Yeast artificial chromosome cloning

Yeast artificial chromosome (YAC) cloning systems enable the cloning of DNA stretches of 50 to well over 2000 kb. This makes it possible to study large intact regions of DNA in detail, by restriction mapping the YAC to produce a physical map and by examining the YAC for coding sequences or genes. YACs are important for their ability to clone the complete sequences of large genes or gene complexes that exceed the size limit for cloning in conventional bacterial cloning vectors like plasmids (up to 10 kb), bacteriophage (15 kb), and cosmids (50 kb). A major advantage of cloning in yeast, a eukaryotc. is that many sequences that are unstable, underrepresented, or absent when cloned into prokaryotic systems, remain stable and intact in YAC clones. It is possible to reinlroduce YACs intact into mammalian cells where the introduced mammalian genes are expressed and used to study the functions of genes in the context of flanking sequences. The correct prolein processing mechanisms are present in the mammalian cells to ensure that a viable protein product is produced.     

Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning

The transformation-competent artificial chromosome vector (TAC) system has been shown to be very useful for efficient gene isolation in Arabidopsis thaliana (Proc. Natl. Acad. Sci. USA 96 (1998) 6535). To adapt the vector system for gene isolation in crops, two new TAC vectors and rice genomic libraries were developed. The new vectors pYLTAC17 and pYLTAC27 use the Bar gene and Hpt gene driven by the rice Act1 promoter as the plant selectable markers, respectively, and are suitable for transformation of rice and other grasses. Two representative genomic libraries (I and II) of an Indica rice variety Minghui63, a fertility restorer line for hybrid rice, were constructed with pYLTAC17 using different size classes of partially digested DNA fragments. Library I and library II consisted of 34,560 and 1.2 x 10(5) clones, with average insert sizes of approximately 77 and 39 kb, respectively. The genome coverage of the libraries I and II was estimated to be about 5 and 11 haploid genome equivalents, respectively. Clones of the library I were stored individually in ninety 384-well plates, and those of the library II were collected as bulked pools each containing 30-50 clones and stored in eight 384-well plates. A number of probes were used to hybridize high-density colony filters of the library I prepared by an improved replicating method and each detected 2-9 positive clones. A method for rapid screening of the library II by pooled colony hybridization was developed. A TAC clone having an 80 kb rice DNA insert was successfully transferred into rice genome via Agrobacterium-mediated transformation. The new vectors and the genomic libraries should be useful for gene cloning and genetic engineering in rice and other crops.     

Isolation and mapping of bacterial artificial chromosome (BAC) clone containing telomere-associated sequence

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High-efficiency yeast artificial chromosome fragmentation vectors

Chromosome fragmentation vectors (CFVs) are used to create deletion derivatives of large fragments of human DNA cloned as yeast artificial chromosomes (YACs). CFVs target insertion of a telomere sequence into the YAC via homologous recombination with Alu repetitive elements. This event results in the loss of all YAC sequences distal to the site of integration. A new series of CFVs has been developed. These vectors target fragmentation to both Alu and LINE human repetitive DNA elements. Recovery of deletion derivatives is ten- to 20-fold more efficient with the new vectors than with those described previously.     

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 manipulation of an infectious clone of the bovine herpesvirus 1 genome maintained as a bacterial artificial chromosome

The complete genome of bovine herpesvirus 1 (BoHV-1) strain V155 has been cloned as a bacterial artificial chromosome (BAC). Following electroporation into Escherichia coli strain DH10B, the BoHV-1 BAC was stably propagated over multiple generations of its host. BAC DNA recovered from DH10B cells and transfected into bovine cells produced a cytopathic effect which was indistinguishable from that of the parent virus. Analysis of the replication kinetics of the viral progeny indicated that insertion of the BAC vector into the thymidine kinase gene did not affect viral replication. Specific manipulation of the BAC was demonstrated by deleting the gene encoding glycoprotein E by homologous recombination in DH10B cells facilitated by GET recombination. These studies illustrate that the propagation and manipulation of herpesviruses in bacterial systems will allow for rapid and accurate characterization of BoHV-1 genes. In turn, this will allow for the full utilization of BoHV-1 as a vaccine vector.     

Yeast artificial chromosomes: tools for mapping and analysis of complex genomes

Libraries of yeast artificial chromosomes (YACs) are representative of complex genomes, and typical YACs contain single fragments of DNA that are up to a megabase or more in size, are stable during growth, and are faithful to genomic DNA. Such YACs may permit (1) the use of complete gene units for a number of research and medical purposes; and (2) the assembly of chromosome-sized contigs in a single map that unifies genetic and physical data.     

