白氏文昌鱼单个体基因组BAC文库的构建

隶属于头索动物亚门的文昌鱼是现存生物中最近似于脊椎动物亚门直接祖先的一个类群, 具有重要的进化地位, 是研究脊椎动物原始祖先的重要材料和模式动物。随着文昌鱼实验室连续繁殖的成功, 全基因组测序成为中国文昌鱼模式化急需完成的工作之一。文章从单条雄性白氏文昌鱼精巢组织中提取高质量的基因组DNA, 经EcoRⅠ限制性内切酶和EcoRⅠ甲基化酶酶切, 脉冲场电泳选择合适酶切DNA片段, 连接线性磷酸化的载体pCC1BAC, 转化大肠杆菌EPI300 E. coli, 构建了含有44 706个克隆的全基因组BAC( Bacterial artificial chromosome)文库, 该文库平均插入片段80 kb,具有9倍的基因组覆盖率, 基本能够满足功能基因等研究需要, 为中国文昌鱼全基因组测序打下基础。     

Construction and characterization of the IGF Arabidopsis BAC library

A bacterial artificial chromosome (BAC) library has been established for Arabidopsis thaliana (ecotype Col-0) covering about seven haploid nuclear genome equivalents. This library, called the Institut für Genbiologische Forschung (IGF) BAC library, consists of 10?752 recombinant clones carrying inserts (generated by partial EcoRI digestion) of an average size of about 100?kb in a modified BAC vector, pBeloBAC-Kan. Hybridization with organellar DNA and nuclear repetitive DNA elements revealed the presence of 1.1% clones with mitochondrial DNA, 0.2% clones with plastid DNA, 3.2% clones with the 180?bp paracentromeric repeat, 1.6% clones with 5S rDNA, and 10.8% clones with the 18S-25S rDNA repeat. With its extensive genome coverage, its rather uniformly sized inserts (80?kb?<85% <120?kb) and low contamination with organellar DNA, this library provides an excellent resource for A. thaliana genomic mapping, map-based gene cloning, and genome sequencing.     

Construction of a BAC library of pearl millet, Pennisetum glaucum

A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from pearl millet (Pennisetum glaucum), and used as a resource for the isolation of microsatellite sequences. The library contains a total of 159,100 clones with an average insert size of 90 kb, and corresponds to 5.8 haploid genome equivalents. The BAC library was pooled for screening by the polymerase chain reaction (PCR) as well as robotically gridded on high-density filters. PCR-based screening of a subset of the library (4.7 haploid genome equivalents) using five sequence-tagged site (STS) and six microsatellite markers identified between 2 and 11 positives superpools (5.4 on average). The frequency of BAC clones carrying inserts of chloroplast DNA was estimated to be less than 1% by hybridisation with a rice chloroplast probe. Received: 30 January 2000 / Accepted: 16 October 2000     

Insight into Trichoderma reesei's genome content, organization and evolution revealed through BAC library characterization

Trichoderma reesei is an important industrial fungus known for its ability to efficiently secrete large quantities of protein as well as its wide variety of biomass degrading enzymes. Past research on this fungus has primarily focused on extending its protein production capabilities, leaving the structure of its 33 Mb genome essentially a mystery. To begin to address these deficiencies and further our knowledge of T. reesei's secretion and cellulolytic potential, we have created a genomic framework for this fungus. We constructed a BAC library containing 9216 clones with an average insert size of 125 kb which provides a coverage of 28 genome equivalents. BAC ends were sequenced and annotated using publicly available software which identified a number of genes not seen in previously sequenced EST datasets. Little evidence was found for repetitive sequence in T. reesei with the exception of several copies of an element with similarity to the Podospora anserina transposon, PAT. Hybridization of 34 genes involved in biomass degradation revealed five groups of co-located genes in the genome. BAC clones were fingerprinted and analyzed using fingerprinted contigs (FPC) software resulting in 334 contigs covering 28 megabases of the genome. The assembly of these FPC contigs was verified by congruence with hybridization results.     

