Highly selective isolation of human DNAs from rodent–human hybrid cells as circular yeast artificial chromosomes by transformation-associated recombination cloning
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T ransformation- a ssociated r ecombination (TAR) can be exploited in yeast to clone human DNAs. TAR cloning was previously accomplished using one or two telomere-containing vectors with a common human repeat(s) that could recombine with human DNA during transformation to generate yeast artificial chromosomes (YACs). On basis of the proposal that broken DNA ends are more recombinogenic than internal sequences, we have investigated if TAR cloning could be applied to the generation of circular YACs by using a single centromere vector containing various human repeats at opposite ends. Transformation with these vectors along with human DNA led to the efficient isolation of circular YACs with a mean size of ≈150 kb. The circular YACs are stable and they can be easily separated from yeast chromosomes or moved into bacterial cells if the TAR vector contains an Escherichia coli F-factor cassette. More importantly, circular TAR cloning enabled the selective isolation of human DNAs from monochromosomal human–rodent hybrid cell lines. Although <2% of the DNA in the hybrid cells was human, as much as 80% of transformants had human DNA YACs when a TAR cloning vector contained Alu repeats. The level of enrichment of human DNA was nearly 3000-fold. A comparable level of enrichment was demonstrated with DNA isolated from a radiation hybrid cell line containing only 5 Mb of human DNA. A high selectivity of human DNA cloning was also observed for linear TAR cloning with two telomere vectors. No human–rodent chimeras were detected among YACs generated by TAR cloning. The results with a circular TAR cloning vector or two vectors differed from results with a single-telomere vector in that the latter often resulted in a series of terminal deletions in linear YACs. This could provide a means for physical mapping of cloned material.Keywords:
Yeast artificial chromosome
Cloning (programming)
In vitro recombination
Cloning vector
Shuttle vector
Cloning (programming)
Expression cloning
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T ransformation- a ssociated r ecombination (TAR) can be exploited in yeast to clone human DNAs. TAR cloning was previously accomplished using one or two telomere-containing vectors with a common human repeat(s) that could recombine with human DNA during transformation to generate yeast artificial chromosomes (YACs). On basis of the proposal that broken DNA ends are more recombinogenic than internal sequences, we have investigated if TAR cloning could be applied to the generation of circular YACs by using a single centromere vector containing various human repeats at opposite ends. Transformation with these vectors along with human DNA led to the efficient isolation of circular YACs with a mean size of ≈150 kb. The circular YACs are stable and they can be easily separated from yeast chromosomes or moved into bacterial cells if the TAR vector contains an Escherichia coli F-factor cassette. More importantly, circular TAR cloning enabled the selective isolation of human DNAs from monochromosomal human–rodent hybrid cell lines. Although <2% of the DNA in the hybrid cells was human, as much as 80% of transformants had human DNA YACs when a TAR cloning vector contained Alu repeats. The level of enrichment of human DNA was nearly 3000-fold. A comparable level of enrichment was demonstrated with DNA isolated from a radiation hybrid cell line containing only 5 Mb of human DNA. A high selectivity of human DNA cloning was also observed for linear TAR cloning with two telomere vectors. No human–rodent chimeras were detected among YACs generated by TAR cloning. The results with a circular TAR cloning vector or two vectors differed from results with a single-telomere vector in that the latter often resulted in a series of terminal deletions in linear YACs. This could provide a means for physical mapping of cloned material.
Yeast artificial chromosome
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In vitro recombination
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We describe a simple method to directly clone any DNA fragment for which a flanking restriction enzyme map is known. Genomic DNA is digested with multiple enzymes cutting outside the fragment to be cloned, selected by electroelution from an agarose gel, and cloned directly into a plasmid vector. It is only necessary to screen 10-1000 colonies and recombinant DNA is ready for immediate molecular analysis without further subcloning. The use of this technique is demonstrated for the cloning of a sequence from within the human alpha-globin complex that was previously shown to be "unclonable" in bacteriophage and cosmid vectors and which is a multiallelic general genetic marker, as well as both beta-globin alleles from an individual with beta-thalassaemia.
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genomic DNA
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A yeast artificial chromosome (YAC) library in Saccharomyces cerevisiae consisting of 30,000 clones with an average insert size of 0.1 megabase pair of human DNA has been generated from primary fibroblast DNA. A YAC vector was modified to enable the recovery of both ends of a human DNA insert in plasmids in Escherichia coli and to confer G418 resistance to mammalian cells. A rapid method for yeast colony hybridization was used that exploits the ability of yeast spheroplasts to regenerate in a thin layer of calcium alginate. This method permits direct replica plating and processing of colonies from the primary transformation plate to nitrocellulose filters. Yeast colony hybridization conditions have been established to identify, within a YAC library of human genomic DNA, artificial chromosomes with homology to human DNA probes of unique single-copy sequence. An artificial chromosome with a 0.1-megabase-pair insert from the human Xq28 region has been identified by hybridization to a DNA probe that detects a unique sequence near the 3' end of the factor VIII gene.
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genomic DNA
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Yeast artificial chromosome
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genomic DNA
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The functional analysis of genes frequently requires manipulation of large genomic regions embedded In yeast artificial chromosomes (YACs). We have designed a yeast–bacteria shuttle vector, pClasper, that can be used to clone specific regions of interest from YACs by homologous recombination. The important feature of pClasper is the presence of the minl-F factor replicon. This leads to a significant increase in the size of the plasmid inserts that can be maintained in bacteria after cloning by homologous recombination in yeast. The utility of this vector lies in its ability to maintain large fragments in bacteria and yeast, allowing for mutagenesis in yeast and simplified preparation of plasmid DNA in bacteria. Using PCR-generated recombinogenic fragments in pClasper we cloned a 27 kb region from a YAC containing the Hoxc cluster and a 130 kb region containing the entire Hoxb cluster. No rearrangements were seen when the recombinants in the shuttle vector were transferred to bacteria. We outline the potential uses of pClasper for functional studies of large genomic regions by transgenic and other analyses.
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SummaryNovelty: A cloning strategy and vectors used to carry out the cloning are described. These may facilitate the isolation of clones containing sequences homologous to another sequence, by using homologous recombination in yeast.Biology: A library of DNA fragments is cloned into a YAC (yeast artificial chromosome) vector in a yeast strain. A DNA fragment or gene cloned into a bacterial vector, which will not replicate in yeast, is introduced in a linear state into the YAC library and any clone which contains a sequence homologous to the linearized DNA can undergo homologous recombination with the YAC clone. The system is set up so that recombinants containing DNA sequences to one side of the YAC clone can be selected on 5-fluoro-orotic acid plates lacking arginine. Clones with sequences on the other side of the YAC are selected on media containing α-amino adipate lacking tryptophan. Since YAC vectors can contain very large regions of DNA, whole genes may be isolated in a YAC vector by only having a small portion of the DNA or a cDNA clone. This may make chromosome walking a more simple process for very large complex human genes.
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Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes.For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged.Some regions of the human genome, however, appear to have a higher propensity than others to rearrange in any host system.Thus, techniques to detect and accurately characterize such rearrangements need to be developed.We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones.This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements.In our examples, highresolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more.Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules.When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA.In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.
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In vitro recombination
Human artificial chromosome
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