A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo

We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the desired DNA fragments or libraries is based on the recombination system of bacteriophage lambda. As an example of the utility of the vector, genes or cDNAs of 18 different proteins were cloned into pBVboostFG and expressed in different hosts. As a proof-of-principle of using the vector for library screening, a chromophoric Thr⁶⁵-Tyr-Gly⁶⁷-stretch of enhanced green fluorescent protein was destroyed and subsequently restored by novel PCR strategy and library screening. The pBVboostFG enables screening of genome-wide libraries, thus making it an efficient new platform technology for functional genomics.     

高覆盖率水稻ＢＡＣ库的构建及抗病基因相关克隆的筛选

利用含Ｘa4、xa5和xa13 3个水稻白叶枯病抗性基因的累加系ＩＲＢＢ５６构建了一个水稻细菌人工染色体文库,该文库包含５５２９６个克隆,平均插入升段为１３２kb。按水稻基因组为４５０Ｍb计,该文库覆盖１４倍基因组,筛选出任一水稻基因或序列的概率为９９．９９％。用均匀分布的３个叶绿体基因和４个线粒体基因克隆作探针筛选文库,结果显示该文库中含细菌器基因组ＤＮＡ同源序列的克隆数小于１％、用分布于水稻３条不同染色体、分别与Ｘa4、xa5和xa13连锁的ＤＮＡ标记筛选文库,分别检测出１１－１０６个阳性克隆,为克隆这些基因打下了基础。该文库对水稻基因组的高度覆盖率和较大的插入片段,非常适合于物理作图和基因的分离和克隆。     

Isolation and characterization of beta- and gamma-crystallin genes from rat genomic cosmid libraries

Two libraries, together containing about 10(6) colonies, have been constructed by cloning different size fractions of a partial Sau3A digest of rat genomic DNA in the cosmid vector pTM. Upon screening with two cDNA clones, one containing alpha A2-crystallin and one containing beta B1-crystallin sequences, 14 cosmid clones were isolated which were beta B1-crystallin-specific; none was found which contained alpha A2-crystallin sequences. The inserts of the beta B1 clones, which range from 35 to 45 kb in length, contain overlapping DNA segments covering more than 60 kb of rat genomic DNA. The composite BamHI restriction map of this region shows a single beta B1-crystallin gene, which is interrupted by several intronic sequences. Five recombinants hybridizing with two different rat lens gamma-crystallin cDNA clones were also isolated from these libraries. Four of these contain 31- to 41-kb inserts, whereas the fifth recombinant contains a 12.2-kb insert. Hybridization analysis with 5' and 3'-specific cDNA fragments indicates that altogether these inserts contain six gamma-crystallin genes, three of which are located on one insert of only 31 kb.     

Construction and Characterization of a Bacterial Artificial Chromosome (BAC) Library of Pacific White Shrimp, Litopenaeus vannamei

The pacific white shrimp (Litopenaeus vannamei) is one of the most economically important marine aquaculture species in the world. To facilitate gene cloning and characterization, genome analysis, physical mapping, and molecular selection breeding of marine shrimp, we have developed the techniques to isolate high-quality megabase-sized DNA from hemocyte nuclear DNA of female shrimp and constructed a bacterial artificial chromosome (BAC) genomic library for the species. The library was constructed in the Hind III site of the vector pECBAC1, consisting of 101,760 clones arrayed in 265 384-well microtiter plates, with an average insert size of 101 kb, and covering the genome approximately fivefold. To characterize the library, 92,160 clones were spotted onto high-density nylon filters for hybridization screening. A set of 18 pairs of overgo probes designed from eight cDNA sequences of L. vannamei genes were used in hybridization screening, and 35 positive clones were identified. These results suggest that the shrimp BAC libraries will provide a useful resource for screening of genomic regions of interest candidate genes, gene families, or large-sized synthetic DNA region and promote future works on comparative genomics, physical mapping, and large-scale genome sequencing in the species.     