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。     

Construction of a full bacterial artificial chromosome （BAC） library of Oryza sativa genome

We have constructed a full BAC library for the superior early indica variety of Oryza sativa,Guang Lu Ai 4.The MAX Efficiency DH10B with increased stability of inserts was used as BAC host cells.The potent pBelo BACII with double selection markers was used as cloning vector.The cloning efficiency we have reached was as high as 98%,and the transformation efficiency was raised up to 10^6 transformants/μg of large fragment DNA.The BAC recombinant transformants were picked at random and analyzed for the size of inserts,which turned out to be of 120 kb in length on average.We have obtained more than 20,000 such BAC clones.According to conventional probability equation,they covered the entire rice genome of 420,000 kb in length.The entire length of inserts of the library obtained has the 5-to 6-fold coverage of the genome.To our knowledge,this is the first reported full BAC library for a complex genome.     

Construction of a bacterial artificial chromosome library containing large Eco RI and Hin dIII genomic fragments of lettuce

 Existing bacterial artificial chromosome (BAC) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5 and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLP^TM markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA. Received: 5 August 1996 / Accepted: 23 August 1996     

A BAC Library Constructed to the Rice Cultivar

"Minghui 63" is the restorer line for a number of the most important commercial rice hybrids varieties in China. To facilitate long-term commitment in genetic analysis and molecular cloning of the superior genes in the genome of "Minghui 63", the authors have constructed a largeinsert genomic DNA library using the bacterial artificial chromosome (BAC) cloning vector (pBe- loBAC 11). Size fractionated Hind m digest of genomic DNA was ligated to the BAC vector, and the ligation mixture was used to transform the bacterial strain DH10B. A total of over 26 000 clones were obtained with the average insert size of about 150 kb, ranging from 90 to 240 kb. These clones thus represent 9 x rice haploid genome equivalents. The library is now being used for physical mapping of several genomic regions for map-based gene cloning.     

Structural analysis of Arabidopsis thaliana chromosome 3. II. Sequence features of the 4,251,695 bp regions covered by 90 P1, TAC and BAC clones.

To deduce the entire sequence of the top arm of the Arabidopsis thaliana chromosome 3, the sequence determination was performed on a total of 90 P1, TAC and BAC clones chosen according to our sequencing strategy. Sequence features of the resulting 4,251,695 bp regions were analyzed with various computer programs for similarity search and gene modeling. As a result, a total of 941 potential protein-coding genes were identified. The average density of the genes identified was 1 gene per 4210 bp. Introns were observed in 73% of the genes, and the average number per gene and the average length of the introns were 3.6 and 159 bp, respectively. These sequence features are essentially identical to those of chromosomes 3 and 5 in our previous reports. The regions also contained 14 tRNA genes when searched by similarity to reported tRNA genes and the tRNA scan-SE program. The sequence data and information on the potential genes are available through the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/kaos/.     

Construction and characterisation of a BAC library from Arabidopsis halleri: Evaluation of physical mapping based on conserved synteny with Arabidopsis thaliana

We constructed and characterized the first large-insert DNA BAC library for Arabidopsis halleri, a close relative of Arabidopsis thaliana. Double size selection of high molecular weight DNA was performed to increase the average insert size. The BAC library consists of 6128 clones of which 87% have an insert size above 125 kb. Organellar DNA contamination is estimated to 1.4%. The coverage of the library is equivalent to 4.5 times the haploid genome (250 Mb), indicating the library is suitable for almost any application. We explored the possibility of generating a physical map of A. halleri using the high conserved synteny existing between this species and A. thaliana. A set of unigenes separated by 50 kb in a 850 kb region of A. thaliana chromosome II was used to probe the library. The A. halleri BAC clones isolated with these probes were grouped into two contigs. Analysis of BAC-end sequences revealed that the two A. halleri genomic contigs were highly colinear with the A. thaliana genome. Therefore, the exploitation of the conserved synteny existing between the two species will greatly facilitate the construction of a raw full physical map of A. halleri.     

