Construction of a rice bacterial artificial chromosome library and identification of clones linked to the Xa-21 disease resistance locus

A bacterial artificial chromosome (BAC) library consisting of 11 000 clones with an average DNA insert size of 125 kb was constructed from rice nuclear DNA. The BAC clones were stable in E. coli after 100 generations of serial growth. Transformation of the BAC clones by electroporation into E. coli was highly efficient and increased with decreasing size of the DNA inserts. The library was evaluated for the presence of organellar, repeated, and telomeric sequences. A very low percentage (<0.3%) of the library consisted of chloroplast and mitochondrial clones. Eighteen BACs were identified that hybridized with an Arabidopsis telomere repeat. Sixteen BACs hybridized with the AA genome-specific repetitive sequence pOs48. Twelve clones were isolated that hybridized with three DNA markers linked to the Xa-21 disease resistance locus. The results indicate that the BAC system can be used to clone and manipulate large pieces of plant DNA efficiently.     

Characterization of a large human transgene following invasin-mediated delivery in a bacterial artificial chromosome

Bacterial artificial chromosomes (BACs) are widely used in transgenesis, particularly for the humanization of animal models. Moreover, due to their extensive capacity, BACs provide attractive tools to study distal regulatory elements associated with large gene loci. However, despite their widespread use, little is known about the integration dynamics of these large transgenes in mammalian cells. Here, we investigate the post-integration structure of a ~260 kb BAC carrying the cystic fibrosis transmembrane conductance regulator (CFTR) locus following delivery by bacterial invasion and compare this to the outcome of a more routine lipid-based delivery method. We find substantial variability in integrated copy number and expression levels of the BAC CFTR transgene after bacterial invasion-mediated delivery. Furthermore, we frequently observed variation in the representation of different regions of the CFTR transgene within individual cell clones, indicative of BAC fragmentation. Finally, using fluorescence in situ hybridization, we observed that the integrated BAC forms extended megabase-scale structures in some clones that are apparently stably maintained at cell division. These data demonstrate that the utility of large BACs to investigate cis-regulatory elements in the genomic context may be limited by recombination events that complicate their use.     

Development of a novel bacterial artificial chromosome cloning system for functional studies

Bacterial artificial chromosome (BAC) cloning systems currently in use generate high quality genomic libraries for gene mapping, identification, and sequencing. However, the most commonly used BAC cloning systems do not facilitate functional studies in eukaryotic cells. To overcome this limitation, we have developed pEBAC190G, a new BAC vector that combines the features of the first generation PAC/BAC vectors with eukaryotic elements that facilitate the transfection, episomal maintenance, and functional analysis of large genomic fragments in eukaryotic cells. A number of different cloning strategies may be used to retrofit genomic fragments from existing libraries into the new vector. The system was tested by the retrofitting of a 170kb NotI genomic fragment from the RPCI-11 BAC library into the NotI site of pEBAC190G. Clones from any eukaryotic genomic library harboured in this vector can be transferred from bacteria directly to eukaryotic cells for functional analysis.     

Generation of Pax2-Cre mice by modification of a Pax2 bacterial artificial chromosome

We generated a transgenic mouse line named E1-Ngn2/Cre that expresses Cre recombinase and GFP under the control of the E1 enhancer element of the gene Ngn2 (Scardigli et al.: Neuron 31:203-217, 2001). Cre-recombinase activity and GFP fluorescence are consistent with the reported expression pattern controlled by the E1-Ngn2 enhancer. Recombination was detected in the progenitor domains p1 and p2 in the ventricular zone of the neural tube and in distinct domains of the pretectum, the dorsal and ventral thalamus, the tegmentum of the mesencephalon, and the hindbrain. In the developing cortex, Cre-recombinase activity is confined to a subpopulation of progenitors predominantly in the region of the ventral and lateral pallium. The E1-Ngn2/Cre mouse line thus provides an excellent novel tool for a region-specific conditional mutagenesis in the developing CNS.     

Construction of a binary bacterial artificial chromosome library of Petunia inflata and the isolation of large genomic fragments linked to the self-incompatibility (S-) locus.

The Solanaceae family of flowering plants possesses a type of self-incompatibility mechanism that enables the pistil to reject self pollen but accept non-self pollen for fertilization. The pistil function in this system has been shown to be controlled by a polymorphic gene at the S-locus, termed the S-RNase gene. The pollen function is believed to be controlled by another as yet unidentified polymorphic gene at the S-locus, termed the pollen S-gene. As a first step in using a functional genomic approach to identify the pollen S-gene, a genomic BAC (bacterial artificial chromosome) library of the S2S2 genotype of Petunia inflata, a self-incompatible solanaceous species, was constructed using a Ti-plasmid based BAC vector, BIBAC2. The average insert size was 136.4 kb and the entire library represented a 7.5-fold genome coverage. Screening of the library using cDNAs for the S2-RNase gene and 13 pollen-expressed genes that are linked to the S-locus yielded 51 positive clones, with at least one positive clone for each gene. Collectively, at least 2 Mb of the chromosomal region was spanned by these clones. Together, three clones that contained the S2-RNase gene spanned approximately 263 kb. How this BAC library and the clones identified could be used to identify the pollen S-gene and to study other aspects of self-incompatibility is discussed.     

