细菌内同源重组法构建靶向Survivin 的腺病毒载体的研究

目的:研究细菌内同源重组法构建靶向Survivin的腺病毒载体及其体外扩增表达。方法:将survivin基因克隆至穿梭质粒载体中,特异性酶切后回收、连接、转化,构建负载survivin片段的重组腺病毒载体。提取重组病毒基因酶切鉴定后,包装成病毒,并扩增到所需滴度。行Western blotting鉴定,观察重组腺病毒载体在真核细胞的表达。结果:(1)重组穿梭质粒的Mlu I酶切鉴定结果显示,酶切结果均与相应的载体及目的片段的大小相符合,基因测序结果基本一致。(2)凝胶电泳产生了两条大约15 kb和8.5kb的片段,由图2可知重组腺病毒质粒酶切充分完全,且回收率较高。(3)重组腺病毒载体转染AD293细胞24 h后已出现细胞病变效应,病变细胞细胞核变大。(4)D260/OD280的值为1.92,表明重组腺病毒纯度较高。(5)AD293细胞病毒上清中有能与抗Survivin单抗反应的蛋白,其相对分子质量与理论值相吻合,阴性对照组无对应条带出现。结论:本方法成功构建表达了含Survivin的重组腺病毒载体,为进一步进行Survivin基因功能的研究提供了实验基础和理论支持。     

用大肠杆菌同源重组获得克隆化重组腺病毒基因组

利用大肠杆菌细胞内质粒间同源重组获得克隆化重组腺病毒基因组 DNA,高效构建携带有外源基因的均一重组腺病毒 .将带有狂犬病毒糖蛋白 (GP)基因和加强型 GFP(enhanced GFP,EGFP)表达盒的重组穿梭质粒 p Ad- Track- CMV/ GP与腺病毒骨架载体质粒 p Ad Easy- 1一起同时电击共转化大肠杆菌 BJ51 83.在 BJ51 83细胞内 ,带有同源序列的重组穿梭质粒与骨架载体可进行同源重组 ,得到以质粒形式存在的克隆化重组腺病毒基因组 p Ad- GP’.以 p Ad- GP’为模板 ,经DNA测序确认 GP基因成功整合入此质粒中的腺病毒基因组 E1区外源基因表达盒中 .线形化的p Ad- GP’转染 2 93细胞后可得到基因组结构均一、在 E1区插入有 GP和 EGFP表达盒的重组腺病毒 ,病毒滴度可达 1× 1 0 8pfu/ ml.电镜下此重组病毒颗粒直径约为 70 nm,略呈球形 ,用荧光显微镜观察感染细胞有很强的 EGFP表达 .实验表明 :利用大肠杆菌同源重组获得克隆化的重组腺病毒基因组 DNA,可高效制备高滴度的均一重组腺病毒     

recA基因通过同源重组途径参与由整合质粒发动的大肠杆菌染色体复制

我们在前文中报道由整合的F'质粒所发动的大肠杆菌染色体的复制依赖于recA基因。本文报道有关recA、recB、recC以及lexA等在染色体复制中的作用,实验结果说明,recA基因通过同源重组途径而不是通过SOS途径参与复制,而且recA基因和Chi热点无关。实验结果还说明,RecBC酶的依赖于ATP的双链DNA外切核酸酶活性和recA基因的作用无关。     

同源重组法制备口蹄疫病毒多基因重组腺病毒

通过细菌内同源重组的方法成功构建了含有O型口蹄疫病毒P1-2A和3C蛋白酶基因和3D基因的重组腺病毒表达载体.首先将P1-2A、3C和3D基因亚克隆连接到穿梭质粒pShuttle-CMV上,再将重组穿梭质粒用PmeI线性化后电转化携带有腺病毒骨架载体pAdeasy-1的大肠杆菌BJ5183感受态菌,经细菌内同源重组产生pAdcmv-p12x3c和pAdcmv-p12x3cd重组腺病毒质粒,经序列测定证实目的基因已正确的插入到腺病毒骨架载体中.重组腺病毒质粒经PacI线性化后转染HEK293细胞,转染1w内细胞出现典型病变.取转染细胞裂解液上清连续传代至第4代时,细胞于24~48h内即病变完全,收取接毒后24h细胞进行PCR和RT-PCR检测,表明目的基因已整合到腺病毒基因组内,且在mRNA水平上有表达.取第4、6、8和10代病毒,用蛋白酶K处理后可扩增出目的基因,证明此重组病毒可稳定存在.本研究为FMDV腺病毒活载体疫苗的研究奠定了基础.     

