Chromosomes formed de novo which originated from the centromeric region of mouse chromosome 7, have been analysed. These new chromosomes were formed by apparently similar large-scale amplification processes, and are organized into amplicons of approximately 30 Mb. Centromeric satellite DNA was found to be the constant component of all amplicons. Satellite DNA sequences either bordered the large euchromatic amplicons (E-type amplification), or made up the bulk of the constitutive heterochromatic amplicons (H-type amplification). Detailed analysis of a heterochromatic megachromosome formed de novo by an H-type amplification revealed that it is composed of a tandem array of 10-12 large (approximately 30 Mb) amplicons each marked with integrated "foreign' DNA sequences at both ends. Each amplicon is a giant palindrome, consisting of two inverted doublets of approximately 7.5-Mb blocks of satellite DNA. Our results indicate that the building units of the pericentric heterochromatin of mouse chromosomes are approximately 7.5-Mb blocks of satellite DNA flanked by non-satellite sequences. We suggest that the formation de novo of various chromosome segments and chromosomes seen in different cell lines may be the result of large-scale E- and H-type amplification initiated in the pericentric region of chromosomes.

A 13,863-base-pair (bp) putative centromeric DNA fragment has been isolated from a human genomic library by using a probe obtained from metaphase chromosomes of human colon carcinoma cells. The abundance of this DNA was estimated to be 16-32 copies per genome. Cotransfection of mouse cells with this sequence and a selectable marker gene (aminoglycoside 3'-phosphotransferase type II, APH-II) resulted in a transformed cell line carrying an additional centromere in a dicentric chromosome. This centromere was capable of binding an anti-centromere antibody. In situ hybridization demonstrated that the human DNA sequence as well as the APH-II gene and vector DNA sequences were located only in the additional centromere of the dicentric chromosome. The extra centromere separated from the dicentric chromosome, forming a stable minichromosome. This functional centromere linked to a dominant selectable marker may be a step toward the construction of an artificial mammalian chromosome.The centromere is a specialized region of the eukaryotic chromosome that is the site of kinetochore formation, a structure that allows the precise segregation of chromosomes during cell division and may play a role in the higher-order organization of eukaryotic chromosomes (1). (6). Isolated metaphase chromosomes were resuspended in 1 ml of buffer (150 mM NaCl/50 mM Tris-HCl, pH 7.5/10 mM MgCl2/5 mM 2-mercaptoethanol) at a DNA concentration of 1 mg/ml and digested with 500 units of EcoRI restriction endonuclease for 1 h. The suspension was diluted with 4 ml of IPP buffer (500 mM NaCI/10 mM Tris-HCl, pH 8.0/0.1% Nonidet P40

We have analysed the replication of the heterochromatic megachromosome that was formed de novo by a large-scale amplification process initiated in the centromeric region of mouse chromosome 7. The megachromosome is organized into amplicons