Combination of overlapping bacterial artificial chromosomes by a two-step recombinogenic engineering method

Recombinogenic engineering or recombineering is a powerful new method to engineer DNA without the need for restriction enzymes or ligases. We report here a general method for using recombineering to combine overlapping bacterial artificial chromosomes (BACs) to build larger, unified BACs. In order to test the feasibility of using recombineering to combine two large DNA fragments (>20 kb), we constructed a unified BAC containing the full-length tyrosinase-related protein-1 (Tyrp-1) gene from two library-derived BACs, one containing the 5′ regulatory elements and the other containing the 3′ coding exons. This was achieved using a two-step homologous recombination method enabled by the bacteriophage λ Red proteins. In the first step, retrieval, a large DNA fragment (~22 kb) was retrieved from one of the original BACs. In the second step, recombination, the retrieved DNA fragment was inserted into the second original BAC to form the unified BAC containing all the desired Tyrp-1 sequence. To further demonstrate the general applicability of our approach, an additional DNA fragment (~20 kb) was inserted into the unified BAC downstream of the coding region. This method should prove very useful for enabling BAC manipulation in a variety of scenarios.     

A multi-step strategy for BAC recombineering of large DNA fragments

Recombineering techniques have been developed to modify bacterial artificial chromosomes (BACs) via bacterial homologous recombination systems, simplifying the molecular manipulations of large DNA constructs. However, precise modifications of a DNA fragment larger than 2-3 kb by recombineering remain a difficult task, due to technical limitations in PCR amplification and purification of large DNA fragments. Here, we describe a new recombineering strategy for the replacement of large DNA fragments using the commonly utilized phage/Red recombination host system. This approach involved the introduction of rare restriction enzyme sites and positive selection markers into the ends of a large DNA fragment, followed by its release from the donor BAC construct and integration into an acceptor BAC. We have successfully employed this method to precisely swap a number of large DNA fragments ranging from 6 to 40 kb between two BAC constructs. Our results demonstrated that this new strategy was highly effective in the manipulations of large genomic DNA fragments and therefore should advance the conventional BAC recombineering technology to the next level.     

A Low-Copy-Number Plasmid for Retrieval of Toxic Genes from BACs and Generation of Conditional Targeting Constructs

Bacterial Artificial Chromosome (BAC) clones are widely used for retrieving genomic DNA sequences for gene targeting. In this study, low-copy-number plasmids pBAC-FB, pBAC-FC, and pBAC-DE, which carry the F plasmid replicon, were generated from pBACe3.6. pBAC-FB was successfully used to retrieve a sequence of a BAC that was resistant to retrieval by a high-copy-number plasmid via λ Red-mediated recombineering (gap-repair cloning). This plasmid was also used to retrieve two other genes from BAC, indicating its general usability retrieving genes from BAC. The retrieved genes were manipulated in generating targeting vectors for gene knockouts by recombineering. The functionality of the targeting vector was further validated in a targeting experiment with C57BL/6 embryonic stem cells. The low-copy-number plasmid pBAC-FB is a plasmid of choice to retrieve toxic DNA sequences from BACs and to manipulate them to generate gene-targeting constructs by recombineering.     

A 191-kb genomic fragment containing the human α-globin locus can rescue α-thalassemic mice

A 191-kb human bacterial artificial chromosome (BAC) containing the human α-globin genomic locus was used to generate transgenic mice that express, exclusively, human α-globin (^huα-globin). Expression of ^huα-globin reaches a level of 36% of that of endogenous mouse α-globin (^muα-globin) on a heterozygous mouse α-thalassemia background (^muα-globin knockout, ^muα^+/−). Hemizygous transgenic mice carrying the ^huα-globin locus on a heterozygous knockout background (^huα^+/0, ^muα^++/−−) demonstrated complementation of most hematologic parameters. By crossing ^huα^+/0, ^muα^++/−− mice, we were able to generate mice entirely dependent on ^huα-globin synthesis. Breeding and fluorescent in situ hybridization studies demonstrate that only mice homozygous for the transgene were able to rescue embryonic lethal homozygous ^muα-globin knockout embryos (^muα^{−−/−−}). Adult rescued mice produce hemoglobin at levels similar to wild-type mice, with partial red cell complementation based on mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and red cell distribution width (RDW) measurements. Significant erythrocythemia above wild-type levels seems to be the main compensatory mechanism for the normalization of the hemoglobin levels in the rescued animals. Our studies demonstrate that the ^huα-globin locus in the 191-kb transgene contains all the necessary elements for the regulated expression of ^huα-globin in transgenic mice. This animal model should be valuable for studying the mechanisms regulating ^huα-globin production and for development of therapeutic strategies for β-thalassemia based on downregulation of α-globin expression. We dedicate this article to the memory of our valued friend and colleague Panayiotis A. Ioannou who passed away during the completion of this work     

Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals

Background  

Vertebrate alpha (α)- and beta (β)-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the α- and β-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil β-globin gene (ω) in the marsupial α-cluster, however, suggested that duplication of the α-β cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous α- and β-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (Ornithorhynchus anatinus), to explore haemoglobin evolution at the stem of the mammalian radiation.     

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     

Generation of a soybean BAC library, and identification of DNA sequences tightly linked to the Rps1-k disease resistance gene

 A soybean bacterial artificial chromosome (BAC) library, comprising approximately 45 000 clones, was constructed from high-molecular-weight nuclear DNA of cultivar Williams 82, which carries the Rps1-k gene for resistance against Phytophthora sojae. The library is stored in 130 pools with about 350 clones per pool. Completeness of the library was evaluated for 21 random sequences including four markers linked to the Rps1 locus and 16 cDNAs. We identified pools containing BACs for all sequences except for one cDNA. Additionally, when screened for possible contaminating BAC clones carrying chloroplast genes, no sequences homologous to two barley chloroplast genes were found. The estimated average insert size of the BAC clones was about 105 kb. The library comprises about four genome equivalents of soybean DNA. Therefore, this gives a probability of 0.98 of finding a specific sequence from this library. This library should be a useful resource for the positional cloning of Rps1-k, and other soybean genes. We have also evaluated the feasibility of an RFLP-based screening procedure for the isolation of BAC clones specific for markers that are members of repetitive sequence families, and are linked to the Rps1-k gene. We show that BAC clones isolated for two genetically linked marker loci, Tgmr and TC1-2, are physically linked. Application of this method in expediting the map-based cloning of a gene, especially from an organism, such as soybean, maize and wheat, with a complex genome is discussed. Received: 12 May 1998/Accepted: 24 August 1998     

Efficient recombination-based methods for bacterial artificial chromosome fusion and mutagenesis

The availability of genomic sequence information and extensive bacterial artificial chromosome (BAC) libraries for both the mouse and human genomes is ushering in a new era in biological research and disease modeling. To facilitate the study of large mammalian genes in vivo, we have developed: i) a simple lambda bacteriophage-based methodology for recombining overlapping bacterial artificial chromosomes (BACs) into a single larger BAC, and ii) a new methodology for targeting "seamless" mutations into BACs. In the first method, overlapping sequence from one BAC is cloned alongside a selectable marker and placed between unique sequence arms from the terminus of the other BAC to create a targeting construct. Two rounds of recombination-based cloning are then performed. The robustness of this methodology is demonstrated herein by using it to obtain a 254 kb BAC containing the entire human androgen receptor (hAR) gene. In the second method, transient expression of three lambda bacteriophage genes to 'pop-in' a targeting cassette is followed by RecA expression from the targeting vector itself to 'pop-out' the vector backbone. This new "hybrid recombineering" method combines the strengths of the lambda bacteriophage and RecA systems, while avoiding their major weaknesses. Application of this method for introduction of a 162 CAG repeat expansion into the hAR 254kb BAC is shown. With "hybrid recombineering", we believe that the power and utility of the classical 'pop-in/pop-out' approach -- so commonly and efficiently employed in yeast for decades -- can now be achieved with BACs.     

The wheat D-genome HMW-glutenin locus: BAC sequencing,gene distribution,and retrotransposon clusters

A bacterial-artificial-chromosome (BAC) clone from the genome of Triticum tauschii, the D-genome ancestor of hexaploid bread wheat, was sequenced and the presence of the two paralogous x- and y-type high-molecular-weight (HMW) glutenin genes of the Glu-D1 locus was confirmed. These two genes occur in the same orientation, are 51,893 bp apart, and the separating DNA includes a 31,000-bp cluster of retrotransposons. A second retrotransposon cluster of 32,000 bp follows the x-type HMW-glutenin gene region. Each HMW-glutenin gene is found within a region of mainly unique DNA sequence which includes multiple additional genes including an active endosperm globulin gene not previously reported in the Triticeae family, a leucine-rich-repeat (LRR) type gene truncated at the 5′ end of the BAC, a kinase gene of unknown activity, remnants of a paralogous second globulin gene, and genes similar to two hypothetical rice genes. The newly identified globulin genes are assigned to a locus designated Glo-2. Comparison to available orthologous regions of the wheat A and B genomes show rapid sequence divergences flanking the HMW-glutenin genes, and the absence of two hypothetical and unknown genes found 5′ to the B-genome x-type ortholog. The region surrounding the Glu-D1 locus is similar to other reported Triticeae BAC sequences; i.e. small gene islands separated by retrotransposon clusters. Electronic Publication     

