几种限制性核酸内切酶的纯化

通常所说的限制性内切酶或限制酶实际上是指Ⅱ类脱氧核糖核酸内切酶。此类酶能够识别双链DNA分子上的特定核苷酸顺序,并在这特定的顺序处将DNA的双链切断。各种不同的限制酶都有它自己的识别顺序,所产生的片段的末端电不尽相同。自从Smith于1970     

大鼠肝含5~1端氨甲酰磷酸合成酶Ⅰ基因的克隆

为研究氨甲酰磷酸合成酶Ⅰ(CPS Ⅰ)基因表达的调控,我们根据CPS Ⅰ编码1—10氨基酸的mR NA顺序,合成一个30碱基的寡聚核苷酸。以它作为探针从大鼠肝基因库中筛选出一个阳性克隆,λ10。对其中4.5kb插入片断的内切酶谱及部分核苷酸序列测定证明它确含5′上游CPS Ⅰ基因DNA序列。     

分子生物学技术讲座（二）——分子克隆中所用的酶

限制性内切酶及其它DNA与RNA修饰酶是分子克隆技术的基本工具。1限制性内切醉和DNA甲基化酶限制性内切酶能特异地结合于一段被称为限制性内切酶识别序列的特定的DNA序列或附近的序列,并在此切割DNA双链。它可以分成3类：Ⅰ类和Ⅲ类在同一蛋白分子中兼有修饰（甲基化）作用及依赖于ATP的限制（切割）活性。Ⅰ类酶结合于识别序列,但随机切割DNA;Ⅲ类酶能在识别序列位点切割DNA。在分子克隆中1类和皿类酶都不常用。D类限制一修饰系统限制性内切醇和修饰（甲基化）酶不在同一蛋白分子中,限制性内切酶能专一地切割识别序列,并不依…     

Hin P_1I底物片段d-ATGCGCAT的固相磷酸三酯法合成

本文报道用固相磷酸三酯法化学合成了限制性内切酶HinP_1Ⅰ的底物片段d-ATGCGCAT。固相载体采用β-丙氨酰基聚苯乙烯树脂(2%交联度)。合成的产物经顺序分析得到证明,同时能被限制性内切酶HinP_1Ⅰ识别并在专一位置上水解。     

60Co-γ射线辐照玉米种子对基因组DNA分子的影响

以不同剂量的60Co-γ射线辐照玉米种子,对各处理组分单株,采用CTAB抽提法提取幼叶组织基因组DNA,并对基因组DNA进行酶切,研究辐照对基因组DNA分子的损伤效应。结果表明:辐照剂量不同,得到的基因组DNA分子在各处理组间存在差异,当辐照剂量>200 Gy时,差异更为明显;辐照不仅能引起DNA分子链的断裂,还能改变核苷酸链上的碱基位置和顺序,使酶切位点发生改变。实验还对同一辐照剂量处理下的不同单株幼叶基因组DNA进行了重复实验,结果是一致的。     

用微型计算机在TSr(Bg1Ⅱ)-1核苷酸顺序中寻找相似序列

<正> 为了在TSr(Bgl Ⅱ-1)核苷酸顺序中寻找有无类似α—顺序的冷点区顺序,我们在微型电子计算机上编制了查找相似序列顺序(WSS程序)。本程序的特点是运行速度快,无重复扫描,可自行选择欲查找的相似百分比和百分比精度,它不仅适用于寻找内切酶的酶切点,而且可以在相当长度的已测序DNA顺序中快速准确地检出碱基位置和数量发生随机变异的DNA相似片段,并直接计算、打印出相似百分比值。     

结构特异性核酸酶FEN-1的功能和结构

FEN-1(flap endo/exonuclease)是一种结构特异性核酸酶,它能识别特定的DNA分叉结构,并切除含有游离5′端的单链核酸. 在DNA复制过程中,FEN-1通过其外切酶、内切酶活力去除了冈崎片段前端RNA引物的最后一个核糖核苷.在DNA修复中,FEN-1以其内切酶活力参与了损伤碱基的修复过程.FEN-1基因含有两个保守区和一个PCNA结合区.     

