Novel bacterial artificial chromosome vector pUvBBAC for use in studies of the functional genomics of Listeria spp

Bacterial artificial chromosome (BAC) vectors are important tools for microbial genome research. We constructed a novel BAC vector, pUvBBAC, for replication in both gram-negative and gram-positive bacterial hosts. The pUvBBAC vector was used to generate a BAC library for the facultative intracellular pathogen Listeria monocytogenes EGD-e. The library had insert sizes ranging from 68 to 178 kb. We identified two recombinant BACs from the L. monocytogenes pUvBBAC library that each contained the entire virulence gene cluster (vgc) of L. monocytogenes and transferred them to a nonpathogenic Listeria innocua strain. Recombinant L. innocua strains harboring pUvBBAC+vgc1 and pUvBBAC+vgc2 produced the vgc-specific listeriolysin (LLO) and actin assembly protein ActA and represent the first reported cloning of the vgc locus in its entirety. The use of the novel broad-host-range BAC vector pUvBBAC extends the versatility of this technology and provides a powerful platform for detailed functional genomics of gram-positive bacteria as well as its use in explorative functional metagenomics.     

基因组细菌人工染色体文库(BAC)的构建及应用

细菌人工染色体 (BAC)是一种承载DNA大片段的克隆载体系统 ,用于人、动物和植物基因组文库构建。BAC具有插入片断大、嵌合率低、遗传稳定性好、易于操作等优点。BAC文库的构建是基因组较大的真核生物基因组学研究的重要基础 ,可用于真核生物重要基因及全基因组物理作图、重要性状基因的图位克隆、基因结构及功能分析。本文主要综述了细菌人工染色体的构建与其鉴定 ,及其在物理图谱构建、图位克隆、转基因技术等研究上的应用。     

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.     

关于细菌人工染色体(BAC)文库载体DNA制备的研究

细菌人工染色体（BAC）文库在基因组研究中起着关键作用。构建BAG文库的一个关键步骤就是BAC载体DNA的制备,制备高质量的BAC载体DNA受到包括酶切,脱磷等诸多因素的影响。以BAC载体pECBAC1为材料,分别采用限制性内切酶BamHⅠ和HK脱磷酶对其进行酶切和脱磷,并结合凝胶回收缩化技术,制备了可用于进一步构建BAC文库的线性载体DNA。并在此基础上,确定了制备BAC载体DNA的适宜条件,其中包括确定适宜限制内切酶用量及酶切时间,脱磷酶种类及浓度和凝胶回收纯化线性载体DNA等关系步骤。     

细菌人工染色体的研究和应用

细菌人工染色体 (Bacterialartificialchromosome ,BAC)是第二代大片段DNA的克隆载体系统。因其嵌合率低 ,遗传稳定性好 ,重组DNA容易分离和制备 ,转化效率高等 ,弥补了YAC的不足 ,很快在基因组研究中处于中心地位。近年来 ,已有多种BAC载体被构建出来 ,这些BAC载体在复杂基因组大片段文库的构建 ,基因的图位克隆 ,基因组物理图谱的构建 ,基因和基因组测序 ,基因组织结构分析 ,染色体组织和进化 ,以及基因的遗传转化和调控研究中得到了广泛的应用。     

A Modular, Positive Selection Bacterial Artificial Chromosome Vector with Multiple Cloning Sites

To construct large-insert libraries for the sequencing, mapping, and functional studies of complex genomes, we have constructed a new modular bacterial artificial chromosome (BAC) vector, pBACe3.6 (GenBank Accession No. U80929). This vector contains multiple cloning sites located within the sacB gene, allowing positive selection for recombinant clones on sucrose-containing medium. A recognition site for the PI-SceI nuclease has also been included, which permits linearization of recombinant DNA irrespective of the characteristics of the insert sequences. An attTn7 sequence present in pBACe3.6 permits retrofitting of BAC clones by Tn7-mediated insertion of desirable sequence elements into the vector portion. The ability to retrofit BAC clones will be useful for functional analysis of genes carried on the cloned inserts. The pBACe3.6 vector has been used for the construction of many genomic libraries currently serving as resources for large-scale mapping and sequencing.     

New Resources for Marine Genomics: Bacterial Artificial Chromosome Libraries for the Eastern and Pacific Oysters (Crassostrea virginica and C. gigas)

Large-insert genomic bacterial artificial chromosome (BAC) libraries of two culturally and economically important oyster species, Crassostrea virginica and C. gigas, have been developed as part of an international effort to develop tools and reagents that will advance our ability to conduct genetic and genomic research. A total of 73,728 C. gigas clones with an average insert size of 152 kb were picked and arrayed representing an 11.8-fold genome coverage. A total of 55,296 clones with an average insert size of 150 kb were picked and arrayed for C. virginica, also representing an 11.8-fold genome coverage. The C. gigas and C. virginica libraries were screened with probes derived from selected oyster genes using high-density BAC colony filter arrays. The probes identified 4 to 25 clones per gene for C. virginica and 5 to 50 clones per gene for C. gigas. We conducted a preliminary analysis of genetic polymorphism represented in the C. gigas library. The results suggest that the degree of divergence among similar sequences is highly variable and concentrated in intronic regions. Evidence supporting allelic polymorphism is reported for two genes and allelic and/or locus specific polymorphism for several others. Classical inheritance studies are needed to confirm the nature of these polymorphisms. The oyster BAC libraries are publicly available to the research community on a cost-recovery basis at     