Identification and Expression Pattern of DNA Polymerase α Gene in a Marine Diatom, Skeletonema costatum

To investigate the potential of DNA polymerase α as a marker for DNA replication in phytoplankton, two gene fragments that showed a high degree of similarity with eukaryotic DNA polymerase α were cloned from two strains of a diatom, Skeletonema costatum (Greville) Cleve. The gene fragments amplified with the polymerase chain reaction were 397 and 396 bp in length, respectively. The deduced amino acid sequences showed 44% to 61% similarity to the corresponding regions of DNA polymerase α sequences of eukaryotic organisms ranging from yeast to humans. The similarity was especially high in three evolutionarily conserved regions within the amplified fragments. Further, hybridization patterns from Southern blotting confirmed that the amplified fragments were an integral part on the genome of S. costatum. In batch cultures abundant messenger of DNA polymerase α appeared in the late exponential phase and the early stationary phase. This pattern suggests that DNA polymerase α expression is associated with actively dividing cells. Received October 27, 1998; accepted November 13, 1998.     

Insertion of modifications in the beta-globin locus using GET recombination with single-stranded oligonucleotides and denatured PCR fragments

We describe the use of the GET recombination system with oligonucleotides or single-stranded polymerase chain reaction (PCR) fragments to insert modifications in the human beta-globin locus without counterselection. The method involves recombination between oligonucleotides or denatured PCR fragments and homologous sequences in the beta-globin gene in a clone of 205-kb bacterial artificial chromosome (BAC), based on the inducible expression of the recE, recT, and gam genes. In this method, oligonucleotides or denatured PCR fragments are electroporated directly into cells carrying both the globin BAC and the pGETrec plasmid, after induction of the GET recombination system. Recombinant BAC clones are identified by PCR, using allele-specific amplification for the mutated sequences. We have used this approach to insert a unique restriction site as well as a common thalassemia mutation (stop codon 39, C-->T) into the human beta-globin locus. We have observed the frequency of recombinant clones to be as high as 1 in 100-200 clones. Therefore, this approach provides a simple and efficient method for introducing point mutations and other fine modifications into BACs, and should greatly facilitate the use of BACs for functional studies and therapeutic applications.     

Rapid BAC selection for tol2-mediated transgenesis in zebrafish

The generation of zebrafish transgenic lines that express specific fluorophores in a cell- or tissue-specific manner is an important technique that takes full advantage of the optical clarity of the embryo. Identifying promoter fragments that faithfully recapitulate endogenous expression patterns and levels is often difficult and using large genomic DNA fragments, such as bacterial artificial chromosomes (BACs), makes the process of transgenesis less reliable. Here we provide a detailed protocol that allows for BAC selection and subsequent rapid modification through recombineering in Escherichia coli, resulting in BACs that can be injected into zebrafish embryos and, aided by tol2-mediated transgenesis, reliably yield stable transgenic lines. A number of BACs can be prepared in parallel, and injection of the BACs containing CFP/YFP/RFP or Gal4 cassettes allows for immediate testing of whether a particular BAC will yield the desired result. Furthermore, since injected embryos often show widespread expression, recombineered BACs provide an alternative to two-color in situ hybridizations: BACs injected into embryos of a different transgenic reporter line thus enable in vivo colocalization studies. Using this protocol, we have generated 66 stable lines for 23 different genes, with an average transgenesis rate above 10%. Importantly, we provide evidence that BAC size shows no apparent correlation to the transgenesis rate achieved and that there are no severe position effects.     

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.     