BAC library development for allotetraploid Leymus (Triticeae) wildryes enable comparative genetic analysis of lax-barrenstalk1 orthogene sequences and growth habit QTLs

Tall-caespitose basin wildrye (Leymus cinereus) and rhizomatous creeping wildrye (Leymus triticoides) are perennial Triticeae relatives of wheat and barley. Quantitative trait loci (QTLs) controlling rhizome proliferation were previously detected on homoeologous regions of LG3a and LG3b in two full-sib families derived from allotetraploid hybrids. Triticeae homoeologous group 3 aligns to rice chromosome 1, which contains the rice lax panicle and maize barrenstalk1 orthogene responsible for induction of axillary branch meristems, but this gene has not been mapped or sequenced in Triticeae. We developed bacterial artificial chromosome (BAC) libraries representing 6.1 haploid equivalents of the tetraploid Leymus genome (10.7 Mb). Overgo probes designed from the lax-barrenstalk1 orthogene hybridized to 12 Leymus BAC clones. Deduced amino-acid sequences from seven BAC clones were highly conserved with the rice, maize, and sorghum lax-barrenstalk1orthogenes. Gene specific primers designed from two of the most divergent BAC clones map to homoeologous regions of Leymus LG3a and LG3b and align with the lax-barrenstalk1 orthogene on rice 1L. Comparisons of genomic DNA sequences revealed two other conserved regions surrounding the Leymus LG3a, rice, and sorghum lax-barrenstalk1 ortholoci, and one of these regions was also present in maize and Leymus LG3b sequences. Comparisons of Leymus LG3a and LG3b lax-barrenstalk1 coding sequences and flanking genomic regions elucidate molecular differences between subgenomes.     

Construction and characterization of a BAC library from a gynogenetic channel catfish Ictalurus punctatus

A bacterial artificial chromosome (BAC) library was constructed by cloning HindIII-digested high molecular weight DNA from a gynogenetic channel catfish, Ictalurus punctatus, into the vector pBeloBAC11. Approximately 53 500 clones were arrayed in 384-well plates and stored at -80°C (CCBL1), while clones from a smaller insert size fraction were stored at -80°C without arraying (CCBL2). Pulsed-field gel electrophoresis of 100 clones after NotI digestion revealed an average insert size of 165 kb for CCBL1 and 113 kb for CCBL2. Further characterization of CCBL1 demonstrated that 10% of the clones did not contain an insert. CCBL1 provides a 7.2-fold coverage of the channel catfish haploid genome. PCR-based screening demonstrated that 68 out of 74 unique loci were present in the library. This represents a 92% chance to find a unique sequence. These libraries will be useful for physical mapping of the channel catfish genome, and identification of genes controlling major traits in this economically important species.     

大片段DNA插入文库的研究进展

基因组DNA大片段插入文库作为基因组学和基因克隆的技术平台,它的发展主要经历了Cosmid文加,YACs文库,BACs文库三个阶段,文中对几种文库作了简单的比较和评价,对大片段文库的应用人了比较详实的介绍。作者根据自己的建库经验,重点介绍了BAC文库的构建。     

Construction of bacterial artificial chromosome library from electrochemical microorganisms

A microbial fuel cell is a device that directly converts metabolic energy into electricity, using electrochemical technology. The analysis of large genome fragments recovered directly from microbial communities represents one promising approach to characterizing uncultivated electrochemical microorganisms. To further assess the utility of this approach, we constructed large-insert (140 kb) bacterial artificial chromosome (BAC) libraries from the genomic DNA of a microbial fuel cell, which had been operated for three weeks using acetate media. We screened the expression of several ferric reductase activities in the Escherichia coli host, in order to determine the extent of heterologous expression of metal-ion-reducing enzymes in the library. Phylogenetic analysis of 16S rRNA gene sequences recovered from the BAC libraries indicates that they contain DNA from a wide diversity of microbial organisms. The constructed bacterial library proved a powerful tool for exploring metal-ion reductase activities, providing information on the electron transport pathway of electrochemical microbial (ECM) organisms.     

