Construction of large DNA segments in Escherichia coli

Recombinant DNA clones containing large pieces of DNA are useful in the study of large genetic units, but these are difficult to make in most bacterial cloning vectors. A strategy is described that uses general and site-specific recombination to construct large pieces of eukaryotic DNA from smaller cloned segments. The large clones are propagated on F factor-based plasmids in Escherichia coli. They can be easily modified to introduce mutations or rearrangements. These techniques were applied to the construction of large DNA segments from the bithorax complex of Drosophila.     

外源DNA直接导入技术在麦类作物育种上的应用(综述)

外源DNA导入是改良作物的有效途径之一。文章概述了该技术在麦类作物上的应用研究进展。对外源DNA的导入途径、导入效果、导入验证和产生变异的机理等方面作了分析。     

Isolation of single-copy human genes from a library of yeast artificial chromosome clones

A recently developed cloning system based on the propagation of large DNA molecules as linear, artificial chromosomes in the yeast Saccharomyces cerevisiae provides a potential method of cloning the entire human genome in segments of several hundred kilobase pairs. Most application of this system will require the ability to recover specific sequences from libraries of yeast artificial chromosome clones and to propagate these sequences in yeast without alterations. Two single-copy genes have now been cloned from a library of yeast artificial chromosome clones that was prepared from total human DNA. Multiple, independent isolates were obtained of the genes encoding factor IX and plasminogen activator inhibitor type 2. The clones, which ranged in size from 60 to 650 kilobases, were stable on prolonged propagation in yeast and appear to contain faithful replicas of human DNA.     

Introduction of human DNA into mouse eggs by injection of dissected chromosome fragments

A procedure has been developed for introducing exogenous DNA into mouse eggs by injection of chromosome fragments. Chromosome fragments were dissected from human metaphase spreads and microinjected into the pronuclei of fertilized mouse eggs. Many of the injected eggs subsequently exhibited normal pre- and postimplantation development. Embryos obtained from eggs injected with centromeric fragments retained human centromeric DNA as demonstrated by in situ hybridization analysis. From eggs injected with noncentromeric fragments, a mouse was obtained whose tail tissue exhibited the presence of human DNA. This procedure should facilitate incorporation of very large (more than 10 megabases) DNA fragments into cells and embryos without the need for cloned sequences.     

Effectiveness of exogenous DNA transfer to chicken embryo cells in vitro and in vivo using retroviral vectors

The frequency of genetic transformation of target cells in a culture is established to be from 9 × 10^?4 to 3 × 10^?3. A transgenic rooster with integration and expression of the lacZ gene in heart, liver, intestine, and testis cells is produced.     

Cloning of human androgen receptor complementary DNA and localization to the X chromosome

The androgen receptor (AR) mediates the actions of male sex steroids. Human AR genomic DNA was cloned from a flow-sorted human X chromosome library by using a consensus nucleotide sequence from the DNA-binding domain of the family of nuclear receptors. The AR gene was localized on the human X chromosome between the centromere and q13. Cloned complementary DNA, selected with an AR-specific oligonucleotide probe, was expressed in monkey kidney (COS) cells and yielded a high-affinity androgen-binding protein with steroid-binding specificity corresponding to that of native AR. A predominant messenger RNA species of 9.6 kilobases was identified in human, rat, and mouse tissues known to contain AR and was undetectable in tissues lacking AR androgen-binding activity, including kidney and liver from androgen-insensitive mice. The deduced amino acid sequence of AR within the DNA-binding domain has highest sequence identity with the progesterone receptor.     

Site-specific cleavage of a yeast chromosome by oligonucleotide-directed triple-helix formation

Oligonucleotides equipped with EDTA-Fe can bind specifically to duplex DNA by triple-helix formation and produce double-strand cleavage at binding sites greater than 12 base pairs in size. To demonstrate that oligonucleotide-directed triple-helix formation is a viable chemical approach for the site-specific cleavage of large genomic DNA, an oligonucleotide with EDTA-Fe at the 5' and 3' ends was targeted to a 20-base pair sequence in the 340-kilobase pair chromosome III of Saccharomyces cerevisiae. Double-strand cleavage products of the correct size and location were observed, indicating that the oligonucleotide bound and cleaved the target site among almost 14 megabase pairs of DNA. Because oligonucleotide-directed triple-helix formation has the potential to be a general solution for DNA recognition, this result has implications for physical mapping of chromosomes.     

