Host-controlled Restriction of T-even Bacteriophages: Relation of Four Bacterial Deoxyribonucleases to Restriction

Escherichia coli strains B and K-12, which restrict growth of nonglucosylated T- even phage (T(*) phage), and nonrestricting strains (Shigella sonnei and mutants of E. coli B) were tested for levels of endonuclease I and exonucleases I, II, and III, by means of in vitro assyas. Cell-free extracts freed from deoxyribonucleic acid (DNA) were examined with three substrates: E. coli DNA, T(*)2 DNA, and T2 DNA. Both restricting and nonrestricting strains had comparable levels of the four nuclease activities and had similar patterns of preference for the three substrates. In addition, mutants of E. coli B and K-12 that lack endonuclease I were as effective as their respective wild types in restricting T(*) phage.     

Characterization of Temperate Bacteriophages of Bacillus subtilis by the Restriction Endonuclease EcoRI: Evidence for Three Different Temperate Bacteriophages

Temperate bacteriophages of Bacillus subtilis were characterized according to host range and digestion of the bacteriophage genome by endonuclease EcoRI. The three bacteriophages, 3T, SPO2, and 105, were all heteroimmune, and the DNA digests showed dissimilar patterns by agarose-ethidium bromide gel electrophoresis.     

Disruption of T-even Bacteriophages by Dimethyl Sulfoxide

Dimethyl sulfoxide (DMSO) disrupted T-even bacteriophages as well as lambda bacteriophage. The component substructures of T2L, T4B01, or T6, in particular heads, were readily isolated after treatment with 67% DMSO (v/v). In contrast, concentrations of DMSO above 50% not only separated heads from tails of bacteriophage lambda but led to degradation of the lambda heads. Examination of the isolated free heads of T-even bacteriophage indicated that a distinct neck substructure was attached to one apex of the head. On some free tails a similar neck substructure was also found at the proximal end of the sheath. The dimensions of this neck substructure were found to be about 130 by 180 A; by virtue of its size and morphological attachment to the free heads, it was concluded that this was a distinct substructure and not an extension of the tail tube.     

鱼类基因组结构研究——Ⅰ.染色体的限制性内切酶显带

本文报道了1种新的鱼类染色体研究方法——限制性内切酶显带技术。我们应用限制性内切酶Bsp631等分别对黄鳝和长春鳊的染色体进行显带处理,结果表明,Bsp631能使这两种鱼的染色体发生稳定的带纹分化:适度的处理产生多重的G-带状带型,而过度的处理则产生特殊的限制性内切酶抗性带型。根据显带结果分析,我们对鱼类染色体限制性内切酶显带的可行性和实用价值进行了讨论。     

Virus-Associated Nucleases: Evidence for Endonuclease and Exonuclease Activity in Rabbitpox and Vaccinia Viruses

The endonucleolytic action of a deoxyribonuclease activity in rabbitpox and vaccinia virus was established by change in sedimentation rate of denatured (3)H-lambda deoxyribonucleic acid substrate. The presence of two deoxyribonuclease activities in pox-virus is confirmed. Exo- and endonuclease activities are unmasked by treatment of purified virus with the detergent Nonidet P-40 and further enhanced by treatment of viral "cores" with trypsin.     

Cofactor Requirement of HpyAV Restriction Endonuclease

Background

Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M) systems in microorganisms.

Principal Findings

We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg⁺⁺. The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity.

Conclusions/Significance

Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.     

Restriction Endonuclease BspD6II,a New Thermophilic Isoschizomer of Eco57I

Russian Journal of Bioorganic Chemistry - From the Bacillus species D6 strain, the restriction endonuclease BspD6II has been isolated and characterized. It recognizes the asymmetric region...     

限制性核酸内切酶与DNA相互作用研究进展

蛋白质对ＤＮＡ识别的模体中,除了锌指结构、螺旋—转角—螺旋、亮氨酸拉链和β带外,近年来发现,Ⅱ型限制性内切酶与ＤＮＡ作用的模体有许多特别之处。通过对ＥｃｏＲＩ、ＢａｍＨＩ、ＥｃｏＲＶ等与ＤＮＡ复合物的空间构象、一级结构分析,发现酶分子存在催化性裂缝,并且氨基端形成臂结构包绕ＤＮＡ;同时ＤＮＡ发生构象变化、螺旋扭结。这些有趣的结构有利于酶对底物的特异性结合和催化作用。     

Endonuclease Specificity and Sequence Dependence of Type IIS Restriction Enzymes

Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage) and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI). We report significant variation of slippage ranging from 1–54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes.     