Direct targeting and rapid isolation of BAC clones spanning a defined chromosome region

To isolate genes of interest in plants, it is essential to construct bacterial artificial chromosome (BAC) libraries from specific genotypes. Construction and organisation of BAC libraries is laborious and costly, especially from organisms with large and complex genomes. In the present study, we developed the pooled BAC library strategy that allows rapid and low cost generation and screening of genomic libraries from any genotype of interest. The BAC library is constructed, directly organised into a few pools and screened for BAC clones of interest using PCR and hybridisation steps, without requiring organization into individual clones. As a proof of concept, a pooled BAC library of approximately 177,000 recombinant clones has been constructed from the barley cultivar Cebada Capa that carries the Rph7 leaf rust resistance gene. The library has an average insert size of 140 kb, a coverage of six barley genome equivalents and is organised in 138 pools of about 1,300 clones each. We rapidly established a single contig of six BAC clones spanning 230 kb at the Rph7 locus on chromosome 3HS. The described low-cost cloning strategy is fast and will greatly facilitate direct targeting of genes and large-scale intra- and inter-species comparative genome analysis.Edwige Isidore and Beatrice Scherrer contributed equally to the work.     

Construction and characterization of the transformation-competent artificial chromosome (TAC) libraries of Leymus multicaulis

Transformation-competent artificial chromosome system is able to clone and transfer genes efficiently in plants.In order to clone genes highly tolerant to barley yellow dwarf virus(BYDV),Aphids,drought and salt from Leymus multicaulis,the two TAC genomic libraries I and II were constructed in vector pYLTAC17 and pYLTAC747H/sacB,which contain about 165000 and 236000 recombinant clones sepa-rately.The genome coverage of the two libraries was totally estimated to be about 3―5 haploid genome equivalents,as size selection of genomic DNA fragments was approximately from 9 to 300 kb.Clones of the genomic libraries were collected as bulked pools each containing 500 clones or so,stored in twelve 96-deep-well plates and then were gridding in triplicate onto a high-density colony hybridization filter with a 3×3 pattern using a GeneTAC?G3 arraying robot after being transferred manually into three 384-well plates.Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacB were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6×6 pattern after a replication of plates.Nineteen positive clones were detected by using the probe glutahione reductase gene of L.multicaulis.TAC libraries constructed here can be used to isolate genomic clones containing target genes,and to carry out genome walking for positional cloning.Once the target TAC clones were isolated,they could be immediately transferred into plant genomes with the Agrobacterium system.     

Stable cosmid vectors that enable the introduction of cloned fragments into a wide range of gram-negative bacteria

A cosmid cloning system has been developed which is useful for the construction of genomic libraries and the introduction of clones into a broad range of bacterial species. The cosmids pMMB33 and pMMB34 allow selective cloning into their unique BamHI site of 36-kb DNA fragments generated by BamHI, Sau3A and MboI partial digestion. This selective cloning is achieved by a strategy that avoids formation of polycosmids without a dephosphorylation step. It uses two unique recognition sites within the vectors for endoncleases that generate blunt-ended DNA fragments for the preparation of left and right cosmid "arms". An alternative method that uses the unique EcoRI and SstI sites and dephosphorylation of the cosmid arms prior to BamHI digestion is also outlined and discussed. The DNA is first cloned with either vector into a rec- E. coli strain, where clones can be maintained stably, and can then be introduced by mobilization into a wide range of Gram-negative species to permit the study of gene expression and complementation. Because mobilization is much more efficient than transformation, the vector has the advantage that it can be transferred between bacterial species that specify different restriction systems, where transformation appears to be inefficient. The vectors have been used to generate gene libraries from the chromosomal DNA of several Pseudomonas and a Thiobacillus species. The genes specifying myo-inositol transport from Pseudomonas strain JD34 have been cloned with this system.     

