Insertion-Duplication Mutagenesis ofNeisseria: Use in Characterization of DNA Transfer Genes in the Gonococcal Genetic Island

Holly L. Hamilton; Kevin J. Schwartz; Joseph P. Dillard

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Insertion-Duplication Mutagenesis ofNeisseria: Use in Characterization of DNA Transfer Genes in the Gonococcal Genetic Island

机译：奈瑟菌的插入-复制诱变：用于淋球菌遗传岛中的DNA转移基因的表征。

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We created plasmids for use in insertion-duplication mutagenesis (IDM) of Neisseria gonorrhoeae. This mutagenesis method has the advantage that it requires only a single cloning step prior to transformation into gonococci. Chromosomal DNA cloned into the plasmid directs insertion into the chromosome at the site of homology by a single-crossover (Campbell-type) recombination event. Two of the vectors contain an erythromycin resistance gene, ermC, with a strong promoter and in an orientation such that transcription will proceed into the cloned insert. Thus, these plasmids can be used to create insertions that are effectively nonpolar on the transcription of downstream genes. In addition to the improved ermC, the vector contains two copies of the neisserial DNA uptake sequence to facilitate high-frequency DNA uptake during transformation. Using various chromosomal DNA insert sizes, we have determined that even small inserts can target insertion mutation by this method and that the insertions are stably maintained in the gonococcal chromosome. We have used IDM to create knockouts in two genes in the gonococcal genetic island (GGI) and to clone additional regions of the GGI by a chromosome-walking procedure. Phenotypic characterization oftraG and traH mutants suggests a role for the encoded proteins in DNA secretion by a novel type IV secretion system.

机译：我们创建了用于淋病奈瑟氏菌的插入复制诱变（IDM）的质粒。该诱变方法的优点在于，在转化为淋球菌之前，仅需一个克隆步骤即可。克隆到质粒中的染色体DNA通过单交换（Campbell型）重组事件直接插入同源位点的染色体中。其中两个载体包含一个红霉素抗性基因 ermC ，该基因具有很强的启动子，其方向应使转录进入克隆的插入片段。因此，这些质粒可用于产生对于下游基因的转录实际上是非极性的插入。除改进的 ermC 外，该载体还包含两个拷贝的奈瑟菌DNA摄取序列，以促进转化过程中的高频DNA摄取。使用各种染色体DNA插入物大小，我们已经确定，即使很小的插入物也可以通过这种方法靶向插入突变，并且插入物在淋球菌染色体中得到稳定的维持。我们已经使用IDM在淋球菌遗传岛（GGI）中的两个基因中创建敲除基因，并通过染色体行走程序克隆了GGI的其他区域。对 traG 和 traH 突变体的表型鉴定表明，编码的蛋白质在新型IV型分泌系统的DNA分泌中发挥了作用。 展开▼

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《Journal of bacteriology》 |2001年第16期|共9页

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Holly L. Hamilton; Kevin J. Schwartz; Joseph P. Dillard;
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中图分类微生物学;

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专利

1. Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion [J] . Ramsey Meghan E., Bender Tobias, Klimowicz Amy K., Molecular Microbiology . 2015,第6期

机译：淋病奈瑟球菌淋球菌遗传岛基因间区域的定向诱变揭示了控制IV型分泌的多种调控机制

2. DNA processing by the MOBH family relaxase TraI encoded within the gonococcal genetic island [J] . Heilers Jan-Hendrik, Reiners Jens, Heller Eva-Maria, Nucleic Acids Research . 2019,第15期

机译：由Mobh系列弛豫酶Trai编码在淋病遗传岛内的DNA处理

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机译：单纯疱疹病毒的分子遗传学。 VII。通过体外诱变产生的温度敏感突变体的表征，DNA合成和γ多肽积累缺陷。

4. TRANSFERABILITY OF TAG SNPS TO CAPTURE COMMON GENETIC VARIATION IN DNA REPAIR GENES ACROSS MULTIPLE POPULATIONS [C] . PAUL I.W. DE BARKER, ROBERT R. GRAHAM, DAVID ALTSHULER, Pacific Symposium on Biocomputing 2006 . 2006

机译：标记SNPS可以捕获多种群DNA修复基因中常见的遗传变异。

5. Characterization of the gonococcal genetic island and peptidoglycan fragment release in Neisseria meningitidis. [D] . Woodhams, Katelynn Lyons. 2013

机译：脑膜炎奈瑟球菌淋球菌遗传岛和肽聚糖片段释放的特征。

6. Insertion-Duplication Mutagenesis of Neisseria: Use in Characterization of DNA Transfer Genes in the Gonococcal Genetic Island [O] . Holly L. Hamilton, Kevin J. Schwartz, Joseph P. Dillard 2001

机译：奈瑟菌的插入-复制诱变：用于淋球菌遗传岛中的DNA转移基因的表征。

7. Insertion-Duplication Mutagenesis of Neisseria: Use in Characterization of DNA Transfer Genes in the Gonococcal Genetic Island [O] . Hamilton, Holly L., Schwartz, Kevin J., Dillard, Joseph P. 2001