Infection by Bacteriophage P1 and Development of Host-controlled Restriction and Modification and of Lysogenic Immunity

Shigella dysenteriae cells were infected with phage P1 or P1cl. The outcome of superinfection of these cells with phage T1.Sh or T1.Sh(P1) or P1cl was studied as a function of time after the initial infection. Cells undergoing either a lytic response or a lysogenic response to the primary infection develop the ability to specifically restrict T1.Sh between 30 and 45 min. Between 15 and 30 min, the cells seem to develop the ability to produce T1.Sh(P1) after infection by T1.Sh. However, reasons are given for believing that this apparent time difference is consistent with a simultaneous development of the two capacities (restriction and modification) within the cell. This development occurs between 30 and 45 min. Cells infected with P1cl and superinfected 45 or more min later with T1.Sh(P1) can yield both P1cl and T1. Cells infected with P1 become resistant to infection by P1cl within 5 to 10 min. It is argued that this early immunity is not necessarily different in mechanism from true lysogenic immunity.     

应用CRISPR/Cas13b编辑技术治疗TNNT2R141W转基因小鼠的扩张型心肌病

本研究旨在应用CRISPR/Cas13b系统对TNNT2R141W转基因扩张型心肌病（dilated cardiomyopathy,DCM）小鼠（DCM小鼠）进行探索性治疗,尝试发现治疗扩张型心肌病的一种新方式,为CRISPR/Cas13b系统在体内应用提供实验基础。随机设计11种Cas13b-TNNT2 gRNA并成功构建表达质粒,把它和人源TNNT2过表达质粒共同转染到293T细胞中,通过实时定量PCR（quantitative real-time PCR,Q-PCR）检测人源TNNT2 mRNA的表达水平。结果显示,gRNA 2引导Cas13b敲低目标基因的效率最高,达到80%（P<0.0001）。把gRNA2表达质粒包装到慢病毒载体中转导出生后1天的DCM小鼠原代心肌细胞,Q-PCR检测结果表明CRISPR/Cas13b系统对人源TNNT2 mRNA的敲低效率达到55%（P<0.01）。把PspCas13b和gRNA2的表达载体分别包装到AAV9病毒载体中,然后将200 μL 约1×1012 AAV9病毒颗粒通过尾静脉注射到4月龄DCM小鼠体内,待注射小鼠发育至5月龄时,Q-PCR检测结果显示,AAV9+DCM组TNNT2R141W表达水平较未注射组对照明显下降至40%（P<0.01）。对5月龄野生型（WT）、DCM（未注射病毒组）和AAV9+DCM（基因组编辑工具注射组）三组小鼠的心脏形态、心功能、心肌纤维化和心力衰竭等表型的观察结合显示：DCM小鼠的心脏形态异常,而AAV9+DCM小鼠心脏形态趋于正常;对三组小鼠的心脏进行超声心动图并对心功能指标进行统计发现,DCM组较WT组小鼠的左心室射血分数（left ventricular percent ejection fraction,LV EF%）、左心室短轴缩短率（left ventricular percent fractional shortening,LV FS%）分别下降了50.4%（P<0.0001）,55.1%（P<0.0001）,而AAV9+DCM组较DCM组小鼠的LV EF%、LV FS%分别上升了66.5%（P<0.01）,77.0%（P<0.01）;通过Q-PCR和天狼星红染色检测三组小鼠的心脏纤维化程度,结果显示DCM组较WT组小鼠的Col3a1和Postn两种纤维化基因,分别高表达5.2倍（P<0.001）、4.5倍（P<0.01）,而AAV9+DCM组较DCM组小鼠两种基因表达分别下降了2.0倍（P<0.05）、1.4倍（NS）,天狼星红染色结果显示纤维化区域明显下降;通过Q-PCR和蛋白质免疫印迹分别检测三组小鼠的心脏心力衰竭基因Nppb mRNA和Nppa蛋白质的表达水平,结果表明DCM组较WT组小鼠Nppb mRNA表达上升14.2倍（P<0.01）,而AAV9+DCM组较DCM组小鼠Nppb mRNA表达明显下降下降2.8倍（P<0.05）,Nppa蛋白质表达趋势与Nppb相同。把gRNA 5和含有R141W突变（gRNA 5T）和正常的TNNT2 mRNA（gRNA 5V）序列分别组合转染到293T细胞中,通过Q-PCR检测两种序列mRNA的表达水平。结果显示,gRNA 5T序列表达效率为30%（P<0.0001）,而并未检测到gRNA 5V mRNA的敲低。本研究通过设计靶向TNNT2R141W mRNA的gRNA,特异性敲低TNNT2R141W转基因小鼠体内突变的mRNA,有效改善了转基因小鼠的心功能,为临床进一步探索扩张型心肌病的治疗奠定了实验室基础。     

