鸡卵黄DNA的存在、提取和特性分析

利用免疫细胞化学技术证实了鸡蛋卵黄球中DNA的存在 .利用Ficoll 40 0密度梯度离心纯化卵黄球并提取了DNA .限制性内切酶分析表明所得卵黄DNA是序列不均一的DNA分子群 .MspⅠ和HpaⅡ分析显示该DNA的甲基化程度极低 .Southern杂交表明卵黄DNA含有基因组DNA的一小部分序列 .并认为卵黄DNA是一种独特的有可能在鸡胚早期发育中发挥功能的DNA .     

含蚕豆叶绿体rRNA基因重组质粒物理图谱的构建

以 pBR322 DNA 为载体,Escherichia coli HB101为受体菌,克隆了含蚕豆叶绿体 rRNA基因的二个 BamHI 片段。应用几种限制性内切酶酶切以及 Southern 印迹法构建了这二个特异片段的物理图谱。重组质粒 pVFB16含有一个4.70kb 的 BamHI 片段,其上含有完整的16S rRNA 基因;重组质粒 pVFB32含有一个5.65kb 的 BamHI 片段,其上含有23S rRNA基因,23S—4.5S/5S rRNA 基因的间隔区及4.5S/5S rRNA 基因。     

MAPPING重组噬菌体中插入片段的“分/合”策略

随着胚胎干细胞基因打靶技术不断推广应用,如何有效地从小鼠基因组DNA文库中筛选得到基因组DNA并进行结构分析(如限制性内切酶酶切图谱分析,即mapping等)已成为一个被普遍关心的问题.设计应用了“分/合”的策略,即先对重组噬菌体插入片段制备一套亚克隆,在对亚克隆进行详细的mapping的基础上,再对完整的大分子噬菌体DNA进行酶切分析,将两者相结合,可以分析得到完整的酶切图谱.应用此策略,简便准确地对所克隆的含有小鼠凝血因子IX基因组DNA的大分子插入片段进行了限制性内切酶酶切图谱分析,结果得到了DNA印迹等的验证.     

单纯疱疹病毒2型特异性DNA片段的克隆和鉴定

用核酸限制性内切酶BamHI对单纯疱疹病毒2型(HSV—2)的DNA进行酶解,回收位于基因组中的反向重复序列区的Bam HIG片段,然后将其克隆在载体质粒PUC 8的Bam HI切点上,进一步用核酸限制性内切酶Eco RI和KPNI对这一重组质粒联合酶解,移去EcoRI—KPNI小片段,经末端修饰后,将其连接得到新的重组质粒pRC102,它含有一小段HSV—2的DNA序列。以此质粒为探针,分别与HSV—1、HSV—2及细胞DNA进行斑点杂交;与HSV—1和HSV—2酶解后的DNA片段进行Southern转印系交。两组实验结果显示,pRC102质粒DNA只与HSV—2 DNA特异性杂交,其HSV—2的型特异性良好。     

平末端DNA随机连接构建重组质粒

目的:拼接DNA片段并克隆。方法:用T4DNA连接酶将DNA片段以平末端随机连接,随后用限制性内切酶切割,琼脂糖电泳分离酶切产物,挑选特定片段纯化回收,与线性化的载体质粒连接,转化大肠杆菌感受态细胞。结果:通过以上步骤,成功拼接了不同DNA片段,构建了含有目的拼接片段的重组质粒。结论:该方法简便、易行、可靠,可作为拼接、克隆DNA的备选方案,在分子生物学研究和基因工程中应用。     

关于细菌人工染色体(BAC)文库载体DNA制备的研究

细菌人工染色体（BAC）文库在基因组研究中起着关键作用。构建BAG文库的一个关键步骤就是BAC载体DNA的制备,制备高质量的BAC载体DNA受到包括酶切,脱磷等诸多因素的影响。以BAC载体pECBAC1为材料,分别采用限制性内切酶BamHⅠ和HK脱磷酶对其进行酶切和脱磷,并结合凝胶回收缩化技术,制备了可用于进一步构建BAC文库的线性载体DNA。并在此基础上,确定了制备BAC载体DNA的适宜条件,其中包括确定适宜限制内切酶用量及酶切时间,脱磷酶种类及浓度和凝胶回收纯化线性载体DNA等关系步骤。     

