人淋巴细胞CD2基因5‘侧翼序列的克隆和鉴定

本文报告以ＣＤ２ｃＤＮＡ５＇端的片段作探针,从人Ｔ淋巴细胞基因组文库筛选阳性重组克隆,经限制性内切酶降解和Ｓｏｕｔｈｅｒｎ杂交分析,证明其中一个阳性克隆的插入片段中含ＣＤ２基因５＇侧翼顺序。经插入片段的亚克隆、限制性内切酶图谱及ＤＮＡ序列分析,鉴定出一含转录起始点及其上游序列的４．ｋｂ片段。将此片段中含转录起始点和两个ＤＮａｓｅＩ高敏感位点的２．５ｋｂ片段定向克隆到以虫萤光素酶为报告基因的表达载体     

油菜细胞质雄性不育系叶绿体DNA特异片段的分子克隆

采用高离子浓度缓冲液法分别提取油菜不育系及保持系的叶绿体DNA。经Sepharcse 4B柱层析纯化后,得到具有较高纯度的叶绿体DNA样品。将其分别用Eco RI、Bam HI、HimdHI、PstI和XhoI 5种限制性内切酶酶解,得到5种限制性内切酶图谱。其中除PstI图谱外,其它4种酶谱均显示出明显的两系间叶绿体DNA结构差异。以pBR 322为载体,分别克隆了不育系Bam HI图谱上的3个特异片段。得到的3个克隆,经克隆杂交及电泳分析后,证实分别带有上述3个目的片段。这些特异片段的特性及其与花粉育性的关系尚在研究中。     

两株蓖麻蚕核型多角体病毒DNA同源性的研究

两株不同来源的蓖麻蚕核型多角体病毒(ArscsNPV和ArNPV)经提纯后,使用SDS—苯酚抽提病毒核酸,并使用限制性内切酶EcoRI,BamHI酶解后,用分子杂交方法与缺口平移标记的ArscsNPV-DNA探针杂交,分析了两株蓖麻蚕NPV病毒核酸的同源性。EcoRI酶解的ArNPV-DNA产生8个片段,其中5个片段能与ArscsNPV-DNA探针杂交。BamHI酶解ArNPV-DNA产生7个片段,其中6个片段能与ArscsNPV-DNA探针杂交。结果表明:两株蓖麻蚕NPV之间病毒核酸具有很高的同源性。使用斑点杂交方法分析了ArscsNPV与ArNPV,柞蚕NPV及家蚕NPV之间的核酸同源性,结果表明:ArscsNPV与ArNPV,柞蚕NPV具有同源性。而与家蚕NPV无核酸同源性。     

一种能表现水稻基因组DNA分化特征的重复DNA顺序

用ＤＮＡ 复性动力学方法克隆到一个水稻中度重复顺序。Ｓｏｕｔｈｅｒｎ 杂交、限制性内切酶分析及序列分析资料表明,该重复顺序在水稻基因组中具有串联重复和散布状态两种存在方式。以该ＤＮＡ 片段作探针,用Ｓｏｕｔｈｅｒｎ 杂交方法分析了多种野生稻种和栽培稻品种的基因组分化特征。某些限制性内切酶消化过的水稻ＤＮＡ,其图谱呈现出多达４０ 条以上的杂交带,包括强杂交带和弱杂交带两种类型。重复实验结果证明,强杂交带表现为ＢＢＣＣ染色体组型特异而弱带则在栽培稻各品种间显示出丰富的多态性,表明该重复顺序片段在水稻理论研究和育种实践中可能具有重要意义     

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。     

油菜叶绿体基因组雄性不育特异DNA片段的定位

实验选用油菜湘矮不育系及同核保持系叶绿体DNA为材料,用4种限制性内切酶EcoRⅠ、BamHⅠ、PstⅠ和SmaⅠ完全酶解。在EcoRⅠ酶解的保持系ctDNA图式中,观察到唯一的差异片段E3.2kb。将此片段连接于载体pUC9进行克隆,经克隆杂交及电泳分析鉴定,获得重组子。以rRNA基因为探针,与EcoRⅠ酶解的保持系ctDNA杂交,其中一条阳性带显示在差异片段E3.2。进一步以E3.2片段为探针,与经过限制酶BamHⅠ、EcoRⅠ、salⅠ、BgⅢ、HindⅢ和PstⅠ酶解后的rRNA基因反杂交。根据rRNA基因图谱,这一与不育性有关的E3.2kb片段被定位于距16S rRNA基因5'端+2.0至+5.4kb的前导序列中。此结果表明,这段可能与花粉育性形成有关的片段定位于叶绿体基因组反向重复区。     

