新型生物催化剂——核酶

起酶作用的RNA和DNA正不断被发现.本文说明酶性DNA和RNA概念的确立过程以及其重要的理论和实际意义.     

真核生物DNA聚合酶δ的研究现状

DNA聚合酶δ(DNA polymerase δ)是真核生物DNA复制的主要复制酶,同时还参与DNA修复,对保持真核生物基因组的结构完整性和遗传稳定性具有重要作用.对DNA聚合酶δ蛋白功能活性及其基因表达机制的研究因受技术上的限制而未能深入研究.由于其重要的生物学功能,目前引起人们的更多关注和重视.文中就该酶的生物学功能、亚基组成、核心酶的分子表达调控以及与其他蛋白相互作用等方面对国内外DNA聚合酶δ的研究进行简要综述.     

DNA分子标记与动物育种

论述了DNA分子标记的分类以及限制性酶切片段长度多态性、随机扩增多态性DNA、扩增片段长度多态性、简单重复序列、单核苷酸多态性等5种主要DNA分子标记的基本原理和优缺点。同时，还分析了它们在动物遗传育种中应用情况和发展前景。     

茶毛虫核型多角体病毒DNA性质的研究

本文报道了对茶毛虫核型多角体病毒蒙山株系(EpNPV-M)核酸性质研究的结果。EpNPV-M含双链DNA分子,含量为6.85μgDNA/mgPIB,提纯的DNA具典型的紫外线吸收特性,琼脂糖凝胶电泳证明DNA分子是大小均一的。DNA的(G+C)％为36.6,限制性片段积加法测得该DNA分子量为75.61×10~6道尔顿,109.57Kb,电镜法测得DNA分子长度为35.8μm,相当于分子量为74.1×10~6道尔顿,107.4Kb.电镜观察证实该病毒含有一些超螺旋环状DNA分子。建立了三种限制性内切酶对该DNA的酶切图谱。三种酶的酶解片断数为:EcoRI,27个;BglⅡ,15个;BamHI,9个。     

中国棉铃虫核多角体病毒(Heliothis armigera NPV)VHA273毒株DNA的酶切分析与比较研究

VHA273毒株原为MNPV型,经纯系中国棉铃虫扩增而得出约10％的SNPV.高度纯化两型多用体,温和碱解后,酚法抽提SNPVDNA和MNPVDNA.限制性内切酶BglⅡ,BamHI,EcoRI和HindⅢ分别平行酶切两种DNA,依次均得出11,8,14和13条电泳带．比较分析DNA的酶切电泳图谱发现,经同一种限制性内切酶酶切,SNPVDNA与MNPVDNA不仅电泳片段数相同,电泳带位也-一对应．讨论提出：该SNPVDNA与MNPVDNA在分子、亚分子水平上是一致的．     

硅藻DNA片段在表达质粒pMZR中再克隆的研究

用部分酶切法切开表达质粒 pMZR 中一个 HindⅢ位点后,插入了一个带 HindⅢ粘性末端的1kb 硅藻 DNA 片段重组 DNA 分子转化 E.coli LE392,对转化子中的重组 DNA 分子用酶切分析后,鉴定了1kb DNA 片段插入 pMZR 的位置。     

耐热FD DNA多聚酶在PCR中的应用条件

探索了用于多聚酶链式反应(PCR)的FD DNA多聚酶的最适反应条件在100μl的反应液中含有25 mmol/L的Tris-HCl(pH8.0),25 mmol/L的(NH_4)_2SO_4,1.5 mmol/L的MgCl_2,200 μg/ml的明胶,dNTP各200 μmol/L,模板量为3.3×10～(-2)Pg,引物量各为290 nmol/L,1～2 U的耐热FD DNA多聚酶,30次循环反应的系统为通用PCR最适反应系统。应用这一反应系统,混迹于大量DNA分子中的特定DNA片段可借助于琼脂糖凝胶电泳专一性地、快速而有效地检出。     