同源重组法制备口蹄疫病毒多基因重组腺病毒

通过细菌内同源重组的方法成功构建了含有O型口蹄疫病毒P1—2A和3C蛋白酶基因和3D基因的重组腺病毒表达载体。首先将P1—2A、3C和3D基因亚克隆连接到穿梭质粒pShuttle—CMV上,再将重组穿梭质粒用PmeI线性化后电转化携带有腺病毒骨架载体pAdeasy—1的大肠杆菌BJ5183感受态菌,经细菌内同源重组产生pAdcmv—p12x3c和pAdcmy—p12x3cd重组腺病毒质粒,经序列测定证实目的基因已正确的插入到腺病毒骨架载体中。重组腺病毒质粒经PacI线性化后转染HEK293细胞,转染1w内细胞出现典型病变。取转染细胞裂解液上清连续传代至第4代时,细胞于24～48h内即病变完全,收取接毒后24h细胞进行。PCR和RT—PCR检测,表明目的基因已整合到腺病毒基因组内,且在mRNA水平上有表达。取第4、6、8和10代病毒,用蛋白酶K处理后可扩增出目的基因,证明此重组病毒可稳定存在。本研究为FMDV腺病毒活载体疫苗的研究奠定了基础。     

修饰的痘苗病毒安卡拉株（MVA）基因组中高频的同源重组

痘苗病毒由于其外源基因容量大,表达产物后加工完善等优势而广泛用于基因工程的研究以及基因治疗,痘苗病毒基因组的同源重组现象为其基因操作带来了方便,也被用于很多痘苗病毒基因结构和功能的研究,痘苗病毒安卡拉株（MVA）是一种修饰的复制限制的痘苗病毒,由于极高的安全性,正在实验室和临床应用的很多领域取代普通的痘苗病毒,为提高重组MVA系统的安全性以及筛选重组MVA的效率,发展了一种暂时选择系统,此系统利用分子内2段同向的相同序列发生同源重组去除选择标记k1l基因,从而消除选择标记对宿主可能的危害。利用此暂时表达系统构建了4个携带编码不同长度外源多蛋白质序列的重组MVA,并估算了每次传代的重组频率,结果显示,MVA同源重组频率虽然比其他痘苗病毒株要低,但仍然是较斋的,将带有k1l基因的重组MVA经3-4次盲传（blind passage),即可获得完全去除选择标记的重组MVA。进一步证明上述利用暂时选择标记k1l基因构建重组MVA的系统具有十分可靠的安全性,适合作为人体活疫苗开发和基因治疗的载体,而且,通过盲传进行筛选,能大大提高去除选择标记的效率,降低鸺建重组MVA的成本。     

Enhancement of extra chromosomal recombination in somatic cells by affecting the ratio of homologous recombination (HR) to non-homologous end joining (NHEJ)

Advancements in somatic cell gene targeting have been slow due to the finite lifespan of somatic cells and the overall inefficiency of homologous recombination. The rate of homologous recombination is determined by mechanisms of DNA repair, and by the balance between homologous recombination (HR) and non-homologous end joining (NHEJ). A plasmid-to-plasmid, extra chromosomal recombination system was used to study the effects of the manipulation of molecules involved in NHEJ (Mre11, Ku70/80, and p53) on HR/NHEJ ratios. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. While a mutant Mre11 and an anti-Ku aptamer did not significantly affect the rate of NHEJ or HR, transient expression of a p53 mutant increased overall HR/NHEJ by 2.5 fold. However, expression of the mutant p53 resulted in increased aneuploidy of the cultured cells. Additionally, we found no relationship between telomerase expression and changes in HR/NHEJ. In contrast, cell synchrony by thymidine incorporation did not induce chromosomal abnormalities, and increased the ratio of HR/NHEJ 5-fold by reducing the overall rate of NHEJ. Overall our results show that attempts at reducing NHEJ by use of Mre11 or anti-Ku aptamers were unsuccessful. Cell synchrony via thymidine incorporation, however, does increase the ratio of HR/NHEJ and this indicates that this approach may be of use to facilitate targeting in somatic cells by reducing the numbers of colonies that need to be analyzed before a HR is identified.     

Ataxia Telangiectasia Mutated (ATM) Is Dispensable for Endonuclease I-SceI-induced Homologous Recombination in Mouse Embryonic Stem Cells

Ataxia telangiectasia mutated (ATM) is activated upon DNA double strand breaks (DSBs) and phosphorylates numerous DSB response proteins, including histone H2AX on serine 139 (Ser-139) to form γ-H2AX. Through interaction with MDC1, γ-H2AX promotes DSB repair by homologous recombination (HR). H2AX Ser-139 can also be phosphorylated by DNA-dependent protein kinase catalytic subunit and ataxia telangiectasia- and Rad3-related kinase. Thus, we tested whether ATM functions in HR, particularly that controlled by γ-H2AX, by comparing HR occurring at the euchromatic ROSA26 locus between mouse embryonic stem cells lacking either ATM, H2AX, or both. We show here that loss of ATM does not impair HR, including H2AX-dependent HR, but confers sensitivity to inhibition of poly(ADP-ribose) polymerases. Loss of ATM or H2AX has independent contributions to cellular sensitivity to ionizing radiation. The ATM-independent HR function of H2AX requires both Ser-139 phosphorylation and γ-H2AX/MDC1 interaction. Our data suggest that ATM is dispensable for HR, including that controlled by H2AX, in the context of euchromatin, excluding the implication of such an HR function in genomic instability, hypersensitivity to DNA damage, and poly(ADP-ribose) polymerase inhibition associated with ATM deficiency.     