人淋巴细胞CD2基因5’侧翼序列的克隆和鉴定

本文报告以ＣＤ２ｃＤＮＡ５’端的片段作探针,从人Ｔ淋巴细胞基因组文库筛选阳性重组克隆,经限制性内切酶降解和Ｓｏｕｔｈｅｒｎ杂交分析,证明其中一个阳性克隆的插入片段中含ＣＤ２基因５’侧翼顺序。经插入片段的亚克隆、限制性内切酶图谱及ＤＮＡ序列分析,鉴定出一含转录起始点及其上游序列的４．０ｋｂ片段。将此片段中含转录起始点和两个ＤＮ（ａｓｅ）Ⅰ高敏感位点的２．５ｋｂ片段定向克隆到以虫萤光素酶为报告基因的表达载体ｐＭＧ３中,并用限制性内切酶对此２．５ｋｂ片段作不同程度缺失,构成一系列突变子。这些重组的表达质粒转染人ＪｕｒｋａｔＴ细胞后,以瞬时表达实验分析各突变子驱动虫萤光素酶基因的表达,结果发现在ＣＤ２基因５’上游具有很弱的启动子活性,初步测定该启动子位于－１．２ｋｂ～－９８ｂｐ域。ＣＤ２基因具有弱启动子、强增强子的特点与Ｔ细胞表面其它抗原分子基因是相似的。     

粪产碱菌(Alcaligenes faecalix)基因文库的构建和nifH基因同源顺序的克隆

采用λ噬菌体置换型载体EMBL4,构建了Alcaligenes faecalis A-15 H1菌株总DNA的基因文库。用Sau3AⅠ限制酶完成部分酶切,取13—20kb大小的片段进行克隆。载体DNA经BamHⅠ和SaiⅠ完全双酶切,左右臂“退火”形成左右臂载体分子后再与外源片段连接。左右臂载体分子与外源片段按照1:1的分子比进行体外连接。用E.coli BHB2688和E.coli BHB2690制备的包装抽提物进行体外包装,所得基因文库效价测定为1.2×10~6 pfu,远远超过理论上所需的库容量。以nif H基因作为探针,经3轮噬菌斑原位杂交,从基因文库中筛选出含有其同源顺序的克隆,并得到了梯度点杂交的验证。对所得重组噬菌体克隆之一进行Southern转移杂交,结果证实,其3.5kb的EcoRⅠ酶切片段为nif H阳性杂交条带。将其克隆到质粒pUC19 DNA上后,转入受体菌JM101中。再次经Southern转移杂交,证明所得重组质粒克隆(pAFH)含有粪产碱菌中的与nifH基因有同源顺序的片段。     

玉米黑粉菌mtDNA的研究 Ⅱ.限制性内切酶酶切图谱

本文报道玉米黑粉菌mtDNA的限制性内切酶酶切图谱。分别将mtDNA的Bam HI各片段制成探针,与mtDNA分别用8种酶酶切后的Southern膜进行杂交,用片段重叠法得出各套片段的排列次序,再将克隆化的Bam HI片段进行第二酶切,按分子量拼排出各酶酶切位点在mtDNA上分布的图谱。此外,片段重叠分析时,还发现玉米黑粉菌mtDNA为环状结构;杂交分析时还发现mtDNA内没有明显的重复序列。DNA总长60.7kb。     

ACS和ACO基因克隆及植物转化

根据已知序列设计PCR引物,分别扩增氨基环丙烷羧酸合酶（ACS）和氨基环丙烷羧酸氧化酶（ACO）基因并克隆到中间载体,经限制性内切酶酶切图谱分析、部分序列分析以及Southern印迹鉴定后,将两个基因单个或相互串联后反向亚克隆至植物表达载体pBI121。经农杆菌LBA4404转化番茄子叶,在抗性培养基上得到8株ACS基因反义转化的生根小苗以ACS基因的酶切小片段作为探针,经Southern印迹分析,证明获得了两株阳性转化株。     

Tn5cos: a transposon for restriction mapping of large plasmids using phage lambda terminase.