黄瓜线粒体中DNA类质粒的分离与鉴定

应用琼酯糖凝胶电泳、S1酶处理及电镜技术，从黄瓜线粒体中分离并鉴定了三种环形DNA类质粒，依其分子由大到小，分别称之为pC1、pC2和pC3。以类质粒凝胶电泳带的荧光扫描值与DNA类质粒的分子长度之比作为类质粒拷贝数的参照值，对三种类质粒进行荧光扫描分析，结果表明pC3拷贝数明显高于pC1和pC2；pC2的拷贝数略高于pC1。     

多酶催化剂的制备及其应用

在生物催化中，很多的复杂反应都需要2个或者多个酶的级联反应催化得到，近几年多酶生物催化开始逐渐取代单酶和细胞发酵，成为酶工程发展的一个热点研究方向。本文对多酶催化剂的制备形式和原理及超分子酶在级联催化中的应用进行了综述。其中超分子酶的制备技术主要包括非定向的固定化技术和定向的基因融合技术、纤维小体支架技术和DNA支架技术等。     

体外定向分子进化的新策略及新应用

体外定向分子进化是发现和改造生物活性分子的重要方法,提供了一种高效的获得多样性的方法。DNA改组(DNA shuffling)是重要的体外分子进化技术,结合高通量筛选能够改造许多重要的医药、工业、环境保护等方面的商业酶。近年来,许多体外分子进化的新策略和新方法层出不穷,得到了良好的发展和应用,其中有代表性的11种是DNA家族改组(DNA family shuffling),部分基因片段改组、单链DNA家族改组(SSDNAs)、简并引物基因改组(DOGS)、基因组改组(Genome Shuffling)、瞬时模板的随机嵌合(RACHITT)、单向引物的随机重组(MURA)、自我复制(CSR)改组、易错环行扩增(error-prone RCA)、基于遗传密码随机切除(COBARDE)、核酸内切酶V(endonuclease V)替代核酸内切酶DNaseI等。     

水稻基因整合平台系统供体质粒pURKMT的构建

提高水稻产量,改良稻米品质是育种学家广泛研究的课题．随着现代生物技术的发展,水稻已成为植物基因工程的重要研究对象．许多实验室已成功地建立了一系列供外源基因转化水稻的系统．但是这些转化系统主要应用Ｔｉ质粒衍生的载体,通过Ｔ－ＤＮＡ左右两端的序列将目的基...     

Evolutionary relationships of human populations from an analysis of nuclear DNA polymorphisms

The genetic relationships of human populations have been studied by comparing gene frequency data for protein and blood-group loci of different populations. DNA analysis now promises to be more informative since not only do the DNA coding sequences have more variation than their corresponding proteins but, in addition, noncoding DNA sequences display more extensive polymorphism. We have now studied the frequency of a group of closely linked nuclear DNA polymorphisms (haplotypes) in the beta-globin gene cluster of normal (beta A) chromosomes of individuals from eight diverse populations. We have found that all non-African populations share a limited number of common haplotypes whereas Africans have predominantly a different haplotype not found in other populations. Genetic distance analysis based on these nuclear DNA polymorphisms indicates a major division of human populations into an African and a Eurasian group.     

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta.

Xeroderma pigmentosum variant (XP-V) is an inherited disorder which is associated with increased incidence of sunlight-induced skin cancers. Unlike other xeroderma pigmentosum cells (belonging to groups XP-A to XP-G), XP-V cells carry out normal nucleotide-excision repair processes but are defective in their replication of ultraviolet-damaged DNA. It has been suspected for some time that the XPV gene encodes a protein that is involved in trans-lesion DNA synthesis, but the gene product has never been isolated. Using an improved cell-free assay for trans-lesion DNA synthesis, we have recently isolated a DNA polymerase from HeLa cells that continues replication on damaged DNA by bypassing ultraviolet-induced thymine dimers in XP-V cell extracts. Here we show that this polymerase is a human homologue of the yeast Rad30 protein, recently identified as DNA polymerase eta. This polymerase and yeast Rad30 are members of a family of damage-bypass replication proteins which comprises the Escherichia coli proteins UmuC and DinB and the yeast Rev1 protein. We found that all XP-V cells examined carry mutations in their DNA polymerase eta gene. Recombinant human DNA polymerase eta corrects the inability of XP-V cell extracts to carry out DNA replication by bypassing thymine dimers on damaged DNA. Together, these results indicate that DNA polymerase eta could be the XPV gene product.     

