Large-insert BAC/YAC libraries for selective re-isolation of genomic regions by homologous recombination in yeast
| Autor: | Pieter J. de Jong, Changjiang Zeng, George A. M. Cross, Natalay Kouprina, Vladimir Larionov, Baoli Zhu, Kazutoyo Osoegawa, Al Cairo, Maarten Hoek |
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| Rok vydání: | 2001 |
| Předmět: |
Yeast artificial chromosome
Chromosomes Artificial Bacterial Genetic Vectors Molecular Sequence Data Trypanosoma brucei brucei Cloning vector Saccharomyces cerevisiae Biology Insert (molecular biology) Genetics Escherichia coli Animals Genomic library Cloning Molecular Chromosomes Artificial Yeast Gene Library Recombination Genetic Bacterial artificial chromosome Hybrid vector Sequence Analysis DNA DNA Protozoan DNA Fingerprinting genomic DNA Homologous recombination Genome Protozoan |
| Zdroj: | Genomics. 77(1-2) |
| ISSN: | 0888-7543 |
| Popis: | We constructed representative large-insert bacterial artificial chromosome (BAC) libraries of two human pathogens (Trypanosoma brucei and Giardia lamblia) using a new hybrid vector, pTARBAC1, containing a yeast artificial chromosome (YAC) cassette (a yeast selectable marker and a centromere). The cassette allows transferring of BACs into yeast for their further modification. Furthermore, the new hybrid vector provides the opportunity to re-isolate each DNA insert without construction of a new library of random clones. Digestion of a BAC DNA by an endonuclease that has no recognition site in the vector, but which deletes most of the internal insert sequence and leaves the unique flanking sequences, converts a BAC into a TAR vector, thus allowing direct gene isolation. Cotransformation of a TAR vector and genomic DNA into yeast spheroplasts, and subsequent recombination between the TAR vector's flanking ends and a specific genomic fragment, allows rescue of the fragment as a circular YAC/BAC molecule. Here we prove a new cloning strategy by re-isolation of randomly chosen genomic fragments of different size from T. brucei cloned in BACs. We conclude that genomic regions of unicellular eukaryotes can be easily re-isolated using this technique, which provides an opportunity to study evolution of these genomes and the role of genome instability in pathogenicity. |
| Databáze: | OpenAIRE |
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