限制性酶

核酸限制性内切酶是识别双链DNA中专一序列的酶,主要从原核生物中提取。限制性内切酶可分为三类:第Ⅰ和第Ⅲ类酶在同一旦白中具有修饰作用(甲基化作用)和需要ATP的限制性裂解的活性。这两种酶都识别底物DNA中非甲基化的序列,不过第Ⅰ类酶的作用是随机的,第Ⅲ类酶则切DNA的专一位点。     

DNA长链分子上核酸内切限制酶识别位置的预测

本文报道了我们自编的能在TRS-80(Ⅰ)型微处理机上执行的用于构造DNA分子的限制性内切酶酶谱的计算机程序。并给出了73种不同专一性的限制性内切酶在噬菌体M13 DNA(6407个核苷酸残基)上的识别位置(或切点位置)处理结果。该程序的最大优点是被检索的核酸序列,其长度在原则上是不受限制的(即不受计算机内存容量的限制),用户可根据需要对输入的序列进行任意的插入和删改。     

嗜热古菌Sulfolobus acidocaldarius核酸内切酶V的重组表达与酶学特征

[目的] 表达纯化嗜酸嗜热硫化叶菌（Sulfolobus acidocaldarius）的核酸内切酶V （Saci_0544）,对其核酸内切酶活性及酶学特征进行探究。[方法] 将Sulfolobus acidocaldarius核酸内切酶V （SacEndoV）在大肠杆菌中进行重组表达,经亲和层析纯化得到目标蛋白;利用带有不同类型损伤的寡核苷酸作为底物,结合变性聚丙烯酰胺凝胶电泳技术,鉴定SacEndoV对相应损伤寡核苷酸底物的剪切活性。[结果] SacEndoV特异性剪切含脱氧肌苷（Deoxyinosine）的损伤DNA底物,明显偏好单链DNA底物。SacEndoV在70-95℃温度范围内酶活性高,酶活性依赖于二价金属离子,Mg²⁺为最佳辅助离子,其最佳反应pH为7.5-8.0,高于200 mmol/L的NaCl会明显抑制其剪切活性。损伤DNA中脱氧肌苷3''端相邻的脱氧核糖核苷酸的结构完整性对于SacEndoV识别并剪切相应底物具有重要影响,脱氧肌苷3''端无碱基位点的存在使得SacEndoV不能够切断损伤DNA。此外,经测定SacEndoV对于含肌苷的损伤RNA底物具有剪切活性。[结论] 本研究证实SacEndoV是一种典型的核酸内切酶V,对含脱氧肌苷的损伤DNA具有特异性的内切酶活性,推测其在Sulfolobus acidocaldarius体内参与脱氧肌苷的切除修复。     

Substrate specificity of restriction endonuclease VspI

The recognition sequence and cleavage point of restriction endonuclease VspI have been determined as 5'-AT decreases TAAT. This enzyme is not isoschizomer of any known restriction endonucleases. DNA pBR322 contains a single VspI recognition sequence in position 3539. Therefore this enzyme may be used for cloning DNA in the VspI site in AmpR-gene of pBR322.     

The site-specific deletion in plasmid pBR322

The formation of a deletion derivative of plasmid pBR322, designated pBR322 delta 1, was observed during cloning of various eukaryotic DNAs, when the BamHI site of the plasmid vector was used for construction of the recombinant molecules. The restriction analysis of six independently isolated pBR322 delta 1 plasmids allowed establishment of their complete identity. Similar deletion derivatives were also formed as a result of transformation of Escherichia coli cells by the linear form of vector pBR322 produced by BamHI cleavage, but not by SalI or HindIII. The endpoints of the deletion in one of the pBR322 delta 1 plasmids occurred at positions 375 and 16666 bp from the EcoRI site, as determined by sequence analysis. Formation of pBR322 delta 1 is most probably due to site-specific recombination between the sequence in the 1666-1670 bp region and the BamHI end of the linear pBR322 molecule. THe deletion was not controlled by the recA system of the host bacteria.     

