水稻(Oryza sativa L.)重复DNA顺序的克隆及序列分析——重复顺序存在意义的探讨

以大肠杆菌启动子选择质粒pKK175-6、pKK232-8为载体,将通过复性动力学方法获得的水稻重复DNA顺序片段进行克隆。阳性克隆菌株表现出对四环素或氯霉素不同程度的抗性。DNA序列分析表明,克隆到的某些水稻重复DNA顺序具有丰富的基因表达调节元件的同源序列。     

大鼠高重复顺序DNA的核苷酸顺序分析及其与灵长类类α-DNA顺序的比较

 啮齿目动物高重复顺序DNA的结构特征,虽已有人进行过分析,并提出大鼠DNA中有酶切位点分布独特的高重复顺序,也有人认为在Sprague-Dawlay大鼠中有类似于α-DNA大小的高重复顺序,并命名为α型(α-type),是否大鼠中也含有类α顺序,并没有人讨论过。我们对Wistar大鼠高重复顺序DNA进行了分离、纯化、分子克隆及核苷酸序列分析,测定了全序列为370bp,它也是具有酶切点分布独特的高重复顺序,但与前者存在着18％的差异性。我们对此顺序与灵长类类α-DNA进行了比较分析,发现二者在几方面都不存在相似之处,如此序列G-C含量占全部碱基组成的37.3％,而类α顺序的G-C含量约在40％以上,一些酶切位点也与类α-DNA完全不相同,另外在此序列的相应位置上也不具有灵长类类α-DNA的三个“冷点区”等等,由此得出大鼠这一高重复顺序并非类α顺序,而类α顺序只是灵长类动物所特有的。     

懒猴高重复顺序DNA的核苷酸顺序分析及其与类α-DNA顺序的比较

为确知懒猴高重复顺序与灵长类类α顺序间的相互关系,我们将懒猴DNA经EcoRI酶切所得到的高重复顺序DNA的最小片段重组到质粒pUC~(12)上,经转化、筛选、鉴定得到含有懒猴高重复顺序片段的克隆,命名为pLS(EcoR Ⅰ)-1,测定其全部核苷酸顺序为641bp,发现该顺序有与类α顺序的相似性,但变异性较大,我们对此进行了讨论。     

水稻重复DNA顺序克隆pRRD3的研究

用ＤＮＡ复性动力学方法克隆到一个水稻（ＯｒｙｚａｓａｔｉｖａＬ．）中度重复ＤＮＡ顺序。不同限制性内切酶消化和Ｓｏｕｔｈｅｒｎ杂交分析显示,这段重复ＤＮＡ顺序以串联加散布的形式存在于水稻基因组中;序列分析表明在它内部含有一个典型的植物启动子序列ＴＧＴＡＴＡＡＡＴＡ;以ｐＲＲＤ３克隆片段作探针,对水稻３４个品种进行拷贝数测定,在野生稻与栽培稻、籼稻与粳稻之间均存在拷贝数上的明显差异;对ＡＡ基因组不同亚型水稻ＤＮＡ进行Ｓｏｕｔｈｅｒｎ杂交分析,得到基因组亚型特异的杂交带谱,说明该重复顺序是研究水稻进化和分类的一个有用探针。     

臭鼩高重复顺序DNA的核苷酸顺序分析及其与树鼩TSr(Bgl Ⅱ)-1顺序的比较

将臭鼩DAN经过Bam H Ⅰ酶切得到的高重复顺序DNA最小片段重组到质粒pAT153上,转化后得到了含有臭鼩BMS(Bam H Ⅰ)-1高重复顺序DNA片段的克隆。再把此片段重组到M_(13)mp19噬菌体DNA上。用末端终止法测得全部苷酸顺序为495个碱基对。对臭鼬BMS(Bam H Ⅰ)-1片段的结构特点进行了分析,并和树鼩TSr(BglⅡ)-1高重复顺序DNA进行了比较。为确定树鼩在分类学上的地位,提供了一定的分子遗传学证据。     

恒河猴高重复顺序DNA的分子克隆

哺乳动物基因组中高重复顺序可用限制性内切酶消化DNA经用电泳分离获得,本文用限制性内切酶Bam HI消化恒河猴DNA分离得到一系列高重复片段。用其中最小片段与质粒pBR 322共价连接,转化到大肠杆菌后得到3个带有重组子的克隆。其中PMBI克隆用Bam HI,Pst I酶解,杂交鉴定,证明此克隆是含有重组的恒河猴高重复片段,此片段大小约为340碱基对。哺乳动物基因组相当繁杂,一般在10~9碱基对(b.p)以上,应用复性动力学方法可将其分成高重复顺序、中重复顺序及单拷贝顺序。高重复顺序在基因组中重复频率很高。可达百万次。它有许多家族,这些家族核苷酸顺序相近,但不相同,目前有证据表明,高重复顺序与基因表达及调控有关,与生物进化有关,而且还与DNA的复制及调控有关,因此具有重要的生理功用。为了得到纯的单一高重复顺序,我们应用分子克隆技术,建立了恒河猴高重复顺序DNA的分子克隆。     