Construction of a bacterial artificial chromosome (BAC) library of Coprinus cinereus

A bacterial artificial chromosome (BAC) library of the genomic DNA of Coprinus cinereus strain MP#2 was constructed using the BAC vector pBACTZ, which carries the C. cinereus trp1 gene. The library consists of 1536 clones. Analysis of inserts in some of the clones suggested that the library covers five times the C. cinereus genome. Screening of the BAC clones using ten markers mapped on nine different chromosomes also indicated that the library is likely to cover the whole length of the genomic DNA. We show an example of transformation of C. cinereus with BACs containing inserts of longer than 170kb.     

Construction and characterization of a bacterial artificial chromosome library of peach

A peach [Prunus persica (L.) Batch] bacterial artificial chromosome (BAC) library of var. Jingyu was constructed. Jingyu is a traditional variety, that displays many of the important agronomic characters of stone fruits. Since peach leaves are rich in polysaccharides, high-molecular-weight (HMW) DNA was extracted from leaf nuclei using a protocol adapted to peach. The HMW DNA embedded in agarose plugs was partially digested by HindIII. After size-selection by pulsed field gel electrophoresis, the selected DNA fragments were ligated to pBeloBAC11 and transformed into E. coli DH10B cells by electroporation. In total 20,736 recombinant clones were obtained. The BAC library has an average insert size of 95 kb and represents approximately 6.7 peach haploid genome equivalents. The BAC clones were stable in E. coli cell after 100 generations. The lack of hybridization to chloroplast and mitochondrial genes demonstrated that the library is predominantly composed of nuclear DNA. The library was screened with two molecular markers, W4 and P20, that are linked to white flesh and nectarine genes of peach, respectively. Ten positive clones were detected. Their fingerprints will be used to determine clone relationships and assemble contigs. This library should be well-suited for the map-based cloning of peach genes and genome physical mapping. Received: 18 January 2000 / Accepted: 29 May 2000     

Construction and characterization of a gridded cattle BAC library

A bovine genomic large-insert bacterial artificial chromosome (BAC) library has been constructed from leukocytes of a Holstein-Friesian male. Size fractionated DpnII-digested genomic DNA was ligated to the dephosphorylated BamH1 ends of a pBACe3.6 vector. Approximately 8.3 x 10(4) individual BAC clones were picked into 384-well plates. Two-hundred and sixty-seven randomly chosen clones were characterized by pulsed-field gel electrophoresis (PFGE). The average insert size was 104 kb with a frequency of clones without inserts of 5.5%. Thirty-four BAC clones were mapped by fluorescence in situ hybridization (FISH) to cattle chromosomes. Three showed signals at more than one location, one of them on the centromeric regions of all autosomes, indicating that the clone contains centromeric repeats. A subset of these BAC clones was used for the development of sequence tagged sites. Both subcloning and direct sequencing of the BACs were used for generating sequence tagged site information. The clones from the library were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Membranes and superpools are available through the Resource Centre of the German Human Genome Project in Berlin (http:// www.rzpd.de).     

Construction and evaluation of a porcine bacterial artificial chromosome library

A porcine bacterial artificial chromosome (BAC) library consisting of 103,488 clones has been constructed. The average insert size in the BAC vector was calculated to be 133 kb based on the examination of 189 randomly selected clones, indicating that the library contained 4.4 genome equivalents. The library can be screened by two-step PCR. The first screening step is performed on 22 superpools, each containing 4704 clones (49 x 96 well plates). In the second screening step, 49 plates comprising a superpool are arrayed in a 7 x 7 matrix and 4D-PCR is performed. Screening of the library superpools by PCR for 125 marker sequences selected from different regions of swine genome revealed 123 sequences, indicating that the library is not biased. Subsequent screenings (4D-PCR) were successfully applied for identification of clones containing each marker sequence. This porcine BAC library and the PCR screening system are useful for isolation of genomic DNA fragments containing desired sequences.