广西蔗区新台糖22号种性退化现状分析及对策措施

目的建立云蔗05-51组培再生体系明确其对抗生素G418的耐受性,为云蔗05-51基因工程遗传改良提供参考。方法以云蔗05-51心叶为外植体,MS基本培养基添加2, 4-D、6-BA和KT,设置质量浓度梯度及不同激素配比,诱导愈伤组织及其分化植株。在云蔗05-51胚性愈伤组织增殖与不定芽分化阶段,培养基中添加不同含量的G418,确定合适的G418筛选质量浓度。以nptⅡ为标记基因,农杆菌介导遗传转化云蔗05-51胚性愈伤组织,筛选获得抗性植株,PCR和NPTⅡ ImmunoStrip检测确定nptⅡ基因的整合与表达。结果云蔗05-51愈伤组织诱导与增殖阶段,适宜的培养基配方为MS基本培养基添加3.0 mg/L 2, 4-D;愈伤组织分化不定芽阶段宜采用MS基本培养基添加1.0 mg/L 6-BA+0.1 mg/L KT。云蔗05-51对G418耐受性筛选结果显示：愈伤组织增殖阶段和分化不定芽阶段G418的适宜筛选质量浓度分别为40 和20 mg/L。G418抗性植株PCR检验表明：标记基因nptⅡ已成功整合到云蔗05-51转基因植株基因组中;NPTⅡ ImmunoStrip检测表明：nptⅡ的蛋白水平得以表达。结论建立了云蔗05-51心叶组培再生体系,明确了40和20 mg/L G418是筛选抗性愈伤和抗性植株的适宜质量浓度。建立了农杆菌介导遗传转化云蔗05-51胚性愈伤组织系统,为有益基因导入甘蔗品种提供了参考。     

Antibody class switching mediated by yeast endonuclease-generated DNA breaks

Antibody class switching in activated B cells uses class switch recombination (CSR), which joins activation-induced cytidine deaminase (AID)-dependent double-strand breaks (DSBs) within two large immunoglobulin heavy chain (IgH) locus switch (S) regions that lie up to 200 kilobases apart. To test postulated roles of S regions and AID in CSR, we generated mutant B cells in which donor Smu and accepter Sgamma1 regions were replaced with yeast I-SceI endonuclease sites. We found that site-specific I-SceI DSBs mediate recombinational IgH locus class switching from IgM to IgG1 without S regions or AID. We propose that CSR evolved to exploit a general DNA repair process that promotes joining of widely separated DSBs within a chromosome.     

Mutagenic processing of ribonucleotides in DNA by yeast topoisomerase I

The ribonuclease (RNase) H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nucleic acid metabolism. RNase H2 is specialized to remove single ribonucleotides [ribonucleoside monophosphates (rNMPs)] from duplex DNA, and its absence in budding yeast has been associated with the accumulation of deletions within short tandem repeats. Here, we demonstrate that rNMP-associated deletion formation requires the activity of Top1, a topoisomerase that relaxes supercoils by reversibly nicking duplex DNA. The reported studies extend the role of Top1 to include the processing of rNMPs in genomic DNA into irreversible single-strand breaks, an activity that can have distinct mutagenic consequences and may be relevant to human disease.     

Modeling greenhouse air humidity by means of artificial neural network and principal component analysis

A back propagation neural network (BPNN) based on principal component analysis (PCA) was proposed for modeling the internal greenhouse humidity in winter of North China. The environment factors influencing the inside humidity include outside air temperature and humidity, wind speed, solar radiation, inside air temperature, open angle of top vent and side vent, and open ration of sunshade curtain, which were all collected as data samples. Through PCA of these data samples, 4 main factors were extracted, and the relationship between the main factors and the original data was discussed. Taking the principal component values as the input of BPNN, the model showed a good performance. A comparison was made between the performances of the BPNN based on PCA and the stepwise regression method with 20 data samples which had not been used to establish the NN model, and the prediction of stepwise regression method was less accurate than the BPNN based on PCA.     

Mitotic recombination within the centromere of a yeast chromosome

Centromeres are the structural elements of eukaryotic chromosomes that hold sister chromatids together and to which spindle tubules connect during cell division. Centromeres have been shown to suppress meiotic recombination in some systems. In this study yeast strains genetically marked within and flanking a centromere, were used to demonstrate that gene conversion (nonreciprocal recombination) tracts in mitosis can enter into and extend through the centromere.     

草莓多酚氧化酶基因全长的克隆及表达载体构建

【目的】为了提高草莓的果实品质和经济价值。【方法】利用改良CTAB法提取五叶草莓叶片总DNA,反向PCR(IPCR)方法克隆基因。【结果】得到了2条完整的草莓多酚氧化酶基因全长3 591bp和1 809bp,分别编码1 196和602个氨基酸,分别命名为FpPPO1与FpPPO_2,GenBank登录号分别为HM063946和HM063947。FpPPO1和FpPPO_2与其他物种中的多酚氧化酶基因比较,氨基酸相似性分别为57%~77%与74%~99%。最后成功构建了多酚氧化酶基因反义植物表达载体pCP430和正义植物表达载体pCP1809。【结论】克隆得到草莓FpPPO1和FpPPO_2基因全长并构建转基因载体,为研究草莓中多酚氧化酶基因的功能奠定了基础。     

Dynamics of DNA double-strand breaks revealed by clustering of damaged chromosome domains

Interactions between ends from different DNA double-strand breaks (DSBs) can produce tumorigenic chromosome translocations. Two theories for the juxta-position of DSBs in translocations, the static "contact-first" and the dynamic "breakage-first" theory, differ fundamentally in their requirement for DSB mobility. To determine whether or not DSB-containing chromosome domains are mobile and can interact, we introduced linear tracks of DSBs in nuclei. We observed changes in track morphology within minutes after DSB induction, indicating movement of the domains. In a subpopulation of cells, the domains clustered. Juxtaposition of different DSB-containing chromosome domains through clustering, which was most extensive in G1 phase cells, suggests an adhesion process in which we implicate the Mre11 complex. Our results support the breakage-first theory to explain the origin of chromosomal translocations.     