Development of an efficient maintenance and screening system for large-insert genomic DNA libraries of hexaploid wheat in a transformation-competent artificial chromosome (TAC) vector

Three large-insert genomic DNA libraries of common wheat, Triticum aestivum cv. Chinese Spring, were constructed in a newly developed transformation-competent artificial chromosome (TAC) vector, pYLTAC17, which accepts and maintains large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. The vector contains the cis sequence required for Agrobacterium-mediated gene transfer into grasses. The average insert sizes of the three genomic libraries were approximately 46, 65 and 120 kbp, covering three haploid genome equivalents. Genomic libraries were stored as frozen cultures in a 96-well format, each well containing approximately 300-600 colonies (12 plates for small library, four for medium-size library and four for large library). In each of the libraries, approximately 80% of the colonies harbored genomic DNA inserts of >50 kbp. TAC clones containing gene(s) of interest were identified by the pooled PCR technique. Once the target TAC clones were isolated, they could be immediately transferred into grass genomes with the Agrobacterium system. Five clones containing the thionin type I genes (single copy per genome), corresponding to each of the three genomes (A, B and D), were successfully selected by the pooled PCR method, in addition to an STS marker (aWG464; single copy per genome) and CAB (a multigene family). TAC libraries constructed as described here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning.     

Expression cloning of oncogenes by retroviral transfer of cDNA libraries.

a cDNA library transfer system based on retroviral vectors has been developed for expression cloning in mammalian cells. The use of retroviral vectors results in stable cDNA transfer efficiencies which are at least 100-fold higher than those achieved by transfection and therefore enables the transfer and functional screening of very large libraries. In our initial application of retroviral transfer of cDNA libraries, we have selected for cDNAs which induce oncogenic transformation of NIH 3T3 fibroblasts, as measured by loss of contact inhibition of proliferation. Nineteen different transforming cDNAs were isolated from a total of 300,000 transferred cDNA clones. Three of these cDNAs were derived from known oncogenes (raf-1, lck, and ect2), while nine others were derived from genes which had been cloned previously but were not known to have the ability to transform fibroblasts (beta-catenin, thrombin receptor, phospholipase C-gamma 2 and Spi-2 protease inhibitor genes). The Spi-2 cDNA was expressed in antisense orientation and therefore is likely to act as an inhibitor of an endogenous transformation suppressor. Seven novel cDNAs with transforming activities, including those for three new members of the CDC24 family of guanine nucleotide exchange factors, were also cloned from the retroviral cDNA libraries. Retroviral transfer of libraries should be generally useful for cloning cDNAs which confer selectable phenotypes on many different types of mammalian cells.     

Metagenomic analysis of Streptomyces lividans reveals host-dependent functional expression

Most functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA in Escherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries in Streptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biologically active clones. To test the functionality of these methods, we constructed and screened two metagenomic libraries in S. lividans. One was constructed with pooled DNA from 14 bacterial isolates cultured from Alaskan soil and the second with DNA directly extracted from the same soil. Functional screening of these libraries identified numerous clones with hemolytic activity, one clone that produces melanin by a previously unknown mechanism, and one that induces the overproduction of a secondary metabolite native to S. lividans. All bioactive clones were functional in S. lividans but not in E. coli, demonstrating the advantages of screening metagenomic libraries in more than one host.     

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.     

Use of conversion adaptors to clone antigen genes in lambda gt11

A strategy has been devised and tested to employ EcoRI conversion adaptors for cloning 5' cohesive-ended restriction fragments into the unique EcoRI site of the lambda gt11 expression vector. Five lambda gt11 chromosomal libraries were constructed with Rickettsia tsutsugamushi genomic DNA digested with restriction enzymes generating five different 5' cohesive ends. Recombinant phage yields as high as 10(7) plaque forming units were achieved without amplification of the five libraries. Sequences encoding epitopes of all eight R. tsutsugamushi polypeptide antigens, previously identified by Western blot analysis, were obtained in the five lambda gt11 expression libraries. Recombinant antigen expression was dependent on lambda gt11 lac promoter induction in 39% of the recombinants assayed. This method significantly improves the efficiency of genomic lambda gt11 library construction by eliminating blunt-ended ligation and simplifying the removal of unligated EcoRI-ended oligonucleotides.     