Integration-free iPS cells engineered using human artificial chromosome vectors

Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost iHAC2. Analyses of pluripotent markers, teratomas and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex virus thymidine kinase were eliminated by ganciclovir treatment, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system.     

Modular bacterial artificial chromosome vectors for transfer of large inserts into mammalian cells

To facilitate the use of large-insert bacterial clones for functional analysis, we have constructed new bacterial artificial chromosome vectors, pPAC4 and pBACe4. These vectors contain two genetic elements that enable stable maintenance of the clones in mammalian cells: (1) The Epstein-Barr virus replicon, oriP, is included to ensure stable episomal propagation of the large insert clones upon transfection into mammalian cells. (2) The blasticidin deaminase gene is placed in a eukaryotic expression cassette to enable selection for the desired mammalian clones by using the nucleoside antibiotic blasticidin. Sequences important to select for loxP-specific genome targeting in mammalian chromosomes are also present. In addition, we demonstrate that the attTn7 sequence present on the vectors permits specific addition of selected features to the library clones. Unique sites have also been included in the vector to enable linearization of the large-insert clones, e. g., for optical mapping studies. The pPAC4 vector has been used to generate libraries from the human, mouse, and rat genomes. We believe that clones from these libraries would serve as an important reagent in functional experiments, including the identification or validation of candidate disease genes, by transferring a particular clone containing the relevant wildtype gene into mutant cells or transgenic or knock-out animals.     

Yeast artificial chromosome segregation from host chromosomes with similar lengths.

We propose a new method for segregation of yeast artificial chromosomes (YACs) from endogenous yeast chromosomes with similar lengths. The method is based on recently developed PNA-assisted rare cleavage (PARC) of genomic DNA. We apply the PARC procedure to YAC-containing samples of yeast DNA in such a way that host chromosomes, which electrophoretically comigrate with the chosen YACs, are selectively digested while YACs remain intact. These data demonstrate that a pool of appropriate PNAs can be used as an efficient tool for the PARC-based isolation of intact purified YACs directly from the host cells.     

Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.     

Bacterial artificial chromosome library of Finegoldia magna ATCC 29328 for genetic mapping and comparative genomics

We constructed a bacterial artificial chromosome (BAC) library of Finegoldia magna ATCC 29328 DNA to facilitate further genome analysis of F. magna. The BAC library contained 385 clones with an average insert size of 55 kb, representing a 10.1-fold genomic coverage. Repeated DNA hybridization using primer sets designed on the basis of BAC-end sequences yielded nine contigs covering 95% of the chromosome and two contigs covering 98% of the plasmid. The contigs were localized on the physical map of F. magna ATCC 29328 DNA. A total of 121 BAC-end sequences revealed 103 unique genes, which had not been previously reported for F. magna. The homolog ORF of albumin-binding protein (urPAB), one of the known virulence factors from F. magna, was sequenced and localized on the physical map. Homology analysis of 121 BAC-end sequences revealed that F. magna is most closely related to clostridia, particularly Clostridium tetani. This close relationship is consistent with the recent classification of peptostreptococci based on 16S rRNA sequence analysis. The BAC library constructed here will be useful for the whole genome sequencing project and other postgenomic applications.     

Construction and targeted retrieval of specific clone from a non-gridded soybean bacterial artificial chromosome library

Although a post-genomic era is emerging for many plants, the bacterial artificial chromosome (BAC) library is still a valuable tool for genomic studies and preservation of precious genetic resources. Construction of non-gridded BAC libraries would dramatically reduce cost and save storage space. A non-gridded BAC library composed of approximately 96,000 insert-containing clones in 80 pools with an average insert size of 75 kb was constructed. This library represented 5.2 genome equivalents. We successfully developed a unique procedure to retrieve positive clones from the non-gridded pools. With this retrieving protocol, the non-gridded library system can be adapted to different species and to serve various research needs.     

Transposable elements for efficient manipulation of a wide range of gram-negative bacteria: promoter probes and vectors for foreign genes

We describe here the construction and use of a series of modified transposons based on the insertion sequence IS1. Like their parent, omegon-Km [Fellay et al., Gene 76 (1989) 215-226], these elements permit efficient insertional mutagenesis of a variety of Gram-negative bacteria. The presence of a functional pBR322 origin of replication within the transposable element facilitates subsequent cloning of the mutated gene. The omegon-Km system was previously shown to function in Pseudomonas putida, Rhizobium leguminosarum and Paracoccus denitrificans. The results we present here demonstrate that its use can be extended to Xanthomonas campestris, a plant pathogen, and to the microaeroduric Zymomonas mobilis. Derivative transposons carrying unique restriction sites for ScaI, NdeI, XbaI and XhoI have been constructed, allowing the cloning and introduction of foreign genes. We have also constructed two derivatives which can be used to generate operon fusions upon insertion and are thus useful for isolating and characterising indigenous promoters. One carries a promoterless chloramphenicol acetyl-transferase (CAT)-encoding gene (cat) and the second, the entire promoterless Escherichia coli lac operon. We demonstrate the utility of the cat promoter probe in X. campestris to target conditional promoters inducible by high salt or subject to repression by glucose.     

