title: Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation.
abstract: Human artificial chromosomes (HACs) provide a unique opportunity to study kinetochore formation and to develop a new generation of vectors with potential in gene therapy. An investigation into the structural and the functional relationship in centromeric tandem repeats in HACs requires the ability to manipulate repeat substructure efficiently. We describe here a new method to rapidly amplify human alphoid tandem repeats of a few hundred base pairs into long DNA arrays up to 120 kb. The method includes rolling-circle amplification (RCA) of repeats in vitro and assembly of the RCA products by in vivo recombination in yeast. The synthetic arrays are competent in HAC formation when transformed into human cells. As short multimers can be easily modified before amplification, this new technique can identify repeat monomer regions critical for kinetochore seeding. The method may have more general application in elucidating the role of other tandem repeats in chromosome organization and dynamics.
authors.affiliation: Laboratory of Biosystems and Cancer, National Cancer Institute, Bethesda, MD, USA.
authors.forename: Tom
authors.initials: T
authors.lastname: Ebersole
authors.forename: Yasuhide
authors.initials: Y
authors.lastname: Okamoto
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Noskov
authors.forename: Natalay
authors.initials: N
authors.lastname: Kouprina
authors.forename: Jung-Hyun
authors.initials: JH
authors.lastname: Kim
authors.forename: Sun-Hee
authors.initials: SH
authors.lastname: Leem
authors.forename: J Carl
authors.initials: JC
authors.lastname: Barrett
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Masumoto
authors.forename: Vladimir
authors.initials: V
authors.lastname: Larionov
mesh_terms: D045744:Cell Line, Tumor; D002503:Centromere / Q000737:chemistry*; D022222:Chromosomes, Artificial, Human; D004247:DNA / Q000096:biosynthesis; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D011995:Recombination, Genetic; D020080:Tandem Repeat Sequences; D013997:Time Factors; D015003:Yeasts / Q000235:genetics
chemical_list: D004247:DNA