一种能识别DNA重复顺序的序列装配方案

提出一种能识别ＤＮＡ重复顺序的序列装配方案。首先通过预筛选和序列联配来判断两处 否真正重叠。然后把来自不同重复区域的所有片段组建成一个重复重叠群（ｒｅｐｅａｔ ｃｏｎｔｉｇ）,而其余片段建成单拷贝重叠群（ｎｏｎｒｅｐｅａｔ ｃｏｎｔｉｇ）。接着重复重叠群被拆分成两个重叠群并与其余的单拷贝重叠群连接成全序列片段顺序。最后按照该全序列片段顺序进行序列多重联配,得到全序列。经过８个目标序列库的验证,表明     

一些水稻的重复DNA顺序在稻属和其它禾本科植物中的多态性

重复DNA顺序是真核生物基因组的特征,很多重复DNA顺序已从小麦、拟南芥菜、燕麦、水稻、玉米等植物的基因组中克隆出来,还发现有一些重复DNA顺序具有基因组特异性,用它们作探针可以分析同属或同科物种的起源和亲缘关系,并建立系统进化树。小卫星DNA或微小卫星DNA所产生的指纹图谱可作为一种遗传学标志来研究系统进化、染色体的精细结构和物种的鉴定。一些中度重复DNA序列还可以作为组织培养株系和细胞杂交筛选的分子标志。稻属已发现并定名的有22个种,根据杂交亲和性、细胞遗传学和生理生化等将它分为6个二倍体组型(AA、BB、CC、DD、EE和FF)和2个四倍体组型(BBCC和CCDD)。现多把禾本科分作5个亚科:竹亚科、稻亚科、早熟禾亚科、画眉草亚科和     

重复顺序与基因表达

一、真核基因组与重复顺序 重复顺序DNA在真核生物基因组中普遍存在,最早研究是在随体中出现。1968年Britten等采用了复性动力学的研究方法获得了可靠的实验证据,并提出了真核基因组中都有高重复顺序,中重复顺序与单考贝DNA顺序。     

非洲绿猴肾细胞株AGMr（HindⅢ）—1高度重复顺序的克隆及初步应用

从非洲绿猴肾细胞株分离高度重复顺序AGMr（HindⅢ）－1基因,以pUC18质粒为载体构建重组质粒pUC18／AGMr（HindⅢ）－1,转化E．coliJM83。酶切分析、SouthernBlot分析和序列分析均证明克隆成功,但所用的第165代Vero细胞株与另一非洲绿猴肾BSC－1细胞株的序列相比较,172个碱基中有6个位点突变。用该重组质粒作探针,对Vero细胞培养的狂犬疫苗作SouthernBlot分析表明,未经纯化的半成品中残余细胞DNA长度约400～5000bp。斑点杂交分析表明,杂交特异性良好,残余细胞DNA最小检出量约4pg。提示AGMr（HindⅢ）－1高度重复顺序可特异地应用于Vero细胞残余DNA检测。     

Capping DNA with DNA

Twelve classes of deoxyribozymes that promote an ATP-dependent "self-capping" reaction were isolated by in vitro selection from a random-sequence pool of DNA. Each deoxyribozyme catalyzes the transfer of the AMP moiety of ATP to its 5'-terminal phosphate group, thereby forming a 5',5'-pyrophosphate linkage. An identical DNA adenylate structure is generated by the T4 DNA ligase during enzymatic DNA ligation. A 41-nucleotide class 1 deoxyribozyme requires Cu(2+) as a cofactor and adopts a structure that recognizes both the adenine and triphosphate moieties of ATP or dATP. The catalytic efficiency for this DNA, measured at 10(4) M(-1) x min(-1) using either ATP or dATP as substrate, is similar to other catalytic nucleic acids that use small substrates. Chemical probing and site-directed mutagenesis implicate the formation of guanine quartets as critical components of the active structure. The observation of ATP-dependent "self-charging" by DNA suggests that DNA could be made to perform the reactions typically associated with DNA cloning, but without the assistance of protein enzymes.     

