噬菌体ψHAU3的cos位点及其与寄主相互作用的功能位点attP和attB的…

ψＨＡＵ３是寄主范围很广的放线菌噬菌体。Ｓｏｕｔｈｅｒｎ杂交实验表明,ψＨＡＵ３可以整合到吸水链霉菌应城变种１０－２２和变铅青链霉菌６６的突变体ＺＸ１的染色体中,形成溶原,其溶原菌自发释放ψＨＡＵ３,不受热激和紫外线照射的诱导。通过比较ψＨＡＵ３衍生噬粒ＰＩＪ８３００的ＤＮＡ酶切片段的中热前、后电泳带谱的区别,将ψＨＡＵ３的ｃｏｓ位点在ＰＩＪ８３００的图谱上得到了定位。还利用Ｓｏｕｔｈｅｒｎ杂交的     

变铅青链霉菌对噬菌体φHAU3抗性与异常修饰之间的相互关系

变铅青链霉菌异常修饰突变菌株ＺＸｌ同时丧失了对噬菌体ＨＡＵ３的抗性,遗传学杂交结果揭示,ＤＮＡ降解和对ＨＡＵ３抗性这两种表型从不发生分离。以ＺＸｌ为宿主和链霉菌低拷贝质粒ＳＣＰ２衍生的载体ｐＩＪ９２２为载体,构建了变铅青链霉菌ＪＴ４６的基因组文库。在８０００多个转化子中获得了一个能使ＺＸ１对ＨＡＵ３显示抗性的杂合质粒,命名为ｐＩＪ８３１０。它除了含有完整的ｐＩＪ９２２外,还携带了大约１１ｋｂ的外源片段。Ｓｏｕｔｈｅｒｎ杂交证实,这个外源片段来源于ＪＴ４６,但在ＺＸ１中完全消失。利用转座子Ｔｎ４５６０对该克隆进行诱变定位,获得了中断噬菌体抗性基因表达的衍生质粒,并已证实Ｔｎ４５６０插入在一个２．５ｋｂ的ＢｄｍＨＩ一ＥｃｏＲＩ片段上。     

变铅青链霉菌对噬菌体ψHAU3抗性与异常修饰之间的相互关系

变铅青链霉菌异常修饰突发菌株ＺＸ１同时丧失了对噬菌体ψＨＡＵ３的抗性。遗传学杂交结果揭示,ＤＮＡ降解和对ψＨＡＵ３抗性这两种表型从不发生分离。以ＺＸ１为宿主和链霉菌低拷贝质粒ＳＣＰ２衍生的载体ｐＩＪ９２２为载体,构建了变铅青链霉菌ＪＴ４６的基因组文库。在８０００多个转化子中获得了一个能使ＺＸ１对ψＨＡＵ３显示抗性的杂合质粒,命名为ｐＩＪ８３１０。它除了含有完整的ｐＩＪ９２２外,还携带了大约１１ｋｂ     

变铅青链霉菌与放线菌噬菌体φHAU3相互关系的研究

变铅青链霉菌ＺＸ１,是由变铅青链霉菌ＪＴ４６经ＮＴＧ诱变后产生的一株修饰基因突变株,与其亲本ＪＴ４６相比,ＺＸ１除了与ＤＮＡ降解有关的基因发生了突变（Ｄｎｄ－,即该突变株的ＤＮＡ在含有Ｆｅ２＋的缓冲液中电泳不受到降解,而野生型变铅青链霉菌在同样条件下则遭到降解）以外,对噬菌体　ＨＡＵ３的抗性也随之消失,这项特征主要表现在噬菌斑大小和成斑单位（效价）上的显著变化。研究结果表明,噬菌体　ＨＡＵ３以同等的频率吸附野生型变铅青链霉菌及其突变菌株ＺＸ１,从　ＨＡＵ３基因组中没有克隆到被变铅青链霉菌识别的特异性靶位点;噬菌体ＨＡＵ３的ＤＮＡ也可以转染野生型变铅青链霉菌原生质,但其释放的噬菌体粒子只能感染突变菌株ＺＸ１,而不能感染野生型变铅青链霉菌。噬菌体ＨＡＵ３在突变株ＺＸ１中的繁殖遵循一步生长曲线,单菌释放量大约为１００,而　ＨＡＵ３感染野生型变铅青链霉菌后,则检测不到噬菌体的释放。     

