Molecular and cytological analysis of a 5.5 Mb minichromosome

Mammalian artificial chromosomes (MACs) provide a new tool for the improvem ent of our knowledge of chromosome structure and function. Moreover, they c onstitute an alternative and potentially powerful tool for gene delivery bo th in cultured cells and for the production of transgenic animals. In the p resent work we describe the molecular structure of MC1, a human minichromos ome derived from chromosome 1. By means of restriction and hybridization an alysis, satellite-PCR, in situ hybridization on highly extended chromatin f ibres, and indirect immunofluorescence, we have established that: (i) MC1 h as a size of 5.5 Mb; (ii) it consists of 1.1 Mb alphoid, 3.5 Mb Sat2 DNA, a nd telomeric and subtelomeric sequences at both ends; (iii) it contains an unusual region of interspersed Sat2 and alphoid DNAs at the junction of the alphoid and the Sat2 blocks; and (iv) the two alphoid blocks and the Sat2- alphoid region bind centromeric proteins suggesting that they participate i n the formation of a functional kinetochore.