FORMATION OF DE-NOVO CENTROMERES AND CONSTRUCTION OF FIRST-GENERATIONHUMAN ARTIFICIAL MICROCHROMOSOMES

We have combined long synthetic arrays of alpha satellite DNA with tel omeric DNA and genomic DNA to generate artificial chromosomes in human HT1080 cells. The resulting linear microchromosomes contain exogenous alpha satellite DNA, are mitotically and cytogenetically stable in th e absence of selection for up to six months in culture, bind centromer e proteins specific for active centromeres, and are estimated to be 6- 10 megabases in size, approximately one-fifth to one-tenth the size of endogenous human chromosomes. We conclude that this strategy results in the formation of de novo centromere activity and that the microchro mosomes so generated contain all of the sequence elements required for stable mitotic chromosome segregation and maintenance. This first-gen eration system for the construction of human artificial chromosomes sh ould be suitable for dissecting the sequence requirements of human cen tromeres, as well as developing constructs useful for therapeutic appl ications.