转座子Tn5096对刺孢吸水链霉菌北京变种PR220转座的诱变

用大肠杆菌／链霉菌穿梭质粒ｐＣＺＡ１６８多次转化农抗１２０产生菌刺孢吸水链霉菌北京变种ＲＦ２２０的原生质体,均未得到转化子。来自吸水链霉菌应城变种１０－２２突变株的链霉菌质粒ｐＩＪ７０２可以转化ＲＦ２２０,但转化频率只有数十个转化子／μｇＤＮＡ。用来自ＲＦ２２０本身的ｐＩＪ７０２对消除ｐＩＪ７０２后的ＲＦ２２０的原生质体进行了再转化,转化率没有明显的提高。用氨苄青霉素和甘氨酸协同处理ＲＦ２２０的菌     

变铅青链霉菌66中对噬菌ΦHAU3显示抗性的基因（ψHAU3￣R）的核苷酸定序和分析

杂合质粒ｐＩＪ８３１０是链霉菌低拷贝质粒ｐＩＪ９２２１携带了１０．８ｋｂ来自变铅青链霉菌ＪＴ４６菌株,编码对噬菌体ΦＨＡＵ３抗性的ＤＮＡ片段。已知Ｔｎ４５６０插到该克隆中３ｋｂ的ＥｃｏＲＩ或３．３ｋｂ的ＢａｍＨＩ片段（这两个片段之间有２．５ｋｂ的重叠区）上,中断了ΦＨＡＵ３抗性基因的表达。已将３．０ｋｂ的ＢｃｏＡＩ片段分别以两种不同的取向克隆到ｐＢｌｕｅｓｃｒｉｐｔＳＫ（＋）上。核苷酸序列分析揭示出一个１．３ｋｂ的ＯＲＦ的存在,其所编码的４７８个氨基酸组成的蛋白与λ噬菌体的ｅａ５９基因所编码的氨基酸序列显示了高度的同源性,其氨基酸序列的同一性高达３７％,相似性达５８％,而λ噬菌体的ｅａ５９基因是编码赖ＡＴＰ和单键ＤＮＡ的核酸内切酶Ｉ的,该区域与λ的复制无关。此外,这段具有高度同源性的ＤＮＡ区域的Ｇ＋Ｃ％只有６０％,低于变铅青霉菌ＤＮＡＧ＋Ｃ％的平均值（７４％）。     

嗜冷细菌弧菌菌株ABE—1中呼吸作用依赖性钠泵偶联位点

嗜冷细菌弧菌菌株ＡＢＥ－１中呼吸作用依赖性钠泵偶联位点ＹＡＳＵＨＩＲＯＴＡＫＡＤＡ．ＮＯＲＩＹＵＫＩＦＵＫＵＮＡＧＡ．ＡＮＤＳＨＯＪＩＳＡＳＡＫＩ研究了嗜冷细菌弧菌菌株ＡＢＺ－１呼吸作用依赖性原钠泵与呼吸链的偶联位点。几种抑制实验表明由呼吸作用依赖性...     

7号淀粉酶链霉菌M1033木糖异构酶基因的启动子活性检测、转录起始点的确定

分别从重组质粒ｐＵＢ１及其Ｍ（１３）亚克隆Ｓ１中将７号淀粉酶链霉菌（ＳｔｒｅｐｔｏｍｙｃｅｓｄｉａｓｔａｔｉｃｕｓＮｏ．７）Ｍ１０３３（以下简称Ｓ．ｄｉ．Ｍ１０３３）木糖异构酶基因的－１９２－＋５８１ｂｐ片段克隆入链霉菌启动子探测质粒ｐＩＪ４０８３中,转化变铅青链霉菌（Ｓ．ｌｉｖｉｄａｎｓ）ＴＫ２４。通过对其邻苯二酚加双氧酶活性的检测表明,该片段具有启动子活性。应用Ｍ１３亚克隆Ｓ１和合成引物Ｐ６延伸制备放射性标记的单链ＤＮＡ探针;通过Ｓ．ｄｉ．Ｍ１０３３的总ＲＮＡ的Ｓ１核酸酶保护实验,确定了其转录的起始位点,并由此探讨了与木糖异构酶基因表达有关的一些因素。     