Characterization of a human chromosome 22 enriched bacterial artificial chromosome sublibrary

Selection of chromosomal sublibraries from total human genomic libraries is critical for chromosome-based physical mapping approaches. We have previously reported a method of screening total human genomic library using flow sorted chromosomal DNA as a hybridization probe and selection of a human chromosome 22-enriched sublibrary from a total human bacterial artificial chromosome (BAC) library (Nucleic Acids Res 1995; 23: 1838–1839). We describe here further details of the method of construction as well as characterization of the chromosome 22-enriched sublibrary thus constructed. Nearly 40% of the BAC clones that have been mapped by fluorescence in situ hybridization (FISH) analysis were localized to chromosome 22. By screening the sublibrary using chromosome 22-specific hybridization probes, we estimated that the sublibrary represents at least 2.5 × coverage of chromosome 22. This is in good agreement with the results from FISH mapping experiments. FISH map data also indicate that chromosome 22-specific BACs in the sublibrary represent all the subregions of chromosome 22.     

A bacterial artificial chromosome contig spanning the major domestication locus Q in wheat and identification of a candidate gene

The Q locus played a major role in the domestication of wheat because it confers the free-threshing character and influences many other agronomically important traits. We constructed a physical contig spanning the Q locus using a Triticum monococcum BAC library. Three chromosome walking steps were performed by complete sequencing of BACs and identification of low-copy markers through similarity searches of database sequences. The BAC contig spans a physical distance of approximately 300 kb corresponding to a genetic distance of 0.9 cM. The physical map of T. monococcum had perfect colinearity with the genetic map of wheat chromosome arm 5AL. Recombination data in conjunction with analysis of fast neutron deletions confirmed that the contig spanned the Q locus. The Q gene was narrowed to a 100-kb segment, which contains an APETALA2 (AP2)-like gene that cosegregates with Q. AP2 is known to play a major role in controlling floral homeotic gene expression and thus is an excellent candidate for Q.     

稻曲病菌UV-2菌株细菌人工染色体文库构建及分析

【目的】稻曲病(Rice false smut)是由稻曲病菌[Villosiclava virens (Cooke) Tak.]引起的严重危害水稻的真菌病害。构建稻曲病菌UV-2的大片段DNA细菌人工染色体(Bacterial artificial chromosome, BAC)文库, 为致病相关基因的鉴定及在图位克隆、比较基因组学等方面的研究奠定基础。【方法】以幼嫩菌丝为材料制备大分子基因组DNA包埋块, 用Hind III部分酶解后经脉冲凝胶电泳筛选, 回收大片段DNA并与pIndigoBAC536-S 载体连接, 连接产物转化大肠杆菌菌株DH10B T1 Phage-Resistant 细胞后进行蓝白斑筛选, 白色菌落捡入384孔板置于?80 °C低温保存。【结果】成功构建UV-2菌株的高质量、高覆盖度的BAC文库, 该文库共含10 368个克隆, 平均插入片段为124.4 kb, 空载率小于1%, 约覆盖该菌基因组的36.8倍。【结论】克服了真菌大分子基因组DNA制备难控制的技术难题, 建立了首个稻曲病菌的BAC文库。该文库已作为一种公共基因组资源向研究者开放(http://GResource.hzau.edu.cn)。     

Melon bacterial artificial chromosome (BAC) library construction using improved methods and identification of clones linked to the locus conferring resistance to melon Fusarium wilt (Fom-2).

Utilizing improved methods, two bacterial artificial chromosome (BAC) libraries were constructed for the multidisease-resistant line of melon MR-1. The HindIII library consists of 177 microtiter plates in a 384-well format, while the EcoRI library consists of 222 microtiter plates. Approximately 95.6% of the HindIII library clones contain nuclear DNA inserts with an average size of 118 kb, providing a coverage of 15.4 genome equivalents. Similarly, 96% of the EcoRI library clones contain nuclear DNA inserts with an average size of 114 kb, providing a coverage of 18.7 genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. High-density filters were screened with two genetic markers FM and AM that co-segregate with Fom-2, a gene conferring resistance to races 0 and 1 of Fusarium wilt. Fourteen and 18 candidate BAC clones were identified for the FM and AM probes, respectively, from the HindIII library, while 34 were identified for the AM probe from filters A, B, and C of the EcoRI library.     

