Large-insert BAC/YAC libraries for selective re-isolation of genomic regions by homologous recombination in yeast.
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.
Zeng
C
C
Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Kouprina
N
N
Zhu
B
B
Cairo
A
A
Hoek
M
M
Cross
G
G
Osoegawa
K
K
Larionov
V
V
de Jong
P
P
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D016172:DNA Fingerprinting; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D018503:Genome, Protozoan; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014346:Trypanosoma brucei brucei / Q000235:genetics
D016054:DNA, Protozoan