苏云金芽孢杆菌δ-内毒素cryIA(c)基因在大肠杆菌和变铅青链霉菌中表达

利用合成的寡聚核苷酸片段,从质粒ｐＯＳ１０００中分离出具有适合克隆位点的苏云金芽孢杆菌（Ｂａｃｉｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ）δ内毒素ｃｒｙＩＡ（ｃ）全长基因。将该基因与大肠杆菌表达载体ｐＫＫ２２３３重组,并引入大肠杆菌ＪＭ１０９中,经ＩＰＴＧ诱导,获得了超量表达的ＣｒｙＩＡ（ｃ）蛋白。将ｃｒｙＩＡ（ｃ）全长基因插入链霉菌表达载体ｐＨＺ１２７２中,得到重组质粒ｐＨＺ１２５６,将该质粒引入变铅青链霉菌（Ｓｔｒｅｐｔｏｍｙｃｅｓｌｉｖｉｄａｎｓ）ＪＴ４６中,经硫链丝菌素诱导,通过Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ测定表明,重组变铅青链霉菌ＪＴ４６（ｐＨＺ１２５６）已表达出相应的ＣｒｙＩＡ（ｃ）蛋白。杀虫试验表明,大肠杆菌和链霉菌所表达出的δ内毒素ＣｒｙＩＡ（ｃ）对小菜蛾均有毒杀作用,其致死率分别为９３％和５７％。     

人肿瘤坏死因子基因在变铅青链霉菌中的克隆与表达

以质粒ｐＩＪ４８６为载体,将来源于质粒ｐＨＴ１的肿瘤坏死坏因子（ＴＮＦ－ａ）ｃＤＮＡ克隆至变铅青链霉菌,以新霉素（３０μｇ／ｍｌ）为选择标记,获得了数百转化子,实验表明Ｎｏ．７转化子ｓ．ｌｉｖｉｄａｎｓ ＴＫ５４－ＨＴ所含重组质粒ｐＩＪＴ７已克隆有ＴＮＦｃＤＮＡ。Ｌ９２９细胞毒实验结果表明该转化子ＴＮＦ表达量可达１０＾８活性单位／升以上,中和实验确证其表达产物为人ＴＮＦ－ａ,ＳＤＳ－ＰＡＧＥ表明克     

Characterization of phi HAU3, a broad-host-range temperate streptomyces phage, and development of phasmids.

phi HAU3 is a temperate Streptomyces phage with cohesive ends and a broad host range that includes Streptomyces hygroscopicus 10-22, a producer of antifungal compounds, but it fails to grow on Streptomyces lividans 66. Two phasmid derivatives were constructed that function as lambda cosmid vectors in Escherichia coli and as phages in Streptomyces spp.     

Streptomyces lividans 66 contains a gene for phage resistance which is similar to the phage λea59 endonuclease gene

The DNA of wild-type Streptomyces lividans 66 is degraded during electrophoresis in buffers containing traces of ferrous iron. S. lividans ZX1, a mutant selected for resistance to DNA degradation, simuiltaneously became sensitive to φHAU3, a wide-host-range temperate bacteriophage. A DNA fragment conferring φHAU3 resistance was cloned; it contains a phage resistance gene whose deduced amino acid sequence is similar to the phage λ Ea59 endonuclease. The S. lividansφHAU3 resistance does not seem to be a classical restriction-modification system, because no host-modified phages able to propagate on the wild-type strain could be isolated. The cloned fragment did not make the host DNA prone to degradation during electrophoresis, indicating that the two phenotypes are controlled by different genes which were deleted together from the chromosome of ZX1.     

Streptomyces coelicolor A3(2) Lacks a Genomic Island Present in the Chromosome of Streptomyces lividans 66

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage HAU3 resistance (HAU3^r) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.     

Development of a gene cloning system for Streptomyces hygroscopicus subsp. yingchengensis, a producer of three useful antifungal compounds, by elimination of three barriers to DNA transfer.