外源DNA导入烤烟后D_1代植株根尖细胞有丝分裂的细胞学观察

以抗赤星病烤烟 CV87为供体、感病烤烟 NC89为受体 ,采用子房注射 ( ZZ)、柱头滴加( DZ)和切柱头后滴加 ( QD)等 3种方法 ,将供体总 DNA导入受体。收获的种子发芽 ,得到的是 D1代个体 ,对其根尖及亲本的根尖进行细胞学观察测。结果显示 ,导入外源 DNA能够提高根尖细胞有丝分裂过程中染色体畸变的发生率。 ZZ、 QD和 DZ的 D1代的染色体畸变发生率 (变异率 ) ,细胞水平上较亲本分别提高 5.50 %、18.10 %和 3.80 % ,个体水平上较亲本分别提高 4 .80 %、13.30 %和 2 .4 0 %。畸变的类型以染色体落后为最多 ,其次是染色体桥 ,染色体断片及其他畸变类型出现很少。     

柴油机进气管喷水节油机理研究

本文提出在柴油机进气管上，安装一个特制的不锈钢化水器，用它喷出一定量的雾化水，参与气缸内燃烧，可提高柴油机的性能．根据柴油机的工况，可以控制所需的较佳喷水量．这套系统经过发动机台装试验与拖拉机实用作业试验表明：喷水节油率达到３％以上，标定功率提高０．５ＰＳ，排气温度下降１０～２０℃，ＮＯｘ降低３０ｇｋｇ－１．喷水试验，在拖拉机上进行了５００ｈ的负荷作业，实测小时节油为０．０６ｋｇ／ｈ，拆机检查，零部件磨损正常，无锈蚀，积炭少，油底壳内机油无水。     

Separation of large DNA molecules by contour-clamped homogeneous electric fields

Electric fields can be manipulated by a method in which multiple electrodes are arranged along a closed contour and clamped to predetermined electric potentials. This method may be applied to a broad range of problems in the separation of macromolecules by gel electrophoresis. DNA molecules as large as 2 megabases can be well separated with a contour-clamped homogeneous electric field alternating between two orientations 120 degrees apart. The pattern of separation is independent of position in the gel, which is an advantage over previous methods. DNA less than 50 kilobases can be separated without distortion even at high voltage with a nonalternating contour-clamped homogeneous field. Decreased band broadening in DNA less than 200 bases can be achieved with a contour-clamped inhomogeneous field.     

SAGP基因克隆及反义表达载体的构建

为了研究不同启动子驱动的反义SAGP抑制内源ADP-Glc PPase小亚基编码基因表达的效果,创造适于外源基因表达的突变体,利用RT-PCR方法,由马铃薯块茎cDNA克隆获得ADP-Glc PPase小亚基编码基因(SAGP)的1 821 bp cDNA。核酸数据库检索和序列比对分析表明,已克隆的SAGP基因cDNA推测编码607个氨基酸组成的多肽,其中第312和411的天冬酰氨均突变为丝氨酸。以pCambia为基础,分别构建了CaMV35S和GBSSI块茎启动子驱动的小亚基cDNA反义结构植物表达载体。     

耐碱Mn-SOD基因克隆与真核表达载体的构建

以高产耐碱Mn-SOD产生菌Bacillussp.110-2总DNA为模板,通过PCR扩增得到耐碱Mn-SOD基因,编码一个完整的开放阅读框,共202个氨基酸,含18个碱性氨基酸,包括12个赖氨酸和6个精氨酸。与地衣芽孢杆菌同源性为99%,与极端耐碱芽孢杆菌(Bacillus halodurans)同源性为78.7%。克隆片段酶切后,插入到巴斯德毕赤酵母表达载体pPIC9K中,获得毕赤酵母真核表达载体pPIC9K-SOD。     

大豆总DNA直接导入法培育优势高蛋白玉米材料研究

利用外源总DNA转导的花粉管通道法和经我们发展的减压渗透法将大豆总DNA导入玉米自交系统48-2和S37，在743份后代材料中筛选出8份高蛋白含量变异材料，它们的蛋白质与受体相比提高20%以上。用种子蛋白SDS-PAGE、谷氨酸-草酰乙酸同工酶及RAPD等分析方法对变异材料及其受体进行了鉴定分析。同时也对此8份变异材料的自交后代进行了筛选和S37在遗传上存在明显的差异。在其自交后代中仍有部分株系保持了主蛋白含量特性。此外，48-2的变异材料的植株形态、花药颜色上有明显变化。上述结果初步证明高蛋白特性是可遗传的，这为选育高蛋白玉米新自交系提供了新材料，也为利用外源DNA直接导入技术创造玉米新种质开辟了一条行这有效的新途径。