细菌人工染色体载体的发展和应用

细茵人工染色体(Bacterial artificial chromosome,BAC)是在大肠杆菌F质粒的基础上建成的高容量、低拷贝的质粒载体.BAC载体已广泛应用于基因组文库的构建及筛选、基因组测序、新基因的发现、图位克隆、BAC微阵列、转基因和动物品种资源保存等方面.本文综述了BAC载体的发展及其应用.     

Construction of a Bacterial Artificial Chromosome Library of TM-1, a Standard Line for Genetics and Genomics in Upland Cotton

A bacterial artificial chromosome （BAC） library was constructed for Gossypium hirsutum acc. TM-1, a genetic and genomic standard line for Upland cotton. The library consists of 147 456 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb. About 96.0% of the clones have inserts over 100 kb. Therefore, this library represents theoretically 7.4 haploid genome equivalents based on an AD genome size of 2 425 Mb. Clones were stored in 384 384- well plates and arrayed into multiplex pools for rapid and reliable library screening. BAC screening was carried out by four-round poiymerase chain reactions using 23 simple sequence repeats （SSR） markers, three sequence-related amplified polymorphism markers and one pair of primers for a gene associated with fiber development to test the quality of the library. Correspondingly, in total 92 positive BAC clones were identified with an average four positive clones per SSR marker, ranging from one to eight hits. Additionally, since these SSR markers have been localized to chromosome 12 （A12） and 26 （D12） according to the genetic map, these BAC clones are expected to serve as seeds for the physical mapping of these two homologous chromosomes, sequentially map-based cloning of quantitative trait loci or genes associated with important agronomic traits.     

Construction of a Bacterial Artificial Chromosome Library of TM-1, a Standard Line for Genetics and Genomics in Upland Cotton

A bacterial artificial chromosome (BAC) library was constructed for Gossyplum hirsutum acc. TM-1, a genetic and genomic standard line for Upland cotton. The library consists of 147 456 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb. About 96.0% of the clones have inserts over 100 kb. Therefore, this library represents theoretically 7.4 haploid genome equivalents based on an AD genome size of 2 425 Mb. Clones were stored in 384 384- well plates and arrayed into multiplex pools for rapid and reliable library screening. BAC screening was carded out by four-round polymerase chain reactions using 23 simple sequence repeats (SSR) markers, three sequence-related amplified polymorphism markers and one pair of pdmere for a gene associated with fiber development to test the quality of the library. Correspondingly, in total 92 positive BAC clones were Identified with an average four positive clones per SSR marker, ranging from one to eight hits. Additionally, since these SSR markers have been localized to chromosome 12 (A12) and 26 (D12) according to the genetic map, these BAC clonee are expected to serve as seeds for the physical mapping of these two homologous chromosomes, sequentially map-based cloning of quantitative trait loci or genes associated with Important agronomic traits.     

水稻双元细菌人工染色体载体系统转化体系的建立

普通双元载体己被广泛碰用于农杆菌介导的植物转化,但这类载体通常只能转移5～20kb的外源DNA片段;而双元细菌人工染色体（BIBAC）载休可以弥补普通双元裁体的不足,通过它已在烟草、番茄等双子叶植物中实现了大片段DNA（150kb）的转移。BIBAC载体在单子叶植物转化中的应用尚未见报道。面于单、双子叶植物间以及大、小片段转化间的转化体系存在明显差异,常规的农杆菌介导的水稻转化体系不能适应BIBAC系统转化的要求。因此,建立适于BIBAC系统的水稻转化体系是十分必要的。通过比较不同的受体材料,不同的预培养、其培养条件,不同的去除农杆菌及选择阳性愈伤的方式等对转化效率的影响,建矿了适合水稻BIBAC系统的转化体系。该体系的技术要点包括：以水稻品种H1493为转化受体：以含毒性辅助质粒pCH32的LBA4404菌株（HP4404）为侵染菌株;预培养的培养拱pH5．6：以N6A代替AAM悬浮农杆菌：侵染菌液浓度为OD600=1．0;共培养温度为24℃;采用过渡（Resting）培养除去农杆菌;采用二步法进行选择等。基于PCR检测、Southern印迹分析的结果表明,BIBAC载体所携带的插入片段及标记基因已整合到转化植株的基因组中。这个体系的建立为在水稻中利用BIBAC系统进行大片段DNA转化奠定基础。     