Streptomyces hygroscopicus 10-22 could not be transformed with any of the commonly used Streptomyces plasmid vectors and was resistant to plaque formation by the Streptomyces phages phi C31 and R4. Repeated selection resulted in the isolation of derivatives of S. hygroscopicus 10-22 that could be transformed with pIJ101- and pJV1-derived cloning vectors and of restriction-deficient derivatives that could accept DNA propagated in Streptomyces lividans 66. These new strains, which include three that still produce the original antibiotics, can be used as hosts for gene cloning. Insertion of nonreplicating vectors by homologous recombination and transposition of Tn4560 were demonstrated in S. hygroscopicus 10-22.     

A linear plasmid temperature-sensitive for replication in Streptomyces hygroscopicus 10-22

Streptomyces hygroscopicus 10-22 harbors a conjugative, autonomously replicating linear plasmid pHZ6 of ca. 70 kb, which shows no obvious homology with chromosomal DNA and is temperature-sensitive for replication, being stable in the host at 28 degrees C but easily lost at 37 degrees C. On a lawn of the wild-type S. hygroscopicus 10-22 cured of pHZ6, pHZ6 elicit pocks. Temperature sensitivity seemed to be a unique property for pHZ6 among six linear plasmids tested, including the well-known linear plasmids SLP2 in Streptomyces lividans 1326 and SCP1 in Streptomyces coelicolor A3(2). The distinct identity of pHZ6 from previously identified pHZ1-pHZ5 was demonstrated by the profile of relevant plasmids in six well-defined strains originated from S. hygroscopicus 10-22.     

吸水链霉菌应城变种的四个内源质粒及其逐个消除的研究

在改良质粒DNA提取方法的基础上,从三种农用抗生素的同一产生菌——吸水链霉菌应城变种中同时发现四个内源质粒,用双向电泳技术确定了它们均为CCC构型,根据分子量从大到小的顺序将它们分别命名为pHZ1、pHZ2、pHZ3和pHZ4,与已知分子量的CCC质粒分子同步电泳估计它们的分子量依次分别为61kb、4.7kb、4.1kb和3.3kb,基于这四个内源质粒中至少部分个体可能为接合性质粒,可在没有某个质粒的衍生菌株的菌坪上形成“麻点”的假设,我们分离和鉴定了三个质粒逐个消除的10-22衍生菌株,并在光学显微镜下确证了二种类型的麻点。     

Streptomyces coelicolor A3(2) lacks a genomic island present in the chromosome of Streptomyces lividans 66

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage phiHAU3 resistance (phiHAU3r) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.     

吸水链霉菌Streptomyces hygroscopicus 17997中噬菌体基因转移系统的建立及其应用

由吸水链霉菌Streptomyces hygroscopicus 17997产生的格尔德霉素geldanamycin(GA)属安莎类抗生素,具有良好的抗肿瘤和抗病毒活性。本文应用链霉菌温和噬菌体ΦC31衍生的KC515载体,在吸水链霉菌S．hygroscopicus 17997中建立并优化了S．hygroscopicus 17997的基因转染体系。利用所建立的基因转染体系,以基因阻断技术从S．hygroscopicus 17997基因文库含有多组PKS基因柯斯质粒中,鉴定了与GA PKS生物合成相关基因的柯斯质粒,该工作为GA生物合成基因簇的克隆奠定了基础。     

Physical map of the linear chromosome of Streptomyces hygroscopicus 10-22 deduced by analysis of overlapping large chromosomal deletions

The chromosomal DNA of Streptomyces hygroscopicus 10-22, a derivative of strain 5102-6, was digested with several restriction endonucleases and analyzed by pulsed-field gel electrophoresis (PFGE). Digestions with AseI gave 11 fragments with a total length of ca. 7.36 Mb. The AseI sites were mapped by analysis of overlapping chromosomal deletions in different mutants and confirmed by Southern hybridizations using partially digested genome fragments and linking cosmids as probes. PFGE analysis of DNA with and without proteinase K treatment, together with the hybridization results, suggested a linear organization with terminal proteins and large terminal inverted repeats. Some deletion mutants had circular chromosomes.