Human artificial chromosomes generated by modification of a yeast artificial chromosome containing both human alpha satellite and single-copy DNA sequences

Abstract

A human artificial chromosome (HAC) vector was constructed from a 1-Mb yeast artificial chromosome (YAC) that was selected based on its size from among several YACs identified by screening a randomly chosen subset of the Centre d'Étude du Polymorphisme Humain (CEPH) (Paris) YAC library with a degenerate alpha satellite probe. This YAC, which also included non-alpha satellite DNA, was modified to contain human telomeric DNA and a putative origin of replication from the human ␤-globin locus. The resultant HAC vector was introduced into human cells by lipidmediated DNA transfection, and HACs were identified that bound the active kinetochore protein CENP-E and were mitotically stable in the absence of selection for at least 100 generations. Microdissected HACs used as f luorescence in situ hybridization probes localized to the HAC itself and not to the arms of any endogenous human chromosomes, suggesting that the HAC 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.As the time rapidly approaches when the complete sequence of a human chromosome will be known, it is striking how little is known about how human chromosomes function. In contrast, the necessary elements for chromosomal function in yeast have been defined for several years. Three important elements appear to be required for the mitotic stability of linear chromosomes: centromeres, telomeres, and origins of replication. The ascertainment of these elements in Saccharomyces cerevisiae provided the basis for the construction of yeast artificial chromosomes (YACs), which have proven to be important tools both for the study of yeast chromosomal function and as large capacity cloning vectors (1-3).

Visible transient expression of EGFP requires intranuclear injection of large copy numbers

“…Human artificial chromosomes (HACs) are presently constructed to understand how chromosomes work and which sequence elements are required for stability and function. [1][2][3][4][5][6] Ultimately, HACs could become a safe and efficient gene transfer tool circumventing DNA-induced cell cycle arrest, replicative loss, integrative repair, and silencing. 7,8 In a series of HAC transfection experiments we recently established that the inclusion of large genomic DNA (100 kb) capped by telomeric repeats improves the de novo formation of HACs based on cloned centromere DNA, making HAC formation more efficient than the conventional procedure to obtain stable clones via integration into host chromosomes (Schindelhauer et al, in preparation).…”

The molecular characterization of maize B chromosome specific AFLPs

“…There are more ARS than seem necessary in the genome replication [26]. Human artificial chromosome (HAC) was constructed successfully as the three functional elements were ar-ranged properly [27], [28]. Likewise, B chromosome could also be constructed by the latent or cryptic functional elements.…”

Chromosome engineering: prospects for gene therapy

“…Following this study, several reports showed that pre-assembled YAC-or PAC-based vectors containing alpha satellite DNA could also form HACs (Figure 1). [6][7][8][9][10] In each case, HACs have been shown to be low in copy number, mitotically stable and to have recruited functionally important centromere proteins. Moreover, they appear to consist of multiple copies of the transfected DNA, assembled by the cell to yield chromosomes in the 1-10 Mb size range.…”