A new cloning strategy for generating multiple repeats of a repetitive polypeptide based on non-template PCR

A new cloning method for generating multiple repeats of amino acids is described which can be used as biomaterials, protein polymers and biomedical applications. Although several traditional methods for cloning multiple repeats are still exploited, these are laborious and complicated because they must go through several consecutive cloning steps. To solve these problems, synthetic gene libraries encoding repetitive patterns were constructed by using non-template PCR. As a result, a ‘length library’ with fourteen different ELP repeating genes was constructed and expressed in a cell-free protein synthesis system. These results showed our novel cloning method is efficient, and has the potential capacity for synthesizing repetitive genes by PCR to be cloned in any commercial expression vectors.     

Pilot studies on the parallel production of soluble mouse proteins in a bacterial expression system

We investigated the parallel production in medium throughput of mouse proteins, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. The methods were scaled up to allow the simultaneous processing of tens or hundreds of protein samples. Scale-up was achieved in two stages. In an initial study, 30 targets were processed manually but with common protocols for all targets. In the second study, these protocols were applied to 96 target proteins that were processed in an automated manner. The success rates at each stage of the study were similar for both the manual and automated approaches. Overall, 15 of the selected 126 target mouse genes (12%) yielded soluble protein products in a bacterial expression system. This success rate compares favourably with other protein screening projects, particularly for eukaryotic proteins, and could be further improved by modifications at the cloning step.     

Cloning in Bacillus subtilis by transfection with bacteriophage vector phi 105J27: isolation and preliminary characterization of transducing phages for 23 sporulation loci

Bacteriophage cloning vector phi 105J27, the construction of which is described in an accompanying paper, has been used for shotgun cloning of sporulation genes in Bacillus subtilis. Various genomic libraries have been constructed and screened for the presence of recombinant phages capable of transducing strains containing sporulation (spo) mutations to Spo+. Of a total of 30 spo loci tested, transducing phages have been isolated for 23, more than half of the known spo loci. Included are nine loci (spo0D, spo0J, spoIIIA, spoIIIE, spoIIIF, spoIVF, spoVB, spoVH and spoVJ) that do not appear to have been cloned previously. Preliminary genetic characterization of some of the new clones by a rapid screening procedure has enabled the status of various sporulation loci to be clarified.     

Molecular analysis of the human MHC class I region using yeast artificial chromosome clones

The cloning of large genomic fragments corresponding to the major histocompatibility complex (MHC) class I region provides the necessary framework for a better understanding of its organization and for the localization of new genes involved in MHC-associated disease. Two human genomic libraries constructed in yeast artificial chromosomes (YACs) have been prepared using complete Not I or Mlu I digestion of source DNA. From these libraries three YAC clones with inserts belonging to the MHC class I region have been isolated. They correspond to exact copies of three genomic fragments of 210, 145, and 50 kilobases (kb), respectively and have been precisely located in the restriction map of the region. Detailed rare-cutter restriction maps of the inserts have been generated. Within these clones we have demonstrated the presence of two class I genes, one of which is HLA-E, and of at least three Hpa II tiny fragment (HTF) islands, corresponding to three putative new transcribed sequences. End clones, which are of particular interest in the extension and refinement of the regional map, have been rescued by systematic subcloning of purified YACs.     

A novel Ti-plasmid-convertible λ phage vector system suitable for gene isolation by genetic complementation of Arabidopsis thaliana mutants

A new λ phage vector system, λTI, has been constructed to facilitate genetic complementation of higher plant mutations. The λTI vectors are stable, and by using the Cre— lox site-specific recombination, are automatically convertible into Ti-plasmid binary vectors which are capable of expressing genes in higher plants. Two λTI vectors were constructed: (i) λTI1, which can generate a Ti-plasmid that contains the cauliflower mosaic virus (CaMV) 35S promoter and is suitable for the expression of cDNA in transformed plants and (ii) λTI2, which can generate a Ti-plasmid with the multicloning site (MCS). cDNA and genomic libraries, which were constructed from the cruciferous plant Arabidopsis thaliana in these λTI vectors, can be probed by large DNA fragments of more than 100 kb, such as yeast artificial chromosomes (YACs), enabling the direct screening of the clones in the chromosome region containing a specified genetic locus. These libraries will certainly become powerful tools for the genetic complementation of Arabidopsis mutant phenotypes by quickly providing transformation-competent clones.