Homologous Desensitization of Muscarinic Cholinergic, Histaminergic, Adrenergic, and Serotonergic Receptors Coupled to Phospholipase C in Cerebellar Granule Cells

Cultured cerebellar granule cells express phospholipase C-coupled muscarinic cholinergic, histaminergic, alpha 1-adrenergic, and serotonergic receptors. In an attempt to study desensitization of these neurotransmitter receptors, cells were prestimulated with saturating concentrations of carbachol, histamine, norepinephrine, or serotonin during the labeling of cells with myo-[3H]inositol and then rechallenged with various receptor agonists for their ability to elicit accumulation of [3H]inositol monophosphate in the presence of lithium. Prestimulation with each of these receptor agonists was found to cause a time-dependent desensitization to subsequent stimulation with the desensitizing agonist. Thus, prestimulation for 0.5, 4, and 18 h decreased carbachol response to 87 +/- 4, 52 +/- 2, and 40 +/- 1% of the control, respectively; histamine response to 37 +/- 2, 24 +/- 2, and 18 +/- 2%, respectively; norepinephrine response to 55 +/- 5, 14 +/- 1, and 10 +/- 1%, respectively; and serotonin response to 36 +/- 1, 18 +/- 1, and 9 +/- 2%, respectively. In all cases, the responses mediated by receptors which were not prestimulated remained virtually unchanged, thus indicating homologous desensitization. Dose-response studies indicate that the desensitization was associated with a major reduction in the maximal extent of agonist-induced responses. The basal accumulation was markedly enhanced following 0.5- and 4-h prestimulation, but returned to near normal after 18-h pretreatment. Biologically active phorbol ester, 4 beta-phorbol 12-myristate 13-acetate, rapidly attenuated basal phospholipase C activity, as well as the responses mediated by carbachol, histamine, norepinephrine, and serotonin, suggesting that activation and translocation of protein kinase C might play a role in the desensitization of phospholipase C-coupled receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Role of the Human Psoralen 4 (hPso4) Protein Complex in Replication Stress and Homologous Recombination

Psoralen 4 (Pso4) is an evolutionarily conserved protein that has been implicated in a variety of cellular processes including RNA splicing and resistance to agents that cause DNA interstrand cross-links. Here we show that the hPso4 complex is required for timely progression through S phase and transition through the G₂/M checkpoint, and it functions in the repair of DNA lesions that arise during replication. Notably, hPso4 depletion results in delayed resumption of DNA replication after hydroxyurea-induced stalling of replication forks, reduced repair of spontaneous and hydroxyurea-induced DNA double strand breaks (DSBs), and increased sensitivity to a poly(ADP-ribose) polymerase inhibitor. Furthermore, we show that hPso4 is involved in the repair of DSBs by homologous recombination, probably by regulating the BRCA1 protein levels and the generation of single strand DNA at DSBs. Together, our results demonstrate that hPso4 participates in cell proliferation and the maintenance of genome stability by regulating homologous recombination. The involvement of hPso4 in the recombinational repair of DSBs provides an explanation for the sensitivity of Pso4-deficient cells to DNA interstrand cross-links.     

Identifying the Impact of Surface Recombination at Electrodes in Organic Solar Cells by Means of Electroluminescence and Modeling

This work reports on combining current‐voltage characteristics, electroluminescence (EL) measurements, and modeling to identify the selectivity of the electrodes in bulk‐heterojunction organic solar cells. Devices with the same photoactive layer but different contact materials are compared and the impact of surface recombination at the contacts on their performance is determined. The open‐circuit voltage, V _OC, depends strongly on the selectivity of the electrodes and it is observed that the EL signal of cells with lower V _OC is dramatically reduced. This is ascribed to an enhanced rate of surface recombination, which is a non‐radiative recombination pathway and does therefore not contribute to the EL yield. In addition, these cells have a lower current in forward direction despite the fact that the surface recombination occurs in addition to the recombination in the bulk. A theoretical model was set up and in the corresponding numerical simulations all three findings (lower V _OC, strongly reduced EL signal and lower forward current) could be clearly reproduced by varying just one single parameter which determines the selectivity of the electrode.     

Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 (SMCHD1) Promotes Non-homologous End Joining and Inhibits Homologous Recombination Repair upon DNA Damage

Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) has been shown to be involved in gene silencing and DNA damage. However, the exact mechanisms of how SMCHD1 participates in DNA damage remains largely unknown. Here we present evidence that SMCHD1 recruitment to DNA damage foci is regulated by 53BP1. Knocking out SMCHD1 led to aberrant γH2AX foci accumulation and compromised cell survival upon DNA damage, demonstrating the critical role of SMCHD1 in DNA damage repair. Following DNA damage induction, SMCHD1 depletion resulted in reduced 53BP1 foci and increased BRCA1 foci, as well as less efficient non-homologous end joining (NHEJ) and elevated levels of homologous recombination (HR). Taken together, these results suggest an important function of SMCHD1 in promoting NHEJ and repressing HR repair in response to DNA damage.     

Chromosome-wide linkage disequilibrium caused by an inversion polymorphism in the white-throated sparrow (Zonotrichia albicollis)

Chromosomal inversions have been of long-standing interest to geneticists because they are capable of suppressing recombination and facilitating the formation of adaptive gene complexes. An exceptional inversion polymorphism (ZAL2^m) in the white-throated sparrow (Zonotrichia albicollis) is linked to variation in plumage, social behavior and mate choice, and is maintained in the population by negative assortative mating. The ZAL2^m polymorphism is a complex inversion spanning >100 Mb and has been proposed to be a strong suppressor of recombination, as well as a potential model for studying neo-sex chromosome evolution. To quantify and evaluate these features of the ZAL2^m polymorphism, we generated sequence from 8 ZAL2^m and 16 ZAL2 chromosomes at 58 loci inside and 4 loci outside the inversion. Inside the inversion we found that recombination was completely suppressed between ZAL2 and ZAL2^m, resulting in uniformly high levels of genetic differentiation (F_ST=0.94), the formation of two distinct haplotype groups representing the alternate chromosome arrangements and extensive linkage disequilibrium spanning ∼104 Mb within the inversion, whereas gene flow was not suppressed outside the inversion. Finally, although ZAL2^m homozygotes are exceedingly rare in the population, occurring at a frequency of <1%, we detected evidence of historical recombination between ZAL2^m chromosomes inside the inversion, refuting its potential status as a non-recombining autosome.     

整合型vgb基因载体的构建和研究

运用染色体-质粒同源基因重组的方法将外源Vitreoscilla血红蛋白基因(vgb)整合在大肠杆菌JM105染色体的苏氨酸操纵子位置上,构建单拷贝的vgb表达载体VGl。VGl保持了vgb基因的生理功能,可在低氧条件下表达Citreoscilla血红蛋白使细胞适应贫氧环境;同时它克服了以质粒作为表达载体时vgb基因剂量和表达量过高带来的生理负担,因此可作为一种适于高密度培养的基因工程宿主菌。实验结果还显示vgb基因的表达使VGl与普通大肠杆菌相比在低氧条件下仍能维持较高的呼吸强度,说明vgb基因产物参与了细胞处于低氧水平时的代谢过程。     

Homologous recombination mediated by two palindromic repeated sequences in the mitochondrial genome of Oryza

Palindromic repeated sequences (PRSs) are distributed in at least ten regions of the mitochondrial (mt) genome of rice and are, apparently, mobile. In the present study, we examined the possibility of homologous recombination via some PRSs during the course of evolution of Oryza. We first performed Southern hybridization of the DNA from 11 species (18 strains) of Oryza in order to identify the distribution of PRSs in the mitochondrial genome of Oryza. The hybridization patterns revealed genome type-specific and/or species-specific variations. We speculated that homologous recombination via some PRSs might have made a contribution to such variations. After subsequent polymerase chain reaction, Southern hybridization and sequencing, we concluded that homologous recombination mediated by two PRSs occurred in the mtDNA of Oryza after divergence of the BB genome type and the other genome types of Oryza. Evidence was obtained that some PRSs were involved in both insertion and recombination events during the evolution of Oryza. Our results indicate, therefore, that PRSs have contributed considerably to the polymorphism of Oryza mtDNAs.     

Identifying the Nature of Charge Recombination in Organic Solar Cells from Charge‐Transfer State Electroluminescence

Charge‐transfer (CT) state electroluminescence is investigated in several polymer:fullerene bulk heterojunction solar cells. The ideality factor of the electroluminescence reveals that the CT emission in polymer:fullerene solar cells originates from free‐carrier bimolecular recombination at the donor‐acceptor interface, rather than a charge‐trap‐mediated process. The fingerprint of the presence of nonradiative trap‐assisted recombination, a voltage‐dependent CT electroluminescence quantum efficiency, is only observed for the P3HT:PCBM system, which is explained by a reduction of the competing bimolecular recombination rate. These results are in agreement with measurements of the illumination‐intensity dependence of the open‐circuit voltage.