寻找克隆基因的探针

基因表达和DNA复制应用到基因克隆的具体方法和技术及其概念在不断发展变化。首先DNA是应用纯化了的限制性内切酶在核酸序列上的特异部位切割下来的DNA片段,许多DNA片段用DNA连接酶把它们拼接成克隆工具。限制性内切酶和DNA连接酶在进行筛选时已加以纯化了,这两种酶在商业上是有价值的。     

共生菌Hebeloma sp.的线形质粒DNA的分子克隆及测序的研究

共生担子菌滑菇 H ebeloma circinas携带两个线形的染色体外 DNA分子 .全部的菌丝 DNA经蛋白酶 K处理后 ,通过琼脂凝胶电泳观察到 :在染色体 DNA旁有两个大小不等的 DNA带 ,命名为 p HCl和 p HC2 ,其分子量分别为 1 0 .3kb和 9.1 kb.用核酸外切酶处理 p HC DNA,确认其 5′端被保护 .对 p HC2用不同的内切酶处理并确定其内切酶图谱 ,对其 3.2 kb H ind 片段进行克隆 ,亚克隆和测序 .结果表明 :其片段有两个开读框 ( open reading frames) ,携带类似于病毒 B型的DNA和 RNA多聚酶基因编码 .p HC2为该菌携带的一个典型的线形质粒 ,这也是首次在共生担子菌中发现的线形质粒 .     

人一β干扰素结构基因的定向点突变

用简单而高效的“缺口双链DNA”的定向点突变方法,将IFN-β结构基因编码17位cys的密码TGT改成编码ser的AGT。变异株用合成的诱变引物作为探针来筛选,由Hinf I酶切电泳图谱分析及DNA顺序的测定得到证实。变异率为11％。在大肠杆菌表达的lFN-βser17的抗病毒活力明显高于在相同宿主菌中表达的lFN-β的抗病毒活力。     

枯草芽孢杆菌C-36内切葡聚糖酶基因的克隆及其在大肠杆菌中融合表达

以自行分离筛选出的天然枯草芽孢杆菌（Bacillus subtilis）C-36的染色体DNA为模板,PCR扩增得到含有内切葡聚糖酶基因的DNA片段,将其克隆到pMD-18T载体中,序列分析表明,克隆得到的DNA片段全长1602bp,编码一个含有499个氨基酸的多肽。与其他芽孢杆菌内切葡聚糖酶基因序列比对,其核苷酸同源率为90％～93％,其编码的氨基酸序列的同源性在90％～98％,已将此基因注册GenBank（DQ782954）。将含内切葡聚糖酶基因的重组克隆质粒进行亚克隆,用Kpn I和EcoR I双酶切后,与相同酶切的表达载体pET-32a相连接,并导入大肠杆菌BL21中表达。蛋白质电泳实验结果表明在6．47×10^4处有表达蛋白带。经测定表达蛋白比酶活力达99．02U／mL,为出发菌C-36（63．78U／mL）的1．55倍。     

Distributions of Topological States in Circular DNA

A distinctive feature of closed circular DNA molecules is their particular topological state, which cannot be altered by any conformational rearrangement short of breaking at least one strand. This topological constraint opens unique possibilities for experimental studies of the distributions of topological states created in different ways. Primarily, the equilibrium distributions of topological properties are considered in the review. It is described how such distributions can be obtained and measured experimentally, and how they can be computed. Comparison of the calculated and measured equilibrium distributions over the linking number of complementary strands, equilibrium fractions of knots and links formed by circular molecules has provided much valuable information about the properties of the double helix. Study of the steady-state fraction of knots and links created by type II DNA topoisomerases has revealed a surprising property of the enzymes: their ability to reduce these fractions considerably below the equilibrium level.     

