Cloning of functional human centromere DNA by YAC technology

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The purpose of this work is to clone functional human centromeric DNA using the technology of the yeast artificial chromosome (YAC).Centromere is the chromosomal domain essential to the segregation of eukaryotic chromosomes. Although DNA is believed to be the primary determinant of the functional centromere structure in mammalian cells, no conclusive information on the essential DNA structure is available until now. To solve this problem, we previously analyzed long range organization of the centromeric satellite DNA of the human chromosome 21, in specific emphasis on the distribution of alphoid DNA containing the recognition sequence of CENP-B,the CENP-B box. We have found two megabase-sized domains of alphoid DNA (alpha21-I and alpha21-II) in the chromosome 21 : alpha21-I,contained many CENP-B boxes at regular intervals and alpha21-II containing very few, if any, CENP-B boxes. In this international collaboration with Dr.P.Hieter, we performed the cloning into YAC of 100 kb size DNA from the alpha21-I and alpha21-II region of the chromosome 21. We found that these alphoid YAC clones were stably maintained in the recombination minus yeast strains. We then introduced the human telomere sequences into the two vector arms of the alphoid YAC clones so that these YAC clones could be maintained in the mammalian cells as mammalian artificial chromosomes (MAC), if they contained functional centromere DNA.We introduced resulting clones into a human cell line by microinjection and lipofection. We have obtained a few transformants with both alpha21-I YAC and alpha21-II YAC.We are currently analyzing intranuclear state of incorporated alphoid YACs.