氯酸[二(2,2'-联吡啶)(4,5-二氮杂芴-9-对硝基苯腙)]合钌(Ⅱ)[Ru(bipy)2DAFND](ClO4)2,采用元素分析、红外光谱、核磁和质谱等手段对其进行了表征。采用电子吸收光谱法、荧光法和粘度法等手段研究了配合物与ctDNA的相互作用,实验结果表明配合物以插入的方式与DNA键合,键合常数为8.91×105L/mol。     

一种玻璃载片共价键固定DNA的新方法

目的：建立了一种简单、快速、高效共价固定DNA到玻璃其质上的方法。方法：玻片用β-（对、间氯甲基苯基）三氯硅乙烷处理在表面自组装形成带有苄氯功能基的单层硅烷膜，然后再用NaI丙酮溶液处理将苄氯基转化为苄碘基。巯基修饰的DNA通过巯基与苄碘基反应形成硫醚键共价偶联到这一自组装的硅烷膜上。结果：放射性分析证实玻璃基质表面共价固定DNA的密度为3.2nmol/m^2。用放射性标记的目标DNA进行杂交试验表明最大杂交密度的0.77nmol/m^2。结论；本方法只需一步偶联反应，反应时间短、偶联效率高、操作简单、不需要严格的试剂，而且制得的DNA芯片有较高的温度稳定性，可方便地用于核酸杂交和探测研究。     

Mg2+对Bloom综合症解旋酶与G4DNA结合的影响研究

利用荧光偏振技术检测了Mg2+对G4DNA、BLM-G4DNA复合物和BLM642-1290解旋酶与G4DNA结合的影响.结果表明,G4DNA荧光偏振值随着Mg2+浓度的增加而增加(P＜0.01);BLM-G4DNA复合物的荧光偏振值随着Mg2+浓度的增加出现下降—升高—下降的变化趋势(P＜0.01);G4DNA与BLM642-1290解旋酶结合的荧光偏振值随着Mg2+浓度的增加而逐渐下降(P＜0.01);分析不同Mg2+浓度下两种分子结合的Kd值,发现Mg2+浓度为3.0 mmol/L时,BLM642-1290解旋酶与G4DNA最容易结合,表明适量Mg2+浓度会促进BLM642-1290与G4DNA的结合,但会引起两种分子结合的形状、流动性和电荷等性质的改变.这些结果可为进一步研究BLM解旋酶对G4DNA的作用机理提供相关资料.     

匍匐茎草本蛇莓RAPD反应体系优化

为了建立蛇莓RAPD反应的最佳反应体系,我们对每个反应因子进行优化研究。在本实验室常用的RAPD反应体系的基础上,根据RAPD扩增效果确定蛇莓基因组DNA的最佳RAPD反应体系。经优化后,我们建立了适合蛇莓的最佳RAPD反应体系:25μL反应体系中含10×Buffer 2.5μL,2 mmol/L Mg2+,1.5 UTaqDNA聚合酶,50 ng模板,0.22 mmol/L dNTPs,0.35μmol/L随机引物S30。本研究结果为下一步野生蛇莓遗传多样评估奠定基础。