Topoisomerase II Regulates the Maintenance of DNA Methylation

The maintenance of DNA methylation in nascent DNA is a critical event for numerous biological processes. Following DNA replication, DNMT1 is the key enzyme that strictly copies the methylation pattern from the parental strand to the nascent DNA. However, the mechanism underlying this highly specific event is not thoroughly understood. In this study, we identified topoisomerase IIα (TopoIIα) as a novel regulator of the maintenance DNA methylation. UHRF1, a protein important for global DNA methylation, interacts with TopoIIα and regulates its localization to hemimethylated DNA. TopoIIα decatenates the hemimethylated DNA following replication, which might facilitate the methylation of the nascent strand by DNMT1. Inhibiting this activity impairs DNA methylation at multiple genomic loci. We have uncovered a novel mechanism during the maintenance of DNA methylation.     

FBH1 Helicase Disrupts RAD51 Filaments in Vitro and Modulates Homologous Recombination in Mammalian Cells

Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.     

Human HEL308 localizes to damaged replication forks and unwinds lagging strand structures

HEL308 is a superfamily II DNA helicase, conserved from archaea through to humans. HEL308 family members were originally isolated by their similarity to the Drosophila melanogaster Mus308 protein, which contributes to the repair of replication-blocking lesions such as DNA interstrand cross-links. Biochemical studies have established that human HEL308 is an ATP-dependent enzyme that unwinds DNA with a 3' to 5' polarity, but little else is know about its mechanism. Here, we show that GFP-tagged HEL308 localizes to replication forks following camptothecin treatment. Moreover, HEL308 colocalizes with two factors involved in the repair of damaged forks by homologous recombination, Rad51 and FANCD2. Purified HEL308 requires a 3' single-stranded DNA region to load and unwind duplex DNA structures. When incubated with substrates that model stalled replication forks, HEL308 preferentially unwinds the parental strands of a structure that models a fork with a nascent lagging strand, and the unwinding action of HEL308 is specifically stimulated by human replication protein A. Finally, we show that HEL308 appears to target and unwind from the junction between single-stranded to double-stranded DNA on model fork structures. Together, our results suggest that one role for HEL308 at sites of blocked replication might be to open up the parental strands to facilitate the loading of subsequent factors required for replication restart.     

Studies on human DNA polymerase epsilon and GINS complex and their role in DNA replication

In eukaryotic cells, DNA replication is carried out by the coordinated action of three DNA polymerases (Pols), Pol α, δ, and ε. In this report, we describe the reconstitution of the human four-subunit Pol ε and characterization of its catalytic properties in comparison with Pol α and Pol δ. Human Pol ε holoenzyme is a monomeric complex containing stoichiometric subunit levels of p261/Pol 2, p59, p17, and p12. We show that the Pol ε p261 N-terminal catalytic domain is solely responsible for its ability to catalyze DNA synthesis. Importantly, human Pol (hPol) ε was found more processive than hPol δ in supporting proliferating cell nuclear antigen-dependent elongation of DNA chains, which is in keeping with proposed roles for hPol ε and hPol δ in the replication of leading and lagging strands, respectively. Furthermore, GINS, a component of the replicative helicase complex that is composed of Sld5, Psf1, Psf2, and Psf3, was shown to interact weakly with all three replicative DNA Pols (α, δ, and ε) and to markedly stimulate the activities of Pol α and Pol ε. In vivo studies indicated that siRNA-targeted depletion of hPol δ and/or hPol ε reduced cell cycle progression and the rate of fork progression. Under the conditions used, we noted that depletion of Pol ε had a more pronounced inhibitory effect on cellular DNA replication than depletion of Pol δ. We suggest that reduction in the level of Pol δ may be less deleterious because of its collision-and-release role in lagging strand synthesis.     