DNA typing from single hairs

The characterization of genetic variation at the DNA level has generated significant advances in gene and disease mapping, and in the forensic identification of individuals. The most common method of DNA analysis, that of restriction fragment length polymorphism (RFLP), requires microgram amounts of relatively undegraded DNA for multi-locus typing, and hundreds of nanograms for single-locus comparisons. Such DNA frequently cannot be obtained from forensic samples such as single hairs and blood stains, or from anthropological, genetic or zoological samples collected in the field. To detect polymorphic DNA sequences from single human hairs, we have used the polymerase chain reaction (PCR), in which specific short regions of a gene can be greatly amplified in vitro from as little as a single molecule of DNA. We have detected genetically variable mitochondrial and nuclear DNA sequences from the root region of shed, as well as freshly-plucked, single hairs; mitochondrial DNA (mtDNA) sequences have been detected in a sample from a single hair shaft. We have used three different means of DNA typing on these samples: the determination of amplified DNA fragment length differences, hybridization with allele-specific oligonucleotide probes, and direct DNA sequencing.     

DNA purification and genetyping： Based on multifunc-tional nanobeads

In this report, a universal protocol for extract-ing genomie DNA from whole blood, saliva, and bacterialculture by using magnetic nanobeads as solid-phase absor-bents was presented. The enrichment of target cells and ad-sorption of DNA have been functionally integrated onto thesurfaces of the earboxyl-modified magnetic nano-beads, andthe DNA segments bound on the surface of the beads can bedirectly used as PCR templates to amplify a target geue. ThePCR products were applied to an oligonueleotide array toperform gene typing. The protocol proves to be simple, rapid,biologically and chemically nonhazardous, and promising forthe mierofabrieation of DNA preparation chip.     

白菜型油菜核育性相关基因片段的克隆与序列分析

通过ＲＡＰＤ标记,所获得的与育性基因紧密连锁的基因片段进行克隆与序列分析,结果表明,该育性基因片段为与油菜小孢子发育早期ＢＰ４调控基因５８％同源,并含有一ＭＡＰＤＳ盒高度同源的保邓列。Ｓｏｕｔｈｅｒｎ杂交结果表明该基因为单拷贝基因。     

Expression of the E. coli uvrA gene is inducible

UvrA+-dependent excision repair is one of the most important systems in Escherichia coli for repairing UV-induced pyrimidine dimers and a variety of other forms of DNA damage. The uvrA protein acts in conjunction with the uvrB and uvrC gene products to introduce a nick at the of a DNA lesion and thus initiate the repair process. We have recently used the Mud(Ap, lac) operon fusion vector to identify a set of genes whose expression is induced by DNA damage. One Mud(Ap, lac) insertion mapped at the uvrA locus and made the cells sensitive to UV light. In this fusion strain, beta-galactosidase expression was induced by DNA-damaging agents in a recA+lexA+-dependent fashion. We were surprised by this result because uvrA+-dependent excision repair is observed both in cells in which protein synthesis has been inhibited and in recA- and lexA- cells, findings which have led to the conclusion that the uvrA gene product is constitutively expressed and not under the control of the complex recA+lexA+ regulatory circuitry (see below). We have investigated this possibility further and describe here the generation and characterization of a set of fusions of the lac genes to the promoter of the uvrA gene. We confirm that the uvrA gene product is induced by DNA damage in a recA+lexA+-dependent fashion.