A method for the rapid restriction mapping of large plasmids has been developed. A 400-bp fragment of phage lambda DNA containing the cos region has been inserted into Tn5. After in vivo transposition of this Tn5cos element into the plasmid of choice, the plasmid is isolated and linearized at its cos site with phage lambda terminase (Ter). Such Ter linearization was about 70% efficient. After partial digestion of the linear molecules with the appropriate restriction enzyme, the products are selectively labelled at the right or left cohesive phage lambda DNA termini by hybridization with digoxygenin (DIG)-11-dUTP-labelled (using terminal transferase) oligodeoxyribonucleotides complementary to the single-stranded cos ends. After pulsed field gel electrophoresis, the labelled fragments are visualized in the dried gel using a DIG-detection kit. The restriction map can be directly determined from the 'ladder' of partial digestion products.     

In vivo DNA cloning with a mini-Mu replicon cosmid and a helper lambda phage

A mini-Mu bacteriophage, containing the cohesive-end packaging site (cos) from a lambda-phi 80 hybrid phage, a high-copy-number plasmid replicon, and a kanamycin-resistance gene for independent selection, was constructed to clone genes in vivo. This mini-Mu element can be derepressed to transpose at a high frequency. DNA segments that become flanked by copies of this mini-Mu element in the same orientation can be packaged by a helper lambda phage. The resulting lambda lysate can be used to infect recipient cells where the injected DNA can circularize by annealing at the cos termini. Drug-resistant transductants obtained carry the mini-Mu-replicon cosmid element with inserts of different nucleotide sequences. These are analogous to recombinant DNA clones generated in vitro with restriction endonuclease cutting and ligase joining reactions replaced by the Mu transposition process. Clones of particular genes were isolated by their ability to complement specific mutations. Both recA+ and recA- recipient cells can be used with equal efficiency. Clones obtained with a helper lambda phage require the presence of the cos site in the mini-Mu replicon. They carry larger inserts than those isolated with the same mini-Mu element and Mu as a helper phage. The mini-Mu replicon-cosmid bacteriophage contains a lac-gene fusing segment for isolating fusions of lac operon DNA to gene control regions in the cloned sequences. Independent clones of a particular gene can be used to prepare a restriction map of the gene and its flanking regions.     

A phosphate group at the cos ends of phage lambda DNA is not a prerequisite for in vitro packaging: an alternative method for constructing genomic libraries using a new phasmid vector, lambda pGY97

It is shown here that the phosphate groups at the cos ends of phage lambda DNA are not a prerequisite for in vitro packaging. Molecules with phosphatase-treated cos ends are packaged in vitro as efficiently as native lambda DNA. This observation can be used for an alternative strategy to improve the efficiency of gene library construction, since cos-cos ligation decreases in vitro encapsidation and infectivity. Dephosphorylated cos ends and a new phasmid vector lambda pGY97 have been used to construct a representative gene bank of alfalfa in a Mcr- (5-methylcytosine restriction deficient) Escherichia coli host strain. These recombinant clones can be propagated as phages or more conveniently as plasmids in recA- E. coli, to prevent possible homologous recombination events between repetitive sequences of the insert that would otherwise interfere with clone stability. The 5-19-kb inserts can be easily recloned as plasmids from the recombinant phasmids with simple EcoRI digestion and re-ligation. This observation also implies that the construction of gene libraries in cosmid vectors can be made more efficient if cos-cos ligates were cleaved by lambda terminase just before in vitro packaging.     

Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones.

We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.     

Analysis of cosmids using linearization by phage lambda terminase

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage λ terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested' with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage λ (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.     

Enhanced recovery and restriction mapping of DNA fragments cloned in a new lambda vector.