The DDN Catalytic Motif Is Required for Metnase Functions in Non-homologous End Joining (NHEJ) Repair and Replication Restart

Metnase (or SETMAR) arose from a chimeric fusion of the Hsmar1 transposase downstream of a protein methylase in anthropoid primates. Although the Metnase transposase domain has been largely conserved, its catalytic motif (DDN) differs from the DDD motif of related transposases, which may be important for its role as a DNA repair factor and its enzymatic activities. Here, we show that substitution of DDN⁶¹⁰ with either DDD⁶¹⁰ or DDE⁶¹⁰ significantly reduced in vivo functions of Metnase in NHEJ repair and accelerated restart of replication forks. We next tested whether the DDD or DDE mutants cleave single-strand extensions and flaps in partial duplex DNA and pseudo-Tyr structures that mimic stalled replication forks. Neither substrate is cleaved by the DDD or DDE mutant, under the conditions where wild-type Metnase effectively cleaves ssDNA overhangs. We then characterized the ssDNA-binding activity of the Metnase transposase domain and found that the catalytic domain binds ssDNA but not dsDNA, whereas dsDNA binding activity resides in the helix-turn-helix DNA binding domain. Substitution of Asn-610 with either Asp or Glu within the transposase domain significantly reduces ssDNA binding activity. Collectively, our results suggest that a single mutation DDN⁶¹⁰ → DDD⁶¹⁰, which restores the ancestral catalytic site, results in loss of function in Metnase.     

Contribution of the intrinsic curvature to measured DNA persistence length

The persistence length of DNA, a, depends both on the intrinsic curvature of the double helix and on the thermal fluctuations of the angles between adjacent base-pairs. We have evaluated two contributions to the value of a by comparing measured values of a for DNA containing a generic sequence and for an "intrinsically straight" DNA. In each 10 bp segment of the intrinsically straight DNA an initial sequence of five bases is repeated in the sequence of the second five bases, so any bends in the first half of the segment are compensated by bends in the opposite direction in the second half. The value of a for the latter DNA depends, to a good approximation, on thermal fluctuations only; there is no intrinsic curvature. The values of a were obtained from measurements of the cyclization efficiency for short DNA fragments, about 200 bp in length. This method determines the persistence length of DNA with exceptional accuracy, due to the very strong dependence of the cyclization efficiency of short fragments on the value of a. We find that the values of a for the two types of DNA fragment are very close and conclude that the contribution of the intrinsic curvature to a is at least 20 times smaller than the contribution of thermal fluctuations. The relationship between this result and the angles between adjacent base-pairs, which specify the intrinsic curvature, is analyzed.     

A procedure for large-scale isolation of RNA-free plasmid and phage DNA without the use of RNase

A preparative procedure for the large-scale isolation of plasmid DNA without the use of RNAse is described. Crude plasmid DNA is prepared using a standard boiling method. High-molecular-weight RNA is removed by precipitation with LiCl, and low-molecular-weight RNA is removed by sedimentation through high-salt solution. The procedure is inexpensive, rapid, simple, and particularly suitable for processing several large-scale preparations simultaneously. A similar procedure has been developed for preparation of lambda-phage DNA.     

Formation of a stable RuvA protein double tetramer is required for efficient branch migration in vitro and for replication fork reversal in vivo

In bacteria, RuvABC is required for the resolution of Holliday junctions (HJ) made during homologous recombination. The RuvAB complex catalyzes HJ branch migration and replication fork reversal (RFR). During RFR, a stalled fork is reversed to form a HJ adjacent to a DNA double strand end, a reaction that requires RuvAB in certain Escherichia coli replication mutants. The exact structure of active RuvAB complexes remains elusive as it is still unknown whether one or two tetramers of RuvA support RuvB during branch migration and during RFR. We designed an E. coli RuvA mutant, RuvA2(KaP), specifically impaired for RuvA tetramer-tetramer interactions. As expected, the mutant protein is impaired for complex II (two tetramers) formation on HJs, although the binding efficiency of complex I (a single tetramer) is as wild type. We show that although RuvA complex II formation is required for efficient HJ branch migration in vitro, RuvA2(KaP) is fully active for homologous recombination in vivo. RuvA2(KaP) is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2(KaP) for forks is decreased compared with wild type. Accordingly, RuvA2(KaP) is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2(KaP) is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2(KaP) is defective for stimulation of RuvB activity and stability of HJ·RuvA·RuvB tripartite complexes. This work demonstrates that the need for RuvA tetramer-tetramer interactions for full RuvAB activity in vitro causes specifically an RFR defect in vivo.