A set of synthetic oligodeoxyribonucleotide primers for DNA sequencing in the plasmid vector pBR322

Seven oligonucleotide primers complementary to the plasmid vector pBR322 at positions adjacent to five of the unique restriction endonuclease cleavage sites (EcoRI, HindIII, BamHI, SalI and PstI) have been chemically synthesized. The polarity of the primers is such that any DNA inserted at one or a combination of two of the above restriction sites may be sequenced by the chain termination method using one of the synthetic DNA primers. One of the primers for sequencing inserts at the PstI site of pBR322 is also complementary to the M13 phage vector designated bla6. This set of universal primers is useful for rapid sequence determination of DNA cloned into pBR322 or M13bla6.     

Alteration of the specificity of PvuII restriction endonuclease.

The restriction endonuclease PvuII which cleaves the sequence CAGCTG, at the position indicated by the arrow, was found to decrease its substrate specificity in the presence of organic solvents. Thirty-three sites, that we have named PvuII sites, were identified on the nucleotide sequence of pBR322 DNA. The new recognition sequences cleaved in pBR322 DNA, at the positions indicated by the arrows, were shown to be AAGCTG, GAGCTG, CNGCTG, CANCTG, CAGNTG, CAGCNG, CAGCTC and CAGCTT. (TAGCTG and the complementary sequence CAGCTA are not present in pBR322 DNA). From these recognition sequences, we deduced that PvuII activity recognizes and cleaves degenerate sequences which differ from the standard PvuII sequence CAGCTG at only one of the recognition site. Any substitution can occur at any one of the six positions in the hexanucleotide sequence. The optimum incubation medium for PvuII activity was found to be: 10-50 mM Tris-HCl, pH 8.5, 12-15 mM MgCl2, 50 mM NaCl, 10% ethanol + 10% dimethylsulfoxide (DMSO).     

Equilibrium binding of four anthracyclines to nucleic acids: thermodynamic properties and sequence selectivity.

The thermodynamic parameters of the interaction of the two anthracyclines 13-dihydrodaunomycin and marcellomycin with calf thymus DNA were examined by equilibrium binding studies. Enthalpy and entropy changes of the binding of both drugs show salt dependence profiles that cannot be rationalized by the polyelectrolyte theory. This feature is common to other anthracycline compounds. The nucleotide sequence binding preferences of daunomycin, adriamycin, 13-dihydrodaunomycin and marcellomycin have been studied by monitoring the degree of protection from cleavage by restriction endonucleases of linearized pBR322. Differential protection of pBR322 DNA against the cleavage of Bgl I and Ava II suggests that these drugs recognize changes in the sequences near the enzyme recognition site. Alterations of the electrophoretic restriction pattern of pBR322 in the presence of anthracyclines are dependent on time and on concentration. These results are discussed in relation to the existence of nucleotide sequences with different affinity for these drugs.     

Ethidium binding sites on plasmid DNA determined by photoaffinity labeling

Photoaffinity labeling of pBR322 with ethidium monoazide (8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride) was used to provide evidence for the sequence specificity of ethidium binding to native DNA. DNA-drug interactions were examined at concentrations of eight covalently bound ethidium drugs per molecule of pBR322 (4363 base pairs). Restriction enzyme cutting was blocked by the covalent binding of a drug molecule at (or near) the enzyme recognition sequence. This phenomenon was observed with all restriction enzymes tested and was not limited to specific regions of the pBR322 molecule. Double-digestion experiments indicated that a drug molecule may bind 2 to 3 base pairs outside the recognition sequence and still block restriction enzyme digestion. Intact plasmid was treated with [3H]ethidium monoazide and digested with restriction enzymes. The amount of covalently-linked ethidium analog was quantitated for different restriction fragments and the G-C content of each fragment was determined from the DNA sequence. In approximately half of the fragments the drug appeared to preferentially bind at a G-C base pair. However, no preference for specific sequences such as 5'-C-G-3' was detected, as had been suggested by previous modeling studies with ethidium bromide. The other fragments were located in specific map regions of the plasmid and did not bind drug with a strict dependence on GC content suggesting that binding specificity may depend on more than one structural feature of the DNA.     

