Rapid creation of BAC-based human artificial chromosome vectors by transposition with synthetic alpha-satellite arrays.
Efficient construction of BAC-based human artificial chromosomes (HACs) requires optimization of each key functional unit as well as development of techniques for the rapid and reliable manipulation of high-molecular weight BAC vectors. Here, we have created synthetic chromosome 17-derived alpha-satellite arrays, based on the 16-monomer repeat length typical of natural D17Z1 arrays, in which the consensus CENP-B box elements are either completely absent (0/16 monomers) or increased in density (16/16 monomers) compared to D17Z1 alpha-satellite (5/16 monomers). Using these vectors, we show that the presence of CENP-B box elements is a requirement for efficient de novo centromere formation and that increasing the density of CENP-B box elements may enhance the efficiency of de novo centromere formation. Furthermore, we have developed a novel, high-throughput methodology that permits the rapid conversion of any genomic BAC target into a HAC vector by transposon-mediated modification with synthetic alpha-satellite arrays and other key functional units. Taken together, these approaches offer the potential to significantly advance the utility of BAC-based HACs for functional annotation of the genome and for applications in gene transfer.
Basu
Joydeep
J
Institute for Genome Sciences and Policy, Duke University CIEMAS Room 2379, 101 Science Drive, Durham, NC 27708, USA. joydeep.basu@duke.edu
Stromberg
Gregory
G
Compitello
George
G
Willard
Huntington F
HF
Van Bokkelen
Gil
G
D001324:Autoantigens / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002503:Centromere / Q000235:genetics*; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D022202:Chromosomes, Artificial, Bacterial; D022222:Chromosomes, Artificial, Human; D004251:DNA Transposable Elements; D004276:DNA, Satellite / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D023281:Genomics / Q000379:methods; D006801:Humans
D001324:Autoantigens; C497926:CENPB protein, human; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone; D004251:DNA Transposable Elements; D004276:DNA, Satellite; D004268:DNA-Binding Proteins