Development of four phylogenetically-arrayed BAC libraries and sequence of the APA locus in Phaseolus vulgaris

The APA family of seed proteins consists of three subfamilies, in evolutionary order of hypothesized appearance: phytohaemagglutinins (PHA), α-amylase inhibitors (αAI), and arcelins (ARL). The APA family plays a defensive role against mammalian and insect seed predation in common bean (Phaseolus vulgaris L.). The main locus (APA) for this gene family is situated on linkage group B4. In order to elucidate the pattern of duplication and diversification at this locus, we developed a BAC library in each of four different Phaseolus genotypes that represent presumptive steps in the evolutionary diversification of the APA family. Specifically, BAC libraries were established in one P. lunatus (cv. ‘Henderson: PHA⁺ αAI⁻ ARL⁻) and three P. vulgaris accessions (presumed ancestral wild G21245 from northern Peru: PHA⁺ αAI ⁺ ARL⁻; Mesoamerican wild G02771: PHA⁺ αAI ⁺ ARL⁺; and Mesoamerican breeding line BAT93: PHA⁺ αAI ⁺ ARL⁻). The libraries were constructed after HindIII digestion of high molecular weight DNA, obtained with a novel nuclei isolation procedure. The frequency of empty or cpDNA-sequence-containing clones in all libraries is low (generally <1%). The Henderson, G21245, and G02771 libraries have a 10× genome coverage, whereas the BAT93 library has a 20× coverage to allow further, more detailed genomic analysis of the bean genome. The complete sequence of a 155 kbp-insert clone of the G02771 library revealed six sequences belonging to the APA gene family, including members of the three subfamilies, as hypothesized. The different subfamilies were interspersed with retrotransposon sequences. In addition, other sequences were identified with similarity to chloroplast DNA, a dehydrin gene, and the Arabidopsis flowering D locus. Linkage between the dehydrin gene and the D1711 RFLP marker identifies a potential syntenic region between parts of common bean linkage group B4 and cowpea linkage group 2     

Genetic and physical mapping of xa13, a recessive bacterial blight resistance gene in rice

 The recessive gene, xa13, confers resistance to Philippine race 6 (PXO99) of the bacterial blight pathogen Xanthomonas oryzae pv oryzae. Fine genetic mapping and physical mapping were conducted as initial steps in an effort to isolate the gene. Using nine selected DNA markers and two F₂ populations of 132 and 230 plants, xa13 was fine-mapped to a genomic region <4 cM on the long arm of rice chromosome 8, flanked by two RFLP markers, RG136 and R2027. Four DNA markers, RG136, R2027, S14003, and G1149, in the target region were used to identify bacterial artificial chromosome (BAC) clones potentially harboring the xa13 locus from a rice BAC library. A total of 11 BACs were identified, forming four separate contigs including a single-clone contig, 29I3, associated with the RG136 STS marker, the S14003 contig consisting of four clones (44F8, 41O2, 12A16, and 12F20), the G1149 contig with two clones, 23D11 and 21H18, and the R2027 contig consisting of four overlapping clones, 42C23, 30B5, 6B7 and 21H14. Genetic mapping indicated that the xa13 locus was contained in the R2027 contig. Chromosomal walking on the R2027 contig resulted in two more clones, 33C7 and 14L3. DNA fingerprinting showed that the six clones of the R2027 contig were overlapping. Clone 44F8 hybridized with a single fragment from the clone 14L3, integrating the R2027 and S14003 contigs into a single contig consisting of ten BAC clones with a total size of approximately 330 kb. The physical presence of the xa13 locus in the contig was determined by mapping the ends of the BAC inserts generated by TAIL-PCR. In an F₂ population of 230 plants, the BAC-end markers 42C23R and 6B7F flanked the xa13 locus. The probes 21H14F and 21H14R derived from BAC clone 21H14 were found to flank xa13 at a distance of 0.5 cM on either side, using a second F₂ population of 132 plants. Thus, genetic mapping indicated that the contig and the 96-kb clone, 21H14, contained the xa13 locus. Received: 15 August 1998 / Accepted: 29 September 1998     

Construction of bacterial artificial chromosome libraries and their application in developing PCR-based markers closely linked to a major locus conditioning bruchid resistance in mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> L. Wilczek)

Bacterial artificial chromosome (BAC) libraries have been widely used in different aspects of genome research. In this paper we report the construction of the first mungbean (Vigna radiata L. Wilczek) BAC libraries. These BAC clones were obtained from two ligations and represent an estimated 3.5 genome equivalents. This correlated well with the screening of nine random single-copy restriction fragment length polymorphism probes, which detected on average three BACs each. These mungbean clones were successfully used in the development of two PCR-based markers linked closely with a major locus conditioning bruchid (Callosobruchus chinesis) resistance. These markers will be invaluable in facilitating the introgression of bruchid resistance into breeding programmes as well as the further characterisation of the resistance locus.