Cloning of soybean leghemoglobin structural gene sequences synthesized in vitro.

Double-stranded soybean leghemoglobin DNA was synthesized from leghemoglobin mRNA isolated from soybean nodules. The dsDNA was inserted into the Bam H1 site of plasmid pBR322 using the poly-dAT-joiner method. A cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The restriction cleavage map and the DNA sequence of a selected part of the inserted DNA are in complete accordance with the amino-acid sequence of soybean leghemoglobin.     

Site and sequence specificity of the daunomycin-DNA interaction

The site and sequence specificity of the daunomycin-DNA interaction was examined by equilibrium binding methods, by deoxyribonuclease I footprinting studies, and by examination of the effect of the antibiotic on the cleavage of linearized pBR322 DNA by restriction endonucleases PvuI and EcoRI. These three experimental approaches provide mutually consistent results showing that daunomycin indeed recognizes specific sites along the DNA lattice. The affinity of daunomycin toward natural DNA increases with increasing GC content. The quantitative results are most readily explained by binding models in which daunomycin interacts with sites containing two adjacent GC base pairs, possibly occurring as part of a triplet recognition sequence. Deoxyribonuclease I footprinting studies utilizing the 160 base pair (bp) tyrT DNA fragment and 61 and 53 bp restriction fragments isolated from pBR322 DNA further define the sequence specificity of daunomycin binding. Specific, reproducible protection patterns were obtained for each DNA fragment at 4 degrees C. Seven protected sequences, ranging in size from 4 to 14 bp, were identified within the tyrT fragment. Relative to the overall tyrT sequence, these protected sequences were GC rich and contained a more limited and distinct distribution of di- and trinucleotides. Within all of the protected sequences, a triplet containing adjacent GC base pairs flanked by an AT base pair could be found in one or more copies. Nowhere in the tyrT fragment did that triplet occur outside a protected sequence. The same triplet occurred within seven out of nine protected sequences observed in the fragments isolated from pBR322 DNA. In the two remaining cases, three contiguous GC base pairs were found. We conclude that the preferred daunomycin triplet binding site contains adjacent GC base pairs, of variable sequence, flanked by an AT base pair. This conclusion is consistent with the results of a recent theoretical study of daunomycin sequence specificity [Chen, K.-X., Gresh, N., & Pullman, B. (1985) J. Biomol. Struct. Dyn. 3, 445-466]. Adriamycin and the beta-anomer of adriamycin produce the same qualitative pattern of protection as daunomycin with the tyrT fragment. Daunomycin inhibits the rate of digestion of pBR322 DNA by PvuI (recognition sequence 5'-CGATCG-3') to a greater extent than it does EcoRI (recognition sequence 5'-GAATTC-3'), a finding consistent with the conclusions derived from our footprinting studies. Our results, as a whole, are the clearest indication to date that daunomycin recognizes a specific DNA sequence as a preferred binding site.     

Cytokinins do not noticeably change the methylation of adenine residues of DNA in cultured Escherichia coli B

6-Benzylaminopurine (6-BAP) (1 mg/ml) does not influence the growth of E. coli B cell cultures or the number of [8-14C] labeled N6-methyladenine (m6A) residues in the total DNA [(100.m6A/(A x m6A) = 1.7]. The growth of bacterial cells in the presence of adenine or cytokinins (6-BAP, kinetin, zeatin) (1 mg/ml) was unaccompanied by significant changes in the intracellular content of plasmid pBR 322. The mode of restriction by endonuclease Cfu I hydrolyzing the Gm6ATC site of plasmids pBR 322 from E. coli B cells grown in the presence of adenine or one of the above-mentioned cytokinins is identical. These plasmids also have identical restriction products Mbo I or Sau 3AI. Thus, the cytokinins under study do not markedly affect the methylation of adenine residues in total DNA of E. coli B cell cultures and the GATC sequence in plasmids pBR 322 isolated from these cells.