Construction of human chromosome 16- and 5-specific circular YAC/BAC libraries by in vivo recombination in yeast (TAR cloning).
Transformation-associated recombination (TAR) in yeast was exploited for the selective isolation of human DNAs as large circular yeast artificial chromosomes (YACs) from two rodent/human hybrid cell lines containing human chromosomes 5 and 16. TAR cloning vectors containing the F-factor origin of replication were constructed for use in these experiments. Presence of the F-factor origin in TAR vectors provides the capability of transferring the YACs generated by in vivo recombination in yeast into Escherichia coli cells and propagating them as bacterial artificial chromosomes (BACs). A high enrichment of human versus rodent YACs was observed during isolation of human DNA from the rodent/human hybrid cell lines. Although <3% of the DNA content in the hybrid cells was human, as many as 75% of the transformants contained human YACs. In contrast to the standard YAC cloning method based on in vitro ligation, no human/mouse chimeras were observed during TAR cloning. The constructed human chromosome 16 YAC library had approximately 2.6x coverage, represented by 4320 YAC clones with an average insert size of 80 kb. YAC clones generated from chromosome 16 were successfully converted into BACs by electroporation of DNA isolated from yeast transformants into E. coli. The BAC clones represent approximately 0.6x chromosomal coverage. Pilot YAC and BAC libraries of chromosome 5 have been also constructed. The chromosomal distribution of YAC/BACs from chromosome 5 and chromosome 16 was evaluated by fluorescence in situ hybridization (FISH). The distribution of FISH signals appeared random along the length of each chromosome. We conclude that TAR cloning provides an efficient means for generating representative chromosome-specific YAC/BAC libraries.
Kouprina
N
N
Laboratory of Molecular Genetics, National Institute of Environmental Health Services, Research Triangle Park, North Carolina, 27709, USA. kouprina@niehs.nih.gov
Campbell
M
M
Graves
J
J
Campbell
E
E
Meincke
L
L
Tesmer
J
J
Grady
D L
DL
Doggett
N A
NA
Moyzis
R K
RK
Deaven
L L
LL
Larionov
V
V
D000818:Animals; D002874:Chromosome Mapping; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D002885:Chromosomes, Human, Pair 16 / Q000235:genetics*; D002895:Chromosomes, Human, Pair 5 / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004270:DNA, Circular / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence; D011995:Recombination, Genetic / Q000235:genetics*; D012377:Rodentia; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004270:DNA, Circular