Human artificial chromosomes generated by modification of a yeast artificial chromosome containing both human alpha satellite and single-copy DNA sequences
Ka. Henning et al., Human artificial chromosomes generated by modification of a yeast artificial chromosome containing both human alpha satellite and single-copy DNA sequences, P NAS US, 96(2), 1999, pp. 592-597
Citations number
52
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
A human artificial chromosome (HAC) vector was constructed from a 1-Mb yeas
t artificial chromosome (YAC) that was selected based on its size from amon
g several YACs identified by screening a randomly chosen subset of the Cent
re d'Etude du Polymorphisme Humain (CEPH) (Paris) YAC library with a degene
rate alpha satellite probe. This YAC, which also included non-alpha satelli
te DNA, was modified to contain human telomeric DNA and a putative origin o
f replication from the human beta-globin locus, The resultant HAC vector wa
s introduced into human cells by lipid-mediated DNA transfection, and HACs
were identified that bound the active kinetochore protein CENP-E and were m
itotically stable in the absence of selection for at least 100 generations.
Microdissected HACs used as fluorescence in situ hybridization probes loca
lized to the HAC itself and not to the arms of any endogenous human chromos
omes, suggesting that the NAC was not formed by telomere fragmentation. Our
ability to manipulate the HAC vector by recombinant genetic methods should
allow us to further define the elements necessary for mammalian chromosome
function.