Centromeres. A journey to the center of the chromosome

The Arabidopsis genome project is the first to give a detailed picture of the centromeres in a higher eukaryote. The centromere, a crucial stretch of DNA buried in the knotty terrain at the center of the chromosome, has generally been ignored by other genome sequencing projects because it is highly repetitive, making it very difficult to sequence accurately. But the discovery of an Arabidopsis mutant whose pollen forms quartets has enabled the plant's centromeric regions to be defined with unprecedented accuracy.     

植物着丝粒的研究进展

着丝粒是许多高等真核生物染色体的重要结构域之一,它的最内层是由串联重复的卫星DNA及其侧翼富集的中度重复元件组成。在整个真核生物类群中,不同物种间着丝粒的DNA序列千差万别,但其功能却相当保守,可确保在有丝分裂和减数分裂过程中染色体的正确分离和传递。近年来,植物着丝粒的结构、功能和进化方面的研究进展较快,故对此进行了综述。     

The centromere paradox: stable inheritance with rapidly evolving DNA

Every eukaryotic chromosome has a centromere, the locus responsible for poleward movement at mitosis and meiosis. Although conventional loci are specified by their DNA sequences, current evidence favors a chromatin-based inheritance mechanism for centromeres. The chromosome segregation machinery is highly conserved across all eukaryotes, but the DNA and protein components specific to centromeric chromatin are evolving rapidly. Incompatibilities between rapidly evolving centromeric components may be responsible for both the organization of centromeric regions and the reproductive isolation of emerging species.     

Elucidation of the small RNA component of the transcriptome

Small RNAs play important regulatory roles in most eukaryotes, but only a small proportion of these molecules have been identified. We sequenced more than two million small RNAs from seedlings and the inflorescence of the model plant Arabidopsis thaliana. Known and new microRNAs (miRNAs) were among the most abundant of the nonredundant set of more than 75,000 sequences, whereas more than half represented lower abundance small interfering RNAs (siRNAs) that match repetitive sequences, intergenic regions, and genes. Individual or clusters of highly regulated small RNAs were readily observed. Targets of antisense RNA or miRNA did not appear to be preferentially associated with siRNAs. Many genomic regions previously considered featureless were found to be sites of numerous small RNAs.     

Chromosome localization of highly repetitive human DNA's and amplified ribosomal DNA with restriction enzymes

Restriction endonucleases cut and partially removed DNA throughout fixed air-dried human metaphase chromosomes. Some enzymes produced a G-banding pattern; some revealed the presence of multiple chromosome-specific classes of highly repetitive DNA in C-band heterochromatin. Enzymes that produced the informative C-band patterns had recognition sequences that were four or five, but not six, base pairs long and did not contain a cytosine-guanine doublet. In both rat and human chromosomes, regions containing amplified ribosomal RNA genes were specifically removed by the restriction endonuclease Msp I.     

中国弓形虫虫株529bp重复序列的PCR扩增、克隆及分析

 【目的】首次对中国来源于不同宿主、不同地域的9个弓形虫虫株(ZS1人株、PY猪株、GY猪株、ZC猪株、NT猪株、ZS人株、SH人株、CN猪株、QHO绵羊株)以及国际标准强毒RH株之间在529 bp重复序列的变异进行研究，从而为进一步的分子诊断和分子遗传学研究以及弓形虫病的防制奠定基础。【方法】抽提基因组DNA后，用PCR方法对10个虫株的529 bp重复序列进行扩增；扩增产物经纯化后克隆于pGEM-T Easy质粒载体，再经菌落PCR及酶切鉴定阳性克隆，然后对阳性克隆进行测序及序列分析。【结果】10个弓形虫虫株的529 bp重复序列都不完全相同，它们之间的核苷酸序列变异范围为0.8%～2.9%。变异主要位于第32～55位碱基之间,这些碱基变异与虫株的宿主来源、地域来源及毒力之间没有相关性。【结论】529 bp重复序列可作为遗传标记，用于弓形虫与其它寄生虫的种间鉴定，但不适合用于研究弓形虫的种内遗传变异。     

Arabidopsis thaliana and Plant Molecular Genetics

Arabidopsis thaliana is a small flowering plant with various properties that make it an excellent organism for experiments in molecular genetics. These properties include having a small nuclear genome, a near absence of dispersed repetitive DNA, and a generation time of 4 to 5 weeks. In addition, mutations that affect hormone synthesis and response, many different enzyme activities, and numerous developmental processes have been identified and characterized.     

A histone acetyltransferase regulates active DNA demethylation in Arabidopsis

Active DNA demethylation is an important part of epigenetic regulation in plants and animals. How active DNA demethylation is regulated and its relationship with histone modification patterns are unclear. Here, we report the discovery of IDM1, a regulator of DNA demethylation in Arabidopsis. IDM1 is required for preventing DNA hypermethylation of highly homologous multicopy genes and other repetitive sequences that are normally targeted for active DNA demethylation by Repressor of Silencing 1 and related 5-methylcytosine DNA glycosylases. IDM1 binds methylated DNA at chromatin sites lacking histone H3K4 di- or trimethylation and acetylates H3 to create a chromatin environment permissible for 5-methylcytosine DNA glycosylases to function. Our study reveals how some genes are indicated by multiple epigenetic marks for active DNA demethylation and protection from silencing.     