可转化人工染色体(TAC)文库载体DNA的制备

对可转化人工染色体(TAC)pYLTAC747NH/sacB文库载体DNA的制备条件进行了较系统的模索和研究.结果表明,该文库载体经碱裂解法提取、QIAGEN Plasmid Mini kit纯化后,可获得较纯净的载体DNA.对其闭环载体DNA分别用不同酶量的Hinid Ⅲ酶切处理,经琼脂糖凝胶电泳检测得出其最佳Hind Ⅲ完全酶切条件为2 U Hind Ⅲ/μg闭环载体DNA、37℃酶切30 min;分别用0.5 MBU和1 MBU HK脱磷酶/μg对其线性载体DNA进行脱磷处理,经电泳和载体自连产物电转化检测表明其适宜的完全脱磷条件为1 MBU HK脱磷酶/μg线性载体DNA,30℃脱磷1 h;将所制备的线性载体DNA与λ DNA/Hind Ⅲ酶切片段进行连接,连接产物转化频率较高,其电转化大肠杆菌DH10B感受态细胞频率可达到9.6×10s.     

细菌人工染色体（BAC）及其分析和修饰方法简介

细菌人工染色体是一种新发展起来的DNA载体系统,它具有容量大、遗传特性稳定、易于操作等优点.在基因文库构建和基因功能分析等方面有广泛的应用.综述了近年来发展起来的对BAC进行分析和修饰的一些方法.     

细菌人工染色体文库的构建方法分析

细菌人工染色体是一种承载大片段DNA的新型载体系统,它具有插入片断大、嵌合率低、遗传稳定性好、易于操作等优点.在高等生物基因组文库的构建和基因功能的分析等方面有广泛应用.BAC文库的构建是基因组较大的真核生物基因组学研究的重要基础.介绍了近年来细菌人工染色体文库构建方法上的研究进展,对其中载体的制备和高分子量DNA的制备这两个关键环节作了较深入的探讨.     

细菌人工染色体文库的构建及应用

细菌人工染色体（BAC）是第二代大片段DNA的克隆载体系统,具有容量大、嵌合率低、遗传特性稳定、转化效率高、插入片段易回收、操作简便等优点,因而被广泛应用于基因组较大的真核生物基因组研究中,并发挥着前所未有的重要作用。本文综述了BAC的发展,利用此载体构建基因组文库的程序和鉴定方法,及其在物理图谱构建、图位克隆、基因组测序、转基因技术等研究中的应用。     

细菌人工染色体基因组文库构建方法的改进

目的:建立一种改进的更简便、易操作的细菌人工染色体(BAC)文库构建方法。方法:在构建猪霍乱沙门氏菌基因组大片段DNA的BAC文库时,对改进的基因组BAC文库构建方法和常规的BAC文库构建方法进行比较。结果:利用改进的方法可简便快速地构建猪霍乱沙门氏菌基因组BAC文库。结论:使用2种方法构建BAC文库,其转化效率,以及在BAC克隆中插入的DNA片段的大小和BAC克隆的稳定性等都相同,从而表明改进的方法更简单、更方便,它能使BAC文库的构建更为高效。     

细菌人工染色体基因组文库构建关键技术研究

基因组文库是进行分子克隆和基因结构与功能研究的基础,完整覆盖的基因组文库的构建,使基因组任何DNA片段的筛选和获得成为可能.细菌人工染色体(Bacterial Artificial Chromosome,BAC)与其它载体系统相比具有转化效率高、嵌合体少、插入片段易回收,高覆盖率、稳定性强,并且具有易分离和操作等特性等优点而被广泛应用.作为物种保护策略的重要部分,构建我国濒危动植物品种基因组BAC文库,对于其遗传资源的保护和研究具有非常重要的作用.在查阅大量国内外相关资料的基础上,就BAC基因组文库构建过程中栽体的制备、高分子量DNA的制备、大片段DNA的回收、连接与电击转化、基因组BAC文库质量鉴定等几个重点、难点问题进行了详细的论述和分析,对于构建高分子量插入片段、高覆盖率和稳定性的基因组文库提供理论基础与技术支撑.     

Genetically Modified Bacterial Strains and Novel Bacterial Artificial Chromosome Shuttle Vectors for Constructing Environmental Libraries and Detecting Heterologous Natural Products in Multiple Expression Hosts

The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery efforts. Our investigators reported previously on the creation and screening of an Escherichia coli library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector. In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in E. coli. To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include Streptomyces lividans and Pseudomonas putida as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities. These tools include derivatives of S. lividans that contain complete and unmarked deletions of the act and red endogenous pigment gene clusters, a derivative of P. putida that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from E. coli to both S. lividans and P. putida by high-throughput conjugation. Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters.     

Genomic Bacterial Artificial Chromosome Library of the Japanese Flounder Paralichthys olivaceus

We have constructed a genomic bacterial artificial chromosome (BAC) library from homozygous cloned Japanese flounder Paralichthys olivaceus using the pBAC-lac vector. This BAC library consists of about 49,100 clones and is deposited in 128 microtiter plates with 384 wells. The average size of inserted DNA was calculated to be 165 kb. The BAC library was determined to cover 9 times the Japanese flounder haploid genome. The Japanese flounder genomic BAC library will be useful for gene isolation as well as quantitative trait loci (QTL) analysis. Received March 1, 2000; accepted May 29, 2000.