DNA序列中限制酶切割位点的分布

本文以人类、小鼠、大鼠和病毒基因组中的DNA序列为材料,分析了其中40种II型限制性核酸内切酶识认位点的数量和分布情况。发现人鼠序列中绝大多数酶的切点数量可以通过序列中单核苷酸或双核苷酸的频率来预测,而切点在序列上的分布也是随机的。例外的情况是人鼠序列中酶EcoRII(识认CCWGG)和MnlI(CCTC)的切点显著偏多,而DpnI*(GATC)的切点显著偏少;MnlI的切点倾向于聚集一处。病毒基因组中酶切位点也基本上是随机分布,但基因组间差异很大,跟人鼠序列差别也大,特别是噬菌体T7中有多达17种酶的切点显著偏少。文中讨论了所得结果对构建限制酶切图谱的理论计算以及对限制酶显带机理的意义,特别指出显带过程在酶的切点达到所要求的浓度时,跟酶的识认片段的专一性、酶切位点的数量都没有关系,而取决于染色体不同区段抵抗酶切破坏的能力。     

中国昆明(KM)小鼠线粒体DNA限制性内切酶图谱的研究

线粒体DNA的限制性内切酶图谱在不同的种系及亚种间存在多态性。我们分别用五种限制性内切酶BamHⅠ、EcoRⅡ、HidⅢ、PstⅠ、MspⅠ对60只中国KM小鼠(雌雄各半)的线粒体DNA进行了酶切电泳分析,结果未发现多态性,且这五种限制性内切酶图谱均与BALB/c小鼠相同。这表明中国KM小鼠与绝大多数实验小鼠一样,均起源于欧州的野鼠M.m.domesticu。未见到KM小鼠经其它亚种野鼠母系遗传污染的迹象。     

Crystal Structure and Directed Evolution of Specificity of NlaIV Restriction Endonuclease

Specificity engineering is challenging and particularly difficult for enzymes that have the catalytic machinery and specificity determinants in close proximity. Restriction endonucleases have been used as a paradigm for protein engineering, but successful cases are rare. Here, we present the results of a directed evolution approach to the engineering of a dimeric, blunt end cutting restriction enzyme NlaIV (GGN/NCC). Based on the remote similarity to EcoRV endonuclease, regions for random mutagenesis and in vitro evolution were chosen. The obtained variants cleaved target sites with an up to 100-fold k_cat/K_M preference for AT or TA (GGW/WCC) over GC or CG (GGS/SCC) in the central dinucleotide step, compared to the only ~ 17-fold preference of the wild-type enzyme. To understand the basis of the increased specificity, we determined the crystal structure of NlaIV. Despite the presence of DNA in the crystallization mix, the enzyme crystallized in the free form. We therefore constructed a computational model of the NlaIV–DNA complex. According to the model, the mutagenesis of the regions that were in the proximity of DNA did not lead to the desired specificity change, which was instead conveyed in an indirect manner by substitutions in the more distant regions.     

农杆菌质粒DNA的间接酶切鉴定

采用常规手段提酶切鉴定法,与普通大肠杆菌质粒小量抽提试剂盒提取农杆菌质粒酶切鉴定法(简称试剂盒法)和农杆菌质粒反导大肠杆菌间接酶切鉴定法(简称间接法)进行对比,发现本试验创新的试剂盒法和间接法可轻松做酶切鉴定,可为农杆菌质粒DNA提取经验不足者参考.     