Construction of an infectious clone of canine herpesvirus genome as a bacterial artificial chromosome

Canine herpesvirus (CHV) is an attractive candidate not only for use as a recombinant vaccine to protect dogs from a variety of canine pathogens but also as a viral vector for gene therapy in domestic animals. However, developments in this area have been impeded by the complicated techniques used for eukaryotic homologous recombination. To overcome these problems, we used bacterial artificial chromosomes (BACs) to generate infectious BACs. Our findings may be summarized as follows: (i) the CHV genome (pCHV/BAC), in which a BAC flanked by loxP sites was inserted into the thymidine kinase gene, was maintained in Escherichia coli; (ii) transfection of pCHV/BAC into A-72 cells resulted in the production of infectious virus; (iii) the BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus CHV/BAC by co-infection with CHV/BAC and a recombinant adenovirus that expressed the Cre recombinase; and (iv) a recombinant virus in which the glycoprotein C gene was deleted was generated by lambda recombination followed by Flp recombination, which resulted in a reduction in viral titer compared with that of the wild-type virus. The infectious clone pCHV/BAC is useful for the modification of the CHV genome using bacterial genetics, and CHV/BAC should have multiple applications in the rapid generation of genetically engineered CHV recombinants and the development of CHV vectors for vaccination and gene therapy in domestic animals.     

Yeast vectors for integration at the HO locus

We have constructed new yeast vectors for targeted integration of desired sequences at the Saccharomyces cerevisiae HO locus. Insertion at HO has been shown to have no effect on yeast growth, and thus these integrations should be neutral. One vector contains the KanMX selectable marker, and integrants can be selected by resistance to G418. The other vector contains the hisG-URA3-hisG cassette, and integrants can be selected by uracil prototrophy. Subsequent growth on 5-FOA permits identification of colonies where recombination between the hisG tandem repeats has led to loss of the URA3 marker and return to uracil auxotrophy. We also describe several new bacterial polylinker vectors derived from pUC21 (ampicillin resistance) and pUK21 (kanamycin resistance).     

Isolation and subregional mapping of a human cDNA clone detecting a common RELP on chromosome 12

Summary Peripheral blood lymphocytes from three patients with Down syndrome (DS; trisomy 21; aged 5–6 years) and three age-matched control children were studied for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs).Cells in G₀ were exposed to bleomycin (20–100 g/ml) for 3 h, and then cultured in medium containing 5-bromodeoxyuridine and phytohemagglutinin for 66 h. By the sister chromatid differential staining method, chromosome analyses were performed on metaphase cells that had divided one, two, or three or more times after treatment. The results indicate that DS cells exposed to bleomycin are hypersensitive to the production of dicentric and ring chromosomes compared to normal cells. Bleomycin also led to a dose-related increase in the frequency of SCEs, but no difference was found between the SCE frequencies in DS or normal lymphocytes exposed to bleomycin.     

Synergy between tetA and rpsL provides high-stringency positive and negative selection in bacterial artificial chromosome vectors

Bacterial artificial chromosome (bacmid) vectors are used to stably propagate large, complex fragments of cloned DNA and are a core technology for functional genomics. The simplest method of analyzing bacmid clones would involve a direct mutagenesis or allele exchange protocol utilizing positive and negative selectable markers. The utility of three different negative selectable markers to function in the context of a bacmid vector was therefore investigated: sacB from Bacillus subtilis, which confers sensitivity to sucrose; tetA from TN10, which confers resistance to tetracycline, osmotic sensitivity, and sensitivity to kanamycin and streptomycin; and rpsL from Escherichia coli, which confers sensitivity to streptomycin. When expressed individually in the context of a bacmid vector, each of these markers confers a similar stringency of negative selection, with plating efficiencies on selective media of 2.3 x 10(-5), 9.4 x 10(-4), and 5.7 x 10(-5), respectively. However coexpression of rpsL and tetA results in a synergistic enhancement of the osmotic, kanamycin, and streptomycin sensitivities, with a stringency of selection of approximately 50- to approximately 1000-fold over that obtained with rpsL or tetA alone and approximately 20-fold more than that obtained using sacB. The combination of rpsL and tetA thus serves as the most efficient positive and negative selectable marker system described to date.