Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes

Abstract

Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome DeltaYq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from DeltaYq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.

Bacterial transfer of large functional genomic DNA into human cells

“…5 Specific alpha satellite DNA arrays from various human chromosomes, such as chromosomes 14,17,21,22, X (and Y inefficiently), have been transferred as purified DNA in order to form centromeres on de novo HACS. [5][6][7][8][9][10][11][12][13] The centromeres formed on the transferred DNA acquire chromatin proteins specific for functional centromeres such as CENP-A a histone H3 variant, only found within active centromere chromatin. 13 Moreover, complete genes can be incorporated into HACs and expressed, as shown for human HPRT (42 kb), 14,15 or GCH1 genes (61 kb).…”

Human Artificial Chromosomes with Alpha Satellite-Based De Novo Centromeres Show Increased Frequency of Nondisjunction and Anaphase Lag

“…Studies from a number of groups have shown that alpha satellite DNA from several human chromosomes is capable of forming de novo centromeres in artificial chromosome assays (11,14,16,18,22,24,34,40,45). Some types of alpha satellite assemble artificial chromosomes that are maintained stably in culture over many cell divisions and contain centromeres that colocalize with kinetochore proteins associated specifically with active centromeres.…”

Segments missing from the draft human genome sequence can be isolated by transformation‐associated recombination cloning in yeast

“…Southern blot hybridization was performed as described by Kouprina et al (1998) with 32 P-labelled probes. Fluorescence in situ hybridization was performed as described elsewhere (Kouprina et al, 2003). Sequencing.…”