Physical mapping across an avirulence locus of Phytophthora infestans using a highly representative, large-insert bacterial artificial chromosome library

The oomycete plant pathogen Phytophthora infestans is the causal agent of late blight, one of the most devastating diseases of potato worldwide. As part of efforts to clone avirulence (Avr) genes and pathogenicity factors from P. infestans, we have constructed a bacterial artificial chromosome (BAC) library from an isolate containing six Avr genes. The BAC library comprises clones with an average insert size of 98 kb and represents an estimated 10 genome equivalents. A three-dimensional pooling strategy was developed to screen the BAC library for amplified fragment length polymorphism (AFLP) markers, as this type of marker has been extensively used in construction of a P. infestans genetic map. Multiple positive clones were identified for each AFLP marker tested. The pools were used to construct a contig of 11 BAC clones in a region of the P. infestans genome containing a cluster of three avirulence genes. The BAC contig is predicted to encompass the Avr11 locus but mapping of the BAC ends will be required to determine if the Avr3 and Avr10 loci are also present in the BAC contig. These results are an important step towards the positional cloning of avirulence genes from P. infestans, and the BAC library represents a valuable resource for largescale studies of oomycete genome organisation and gene content.     

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.     

Yeast artificial chromosome mapping of the cystinosis locus on chromosome 17p by fluorescence in situ hybridization

The gene locus for cystinosis has been mapped between markers D17S1583 and D17S1584 on the short arm of chromosome 17. Using markers encompassing the cystinosis region, we assigned different yeast artificial chromosome (YAC) clones previously identified by sequence tagged site (STS) screening to 17p13.3. Three of the clones hybridized to the target 17p gene region; one of these was chimeric, hybridizing both to chromosomes 3p and 5q; two of the YACs did not contain sequences of 17p13.3. Our physical mapping has identified candidate YACs as a first step towards a positional cloning approach. Received: 28 February 1996 / Revised: 3 May 1996     

Construction and characterization of a bacterial artificial chromosome library of apple

 A bacterial artificial chromosome (BAC) library has been constructed from apple (Malus×domestica Borkh.) using the variety “Florina”, which is resistant to scab (Venturia inaequalis) by virtue of the Vf gene. Since apple leaves are rich in polyphenols, high-molecular-weight DNA was extracted from leaf nuclei with a protocol adapted to apple. The nuclei were then embedded in agarose microbeads and partially digested by varying ratios of EcoRI to EcoRI methylase. The resulting DNA fragments were size-selected by pulsed-field gel electrophoresis, ligated to the BAC cloning vector pECBAC1 and transformed into Escherichia coli cells by electroporation. A total of 36 864 recombinant clones (BACs) were obtained. The library has an average insert size of 120 kb and represents approximately 5×apple haploid-genome equivalents. It was screened with six cDNA probes using the chemiluminescent DIG system. An average of 4.4 clones was detected for each locus. The apple BAC library will be used to isolate the Vf scab resistance gene through map-based cloning. In this connection the library was screened with a marker closely linked to the Vf gene and six positive clones have been isolated. This library should thus be well suited for map-based gene cloning, in particular for the isolation of the Vf gene and for the construction of a physical map of the apple genome. Received: 19 February 1998 / Accepted: 30 April 1998     

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.     

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 Schistosoma mansoni bacterial artificial chromosome library

A bacterial artificial chromosome (BAC) library has been established from genomic DNA isolated from the trematode parasite of human, Schistosoma mansoni. This library consists of more than 21,000 recombinant clones carrying inserts in the pBeloBAC11 vector. The mean insert size was 100 kb, representing an approximate 7.95-fold genome coverage. Library screening with eight chromosome-specific or single-copy gene probes yielded between 1 and 9 positive clones, and none of those tested was absent from the library. End sequences were obtained for 93 randomly selected clones, and 37 showed sequence identity to S. mansoni sequences (ESTs, genes, or repetitive sequences). A preliminary analysis by fluorescence in situ hybridization localized 8 clones on schistosome chromosomes 1 (2 clones), 2, 3, 5, Z, and W (3 clones). This library provides a new resource for the physical mapping and sequencing of the genome of this important human pathogen.     

Construction and characterization of a common bean bacterial artificial chromosome library

We have constructed a common bean (Phaseolus vulgaris L.) bacterial artificial chromosome (BAC) library consisting of 33 792 clones and an estimated 3- to 5-fold coverage of the common bean genome. Leaf nuclei were used as the source for high-molecular-weight DNA, and an endonuclease/methylase competition assay was employed to partially cleave the DNA. The library was screened with a number of nuclear and mitochondrial probes. Each nuclear probe identified at least two BACs with an average insert size of ca. 100 kb. Only 26 clones were identified after hybridizing with mitochondrial probes, indicating contamination with organellar sequences is low. Numerous clones could be identified after screening the library with two repetitive probes flanking the nuclear fertility restorer Fr. Intriguingly, 12 clones appeared to hybridize to both markers, and restriction analysis of these clones revealed that they can be assembled into maximally four contigs, suggesting that these repetitive probes may be useful for the physical mapping of the Fr locus.