In this paper we describe a modification to the lambda vector EMBL3 which greatly expedites the construction of restriction maps of cloned DNA sequences. In the modified vector, EMBL3cos, all the phage coding sequences are placed to the right of the cloning sites so that the left cohesive end is separated by only 200bp, rather than 20kb (as in conventional lambda vectors), from the inserted DNA fragment. We show that reliable restriction maps can be rapidly constructed from partial digests of clones made in this vector by labelling the left cohesive end with a complementary 32P-labelled oligonucleotide. In addition, we quantify the restriction of clones containing human DNA by the McrA and McrB systems of E. coli and show that the use of Mcr- plating strains can increase the yield of recombinant phage up to tenfold, to give cloning efficiencies of greater than or equal to 10(7) pfu/microgram of human DNA.     

Composite human VK genes and a model of their evolution.

A phage library and two cosmid libraries were screened for human VK genes. Two recombinant phage and four cosmid clones were analysed in detail by restriction mapping and sequencing. Each one contained a single VKI sequence. Two of these six sequences are potentially functional VK genes and four are pseudogenes. Two pseudogenes derived from different genomic DNAs are highly homologous and are therefore either allelic variants or the products of a recent duplication event. Comparisons of our sequences with all fully determined human VKI amino acid and DNA sequences reveal identical segments which at first sight appear like minigenes. But these segments do not coincide with the subregions and some of the segments include both, framework and complementarity determining regions (FR, CDR, ref. 2). The findings may be explained by an evolutionary model generating composite genes by gene conversion and selection.     

Efficient simplified cosmid cloning: construction and characterization of cosmid vectors that carry the two cohesive end sites of lambda phages arrayed in tandem

We constructed a series of cosmid vectors that carry the two cohesive end sites (cos) of lambda phage, arrayed in tandem, which enabled us to clone fragments of genomic DNA of up to 50 kb without a vector background. An equimolar mixture of the left and right vector arms of equal length was prepared from the vector DNA, simply by treating the DNA sequentially with three enzymes, restriction enzyme PvuII, alkaline phosphatase, and restriction enzyme BamHI (or BglII), without purification by agarose gel electrophoresis. After phenol extraction and ethanol precipitation, the equimolar mixture of the vector arms, which carried a single cos oriented from left to right, was directly ligated with insert DNA without further manipulation. We established conditions for cosmid cloning, using two kinds of DNA fragment of 40-50 kb, prepared from mouse L cell genomic DNA, as insert DNAs, namely, three cloned BamHI fragments and Sau3AI fragments, size-selected on a sucrose density gradient. The most important parameters affecting the cloning efficiency were the quality of the insert DNA and the molar ratio of the insert and vector arms. We achieved cloning efficiencies of 3.6 X 10(6)-1.3 X 10(7) colony forming units (cfu)/micrograms of insert DNA and 1.7 X 10(5)-1.0 X 10(6) cfu/micrograms of insert DNA, using the cloned BamHI fragments and the Sau3AI fragments, respectively. We examined more than 5000 clones and found that they all contained insert DNA.     

Restriction mapping of recombinant lambda DNA molecules using pulsed field gel electrophoresis

We have integrated pulsed field gel electrophoresis with the partial digestion strategy of Smith and Birnstiel (1976, Nucleic Acids Res. 3,2387-2398) to generate a rapid and accurate method of restriction endonuclease mapping recombinant lambda DNA molecules. Use of pulsed field gels dramatically improves the accuracy of size determination and resolution of DNA restriction fragments relative to standard agarose gels. Briefly, DNA is partially digested with restriction enzymes to varying extents and then hybridized with a radiolabeled oligonucleotide which anneals specifically to one of the lambda cohesive (cos) ends, effectively end labeling only those digestion products containing that cos end. In this study, we have used an oligonucleotide hybridizing to the right cos end. DNA is then fractionated by pulsed field gel electrophoresis, the gel dried down, and cos end containing fragments visualized by autoradiography. Fragment sizes indicate the distances from the labeled cos end to each restriction site for the particular restriction enzyme employed. This procedure requires only minimal quantities of DNA and is applicable to all vectors utilizing lambda cos ends.