机译：奈瑟菌的插入-复制诱变：用于淋球菌遗传岛中的DNA转移基因的表征。

8. Somatic Cell and Molecular Genetics Approach to DNA Repair and Mutagenesis [R] . Thompson, L. H. 1985

机译：体细胞和分子遗传学方法进行DNa修复和诱变

1. 男性泌尿生殖道感染患者分离奈瑟菌的16S rDNA和PIA基因检测 [J] . 姜敏敏 ,王涛 ,王和 . 江苏医药 . 2012,第005期

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3. 中国脑膜炎奈瑟菌中插入序列1301的分布与分子流行病学研究 [J] . 庞杏林 ,侯配斌 ,高源 . 中华流行病学杂志 . 2009,第005期

4. 可变数目串联重复序列技术用于中国C群脑膜炎奈瑟菌基因分型的研究 [J] . 田国忠 ,邵祝军 ,李马超 . 中华流行病学杂志 . 2006,第007期

5. 呼吸道和男性泌尿生殖道非脑膜炎球菌与非淋球菌奈瑟菌感染的实验诊断与临床研究 [J] . 王丹霓 ,王和 ,罗振华 . 贵州医药 . 2009,第003期

6. 多位点分型技术应用于浙江省脑膜炎奈瑟菌基因分型研究 [C] . ZHU Shui-rong ,朱水荣 . 第六届传染病防控技术研究与应用技术论坛 . 2015

7. 淋病奈瑟菌耐药性与毒力岛的研究 [A] . 崔海燕 . 2008

1. 用于在植物体的基因组中无需插入复制子即可编辑基因组的基于病毒的复制子及其用途 [P] . 中国专利： CN112689678A . 2021-04-20

2. 一种淋病奈瑟菌免疫阻遏突变基因Δrmp及突变方法 [P] . 中国专利： CN102382844B . 2012.11.21

3. METHOD FOR COLORING DNA OF LIVING MAMMALIAN CELLS, CELL WITH COLORED DNA, PRONUCLEI COLORING PROCESS, LIVE COLORED MAMMALIAN PRONUCLEAR CELL, METHOD OF REMOVING GENETIC MATTER FROM CELL, GENETIC MATERIAL OBTAINED, PROCESS FOR TRANSFERRING IN PRONUCLEUS, GENETICALLY MODIFIED LIVING MAMMAL CELL, AND PROCESS FOR PRODUCING PHOTOGRAPHIC REPRESENTATION OF SPERMATOZOID MOVEMENT [P] . 外国专利： FR2550218A1 . 1985-02-08

机译：活体哺乳动物细胞的DNA染色方法，有色DNA的细胞，原核染色过程，活的有色哺乳动物原核细胞，从细胞中去除遗传物质的方法，获得的遗传材料，转移的过程，向母体和母体的遗传过程制作精子运动的照相代表

4. rna targeting dna; DNA polynucleotide; recombinant expression vector; genetically modified host cell in vitro; variant site-directed modification polypeptide; chimeric site-directed modification polypeptide; rna polynucleotide; genetically modified cell; transgenic non-human organism whose genome comprises a transgene; composition; site specific modification method of a target DNA; site specific transcriptional modulation method within a target DNA; site-specific modification method in target DNA; method of promoting site-specific cleavage and modification of a target DNA in a cell; method of producing a genetically modified cell in a subject; method of modifying target DNA in a genetically modified cell; kit; target dna target kit; method of selectively modulating transcription of a target DNA into a host cell; isolated nucleic acid; genetically modified host cell; and nucleic acid library [P] . 外国专利： BR112014029441A2 . 2017-06-27

机译：针对dna的rna; DNA多核苷酸;重组表达载体体外转基因宿主细胞;变体定点修饰多肽;嵌合定点修饰多肽; rna多核苷酸;转基因细胞基因组包含转基因的转基因非人类生物;组成;靶DNA的位点特异性修饰方法;靶DNA内的位点特异性转录调节方法;靶DNA的位点特异性修饰方法;促进细胞中靶DNA的位点特异性切割和修饰的方法;在受试者中产生基因修饰的细胞的方法;在转基因细胞中修饰靶DNA的方法;套件目标dna目标试剂盒;选择性调节靶DNA转录到宿主细胞中的方法;分离的核酸转基因宿主细胞;和核酸文库

5. Brief description of the coloration of the dna of living cells of mammals, cell, the dna is coloured, the coloration of pronuclear, the living cell pronuclear of coloree, of a material genetic of a cell, genetic material obtained, in order to transfer it into a pronucleus, the living cell genetically modified, and method of production of a photographic representation of the movement of the spermatozoa [P] . 外国专利： FR2550218B1 . 1989-01-20

机译：哺乳动物活细胞，细胞的dna的着色的简要说明，细胞的dna是着色的，原核的着色，色素的活细胞的原核的着色，细胞的物质遗传，获得的遗传物质，以便转移进入原核，经基因修饰的活细胞，以及产生精子运动的照片的方法

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Insertion-Duplication Mutagenesis ofNeisseria: Use in Characterization of DNA Transfer Genes in the Gonococcal Genetic Island

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