Rearranging the centromere of the human Y chromosome with phiC31 integrase.
We have investigated the ability of the integrase from the Streptomyces phiC31 'phage to either delete or invert 1 Mb of DNA around the centromere of the human Y chromosome in chicken DT40 hybrid somatic cells. Reciprocal and conservative site-specific recombination was observed in 54% of cells expressing the integrase. The sites failed to recombine in the remaining cells because the sites had been damaged. The sequences of the damaged sites indicated that the damage arose as a result of repair of recombination intermediates by host cell pathways. The liability of recombination intermediates to damage is consistent with what is known about the mechanism of serine recombinase reactions. The structures of the products of the chromosome rearrangements were consistent with the published sequence of the Y chromosome indicating that the assembly of the highly repeated region between the sites is accurate to a resolution of about 50 kb. Mini-chromosomes lacking a centromere were not recovered which also suggested that neo-centromere formation occurs infrequently in vertebrate somatic cells. No ectopic recombination was observed between a phiC31 integrase attB site and the chicken genome.
Malla
Sunir
S
Institute of Genetics, Queen's Medical Centre, Nottingham NG7 2UH, UK.
Dafhnis-Calas
Felix
F
Brookfield
John F Y
JF
Smith
Margaret C M
MC
Brown
William R A
WR
D000818:Animals; D001435:Bacteriophages / Q000201:enzymology; D002503:Centromere; D002645:Chickens / Q000235:genetics; D041322:Chromosomes, Human, Y; D005818:Genetic Engineering; D006801:Humans; D006822:Hybrid Cells; D019426:Integrases / Q000378:metabolism*; D009154:Mutation; D011995:Recombination, Genetic; D013302:Streptomyces / Q000821:virology
D019426:Integrases