Genotyping of Staphylococcus epidermidis by Small-Fragment Restriction Endonuclease Analysis and Pulsed-Field Gel Electrophoresis of Genomic Restriction Fragments

Small-fragment restriction endonuclease analysis (SF-REA) was established as a typing tool for Staphylococcus epidermidis. A total of 60 isolates comprising 48 epidemiologically nonrelated strains and 12 putatively linked isolates from 7 patients in 2 wards were analyzed. Nonrelated isolates were characterized by unique fingerprints when DNA was cleaved with EcoRI or ClaI, electrophoretically separated in a polyacrylamide gel, and silver stained. Three blood culture isolates from one patient in an intensive care unit, 4 isolates obtained from a child over a span of 2 weeks, and 5 isolates from 5 newborns in the same ward were grouped into 3 DNA pattern types, indicating identity of sequential isolates from 2 patients and nosocomial transmission of one Staphylococcus epidermidis strain between 5 babies. Results from pulsed-field gel electrophoresis of SmaI and SacII DNA digests and conventional marker systems such as antibiogram and plasmid profile were in accordance with these interpretations, whereas slight variation was observed in the biotypes of several strains. From the results of this study, we conclude that SF-REA is a precise and efficient method for the genotypic characterization of Staphylococcus epidermidis strains that can be used as a rapid and reliable typing tool.     

洋葱伯克霍尔德氏菌HMW DNA的制备及酶切研究

高质量粘粒基因组文库构建的关键是HMW DNA的长度至少为粘粒载体容量的10倍,通常粘粒载体的容量为30～50 kb,因此提取的HMW DNA应不小于500 kb.HMW DNA在制备时不能受到任何物理的剪切力,以免DNA断裂和损伤.利用琼脂糖凝胶包埋制备的DNA胶块经裂解和纯化后发现其DNA长度远大于500 kb,明显优于商品化试剂盒提取和酚抽提法.用BamHⅠ、Sau3AⅠ、XbaⅠ和HindⅢ对DNA胶块进行不完全酶切研究表明,构建文库常用的Sau3AⅠ并不适合胶块内酶切反应,而BamHⅠ酶切B.cepacia HMW-DNA效果较好,产生的DNA片段集中在20～50 kb之间,完全适合粘粒基因组文库的构建,为B.cepacia大插入片段基因组文库的构建以及功能基因组的研究奠定了良好的理论基础.     

鲤鱼肝组织线粒体DNA的限制性内切酶分析

用EcoRⅠI,HindⅢ,PstⅠ,BglⅡ,BamHⅠ,Xho Ⅰ, Xba Ⅰ, Sal Ⅰ和Kpn Ⅰ共9种限制性内切酶酶切对鲤鱼肝组织的mtDNA进行酶解,利用Gel-Pro Analyzer算出各酶切片段长度及mtDNA大小,测出其大小约为16．61kb(1kb=1×103b),另外,对电泳条件的优化进行了探讨,并将实骚结果与以前报道的有关酶切实验结果进行了比较,表明鲤鱼mtDNA的长度及限制性酶切位点均存在地域差异．     

Restriction Endonuclease Analysis of Highly Repetitive DNA as a Phylogenetic Tool

Multiple band patterns of DNA repeats in the 20–500-nucleotide range can be detected by digesting genomic DNA with short—cutting restriction endonucleases, followed by end labeling of the restriction fragments and fractionation in nondenaturing polyacrylamide gels. We call such band patterns obtained from genomic DNA ``taxonprints' (Fedorov et al. 1992). Here we show that taxonprints for the taxonomic groups studied (mammals, reptiles, fish, insects—altogether more than 50 species) have the following properties: (1) All individuals from the same species have identical taxonprints. (2) Taxonprint bands can be subdivided into those specific for a single species and those specific for groups of closely related species, genera, and even families. (3) Each restriction endonuclease produces unique band patterns; thus, five to ten restriction enzymes (about 100 bands) may be sufficient for a statistical treatment of phylogenetic relationships based on polymorphisms of restriction endinuclease sites. We demonstrate that taxonprint analysis allows one to distinguish closely related species and to establish the degree of similarity among species and among genera. These characteristics make taxonprint analysis a valuable tool for taxonomic and phylogenetic studies. Received: 10 February 1997 / Accepted: 10 March 1997