Eukaryotic DNA polymerases in DNA replication and DNA repair

DNA polymerases carry out a large variety of synthetic transactions during DNA replication, DNA recombination and DNA repair. Substrates for DNA polymerases vary from single nucleotide gaps to kilobase size gaps and from relatively simple gapped structures to complex replication forks in which two strands need to be replicated simultaneously. Consequently, one would expect the cell to have developed a well-defined set of DNA polymerases with each one uniquely adapted for a specific pathway. And to some degree this turns out to be the case. However, in addition we seem to find a large degree of cross-functionality of DNA polymerases in these different pathways. DNA polymerase α is almost exclusively required for the initiation of DNA replication and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint control has been assigned to this enzyme. DNA polymerase δ functions as a dimer and, therefore, may be responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase ζ, for mutagenesis. The function of DNA polymerase ɛ in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase δ or ɛ suffices for the repair of UV-induced damage. The role of DNA polymerase β in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase δ appears to fullfill that function. Received: 20 April 1998 / Accepted: 8 May 1998     

DNA supercoiling by DNA gyrase

Using purified DNA gyrase to supercoil circular plasmid pBR322 DNA, we examined how the linking number attained at the steady state (‘static head’) varies with the concentrations of ATP and ADP, both in the absence and presence of spermidine. In the absence of spermidine at total adenine nucleotide concentrations between 0.35 and 1.4 mM, the static-head linking number was independent of the sum concentration of ATP and ADP, but depended strongly on the ratio of their concentrations. We established that the same linking number was attained independent of the direction from which the steady state was approached. The decrease in linking number at static head is more extensive when spermidine is present in the incubation, but remains a function of the [ATP]-to-[ADP] ratio. These results are discussed in terms of various kinetic schemes for DNA gyrase. We present one kinetic scheme that accounts for the experimental observations. According to this scheme our experimental results imply that there is significant slip in DNA gyrase when spermidine is absent. It is possible that spermidine acts through adjustment of the degree of coupling of DNA gyrase.     

DNA topoisomerases and DNA repair

DNA topoisomerases are enzymes that can modify, and may regulate, the topological state of DNA through concerted breaking and rejoining of the DNA strands. They have been believed to be directly involved in DNA excision repair, and perhaps to be required for the control of repair as well. The vicissitudes of this hypothesis provide a noteworthy example of the dangers of interpreting cellular phenomena without genetic information and vice versa.     

DNA knots and DNA supercoiling

Comment on: Witz G, et al. Proc Natl Acad Sci USA 2011; 108:3608-11.     

DNA杂交与DNA指纹技术

Southern印迹杂交和DNA指纹技术在分子生物学研究以及疾病的诊断、亲缘关系鉴定、犯罪分子确认等过程中发挥了重要作用。回顾了2种技术的发明、发展历程和在生命科学研究中的作用,并探讨了可能的发展方向,从中可以从一个侧面了解分子生物学的发展历程和体会科学家的智慧在科学技术发展中所起的重要作用。     

DNA supercoiling inhibits DNA knotting

Despite the fact that in living cells DNA molecules are long and highly crowded, they are rarely knotted. DNA knotting interferes with the normal functioning of the DNA and, therefore, molecular mechanisms evolved that maintain the knotting and catenation level below that which would be achieved if the DNA segments could pass randomly through each other. Biochemical experiments with torsionally relaxed DNA demonstrated earlier that type II DNA topoisomerases that permit inter- and intramolecular passages between segments of DNA molecules use the energy of ATP hydrolysis to select passages that lead to unknotting rather than to the formation of knots. Using numerical simulations, we identify here another mechanism by which topoisomerases can keep the knotting level low. We observe that DNA supercoiling, such as found in bacterial cells, creates a situation where intramolecular passages leading to knotting are opposed by the free-energy change connected to transitions from unknotted to knotted circular DNA molecules.     

Active DNA demethylation and DNA repair

DNA methylation on cytosine is an epigenetic modification and is essential for gene regulation and genome stability in vertebrates. Traditionally DNA methylation was considered as the most stable of all heritable epigenetic marks. However, it has become clear that DNA methylation is reversible by enzymatic “active” DNA demethylation, with examples in plant cells, animal development and immune cells. It emerges that “pruning” of methylated cytosines by active DNA demethylation is an important determinant for the DNA methylation signature of a cell. Work in plants and animals shows that demethylation occurs by base excision and nucleotide excision repair. Far from merely protecting genomic integrity from environmental insult, DNA repair is therefore at the heart of an epigenetic activation process.