一种适宜拟南芥PCR检测的DNA提取方法

[目的]介绍一种适于拟南芥PCR检测的DNA快速提取方法。[方法]通过对常规DNA提取方法的改进,获得可以快速大批量地提取拟南芥DNA样品的方法,并以随机抽取的拟南芥转基因株系和突变株系为样品进行验证。[结果]经过琼脂糖凝胶电泳及紫外吸收检测,DNA样品完整且污染少,PCR扩增目的片段结果良好,适于作为PCR反应的模板。经过对随机抽取的拟南芥转基因株系和突变株系的PCR检测,阳性植株目的基因扩增条带清晰,无假阳性,试验结果理想。[结论]该方法适用于拟南芥DNA样品的快速提取、PCR检测及拟南芥突变体筛选工作。     

一种适宜拟南芥PCR检测的DNA提取方法（摘要）（英文）

[目的]介绍一种适于拟南芥PCR检测的DNA快速提取方法。[方法]通过对常规DNA提取方法的改进（省去液氮研磨和苯酚提取的步骤）,获得可以快速大批量地提取拟南芥DNA样品的方法。于1.5ml Eppendorf管内加入400μl DNA提取液[内含200 mmol/LTri（pH值7.5）,25 mmol/LEDTA（pH值8.0）,250 mmol/LNaCl,0.5% SDS（W/V）],剪取拟南芥叶片材料少许（1片以下）加入400μl DNA提取液,用微型研磨棒将叶片捣碎至溶液成绿色,放置3 ～5 min;加入400μl氯仿/异戊醇（体积比24：1）,混合均匀,12 000 r/min离心5 min;取上清液至另一1.5 ml Eppendorf管内,加入300μl异丙醇,颠倒混合均匀,室温下放置5 min,12 000 r/min离心5 min;弃去上清液,以70%乙醇漂洗,放置晾干,加入100μl灭菌超纯水溶解,4℃放置备用。以随机抽取的拟南芥转基因株系和突变株系为样品进行验证。[结果]经过琼脂糖凝胶电泳及紫外吸收检测,DNA样品完整且污染少,PCR扩增目的片段结果良好,适于作为PCR反应的模板。经过对随机抽取的拟南芥转基因株系和突变株系的PCR检测,阳性植株目的基因扩增条带清晰,无假阳性,试验结果理想。[结论]该方法适用于拟南芥DNA样品的快速提取、PCR检测及拟南芥突变体筛选工作。     

反义抑制MnSOD转基因拟南芥构建及其抑制效果的分析

为阐明植物的线粒体MnSOD在逆境适应性反应中的作用,本研究利用分子生物学技术,以拟南芥Mn-SOD基因上游序列构建其反义序列表达载体,转化拟南芥获得转基因植株。经Northern b lot鉴定发现转基因拟南芥MnSOD mRNA水平降低,NBT法检测转基因拟南芥MnSOD活性下降,表明该反义DNA序列对MnSOD基因表达具有明显的抑制效果。     

基于基因组原位杂交快速构建黄瓜变种间核型

【目的】利用基因组荧光原位杂交（genomic in situ hybridization,GISH）技术,对黄瓜（Cucumis sativus L.,2n=2x=14）种内两个变种（栽培黄瓜C. sativus var. sativus和野生黄瓜C. sativus var hardwickii）进行中期染色体分析,建立黄瓜变种染色体核型的快速分析方法,为黄瓜细胞分子遗传学研究提供基础。【方法】以栽培黄瓜‘9930’和野生黄瓜C. sativus var. hardwickii为材料,利用CTAB法提取栽培黄瓜‘9930’的基因组总DNA,采用缺刻平移法,将栽培黄瓜‘9930’基因组DNA和45S rDNA分别利用地高辛和生物素标记为探针,与栽培黄瓜‘9930’和野生变种C.sativus var. hardwickii的中期染色体进行荧光原位杂交,根据杂交结果显示的栽培黄瓜与野生变种每条染色体GISH荧光带型的不同,结合45S rDNA位点信号特征,区分栽培黄瓜与野生变种的每条染色体,并进行核型分析。【结果】荧光原位杂交结果显示,GISH信号并非平均分布于所有染色体上,而是在不同染色体的特定部位产生独特的信号,且两个变种间中期染色体的GISH信号模式差异显著。在栽培黄瓜‘9930’有丝分裂中期染色体上,除了6号染色体仅在短臂末端和近着丝粒处产生GISH信号外,其他染色体上的GISH信号集中分布于染色体的两端和近着丝粒的一侧或两侧,且每条染色体的信号特征差异明显;45S rDNA信号主要分布于‘9930’的第1、2、3、4和7号染色体的近着丝粒处,有3对强信号和2对弱信号。在野生黄瓜C. sativus var. hardwickii有丝分裂中期染色体上,杂交信号的位置及强弱与栽培黄瓜‘9930’表现明显不同,近着丝粒处均有GISH信号,但仅在第1、2、4和5号染色体的一端产生GISH信号,45S rDNA信号仅出现在第1、2和3号染色体上,表现为第1号染色体上信号极强,第2和3号染色体上信号极微弱。这些结果显示,以栽培黄瓜基因组DNA为探针的荧光原位杂交能反应出两个变种中期染色体独特的信号分布模式,通过信号的分布模式和强弱,结合45S rDNA位点信号的特异分布,可对每条染色体进行清晰地鉴别,并据此建立了两个变种的核型模式。比较前人发表的黄瓜已有重复序列的分布图,发现GISH揭示的信号分布主要位于黄瓜染色体串联重复序列区域。【结论】黄瓜基因组原位杂交能一次性快速显示基因组串联重复序列的分布图,能有效地用于不同黄瓜变种的快速核型分析;同时发现染色体上串联重复序列的分布及强弱在黄瓜变种间表现出明显的分化。     

一种简易的拟南芥幼苗微量DNA提取方法

报道了一种简易的拟南芥幼苗微量DNA提取方法。陔方法可在常温下进行DNA提取，而且样品用量少，仅需10mg左右，用100μL重蒸水回溶DNA沉淀所获得的粗提液可直接用于PCR检测。结果表明：应用该法提取的DNA完整性好，PCR效果佳，适用于拟南芥转基因植株鉴定。     

Genomic and genetic definition of a functional human centromere

The definition of centromeres of human chromosomes requires a complete genomic understanding of these regions. Toward this end, we report integration of physical mapping, genetic, and functional approaches, together with sequencing of selected regions, to define the centromere of the human X chromosome and to explore the evolution of sequences responsible for chromosome segregation. The transitional region between expressed sequences on the short arm of the X and the chromosome-specific alpha satellite array DXZ1 spans about 450 kilobases and is satellite-rich. At the junction between this satellite region and canonical DXZ1 repeats, diverged repeat units provide direct evidence of unequal crossover as the homogenizing force of these arrays. Results from deletion analysis of mitotically stable chromosome rearrangements and from a human artificial chromosome assay demonstrate that DXZ1 DNA is sufficient for centromere function. Evolutionary studies indicate that, while alpha satellite DNA present throughout the pericentromeric region of the X chromosome appears to be a descendant of an ancestral primate centromere, the current functional centromere based on DXZ1 sequences is the product of the much more recent concerted evolution of this satellite DNA.     

柔嫩艾美耳球虫端粒DNA重复序列的South-ern印迹杂交分析

为进一步确定柔嫩艾美耳球虫(E.tenella)端粒DNA的重复序列,为下一步端粒酶活性检测奠定基础,根据已克隆发表的E.tenella端粒DNA重复序列信息,设计了由4个端粒重复序列串联的寡聚核苷酸探针(TTTAGGG)4,并以生物素地高辛标记。将该探针与经BAL31-EcoRⅠ酶切后的E.tenella基因组DNA进行Southern印迹杂交分析。结果显示:E.tenella基因组DNA与探针杂交获得了清晰的杂交条带,随BAL31酶切时间的延长,杂交信号逐渐减弱,进一步证明了E.tenella端粒DNA重复序列为5-′TTTAGGG-3′,且此重复序列在E.tenella染色体的末端。     

UV-C辐射对拟南芥基因组完整性及蛋白表达的影响

[目的]探究UV-C辐射对基因组完整性及蛋白表达的影响。[方法]CTAB法提取拟南芥基因组DNA,琼脂糖凝胶电泳检测基因组DNA的完整性。用TCA/丙酮法提取叶片总蛋白,通过SDS-PAGE凝胶检测蛋白表达变化。[结果]拟南芥基因组DNA随UV-C辐射时间的延长断裂明显增强、并诱导产生1条分子量约为66 kDa的蛋白条带。[结论]UV-C辐射可以引起拟南芥基因组DNA的断裂,对蛋白也有一定程度的影响。     

拟南芥bZIP23基因过量表达载体的构建及过表达植株的筛选

[目的]以拟南芥为材料克隆bZIP23基因,构建bZIP23基因的过量表达载体和筛选过表达植株,为验证其功能奠定基础.[方法]提取拟南芥总RNA和RT-PCR克隆bZIP23基因,用限制性内切酶切割和T4 DNA连接酶连接,使bZIP23基因连接到35S强启动子的pART27载体上;将连接产物转化到Trans1-T1感受态细胞中,筛选阳性单克隆进行菌落PCR鉴定并测序验证,获得重组质粒.将该重组质粒电激转化至根瘤农杆菌GV3101菌株,浸花法转化拟南芥野生型植株.[结果]通过单菌落PCR鉴定和DNA测序结果显示,bZIP23基因与35S过量表达载体已连接,获得了重组载体;抗性筛选与遗传鉴定获得相应的转基因过量表达阳性植株.[结论]构建的过量表达载体及筛选得到的过量表达植株为验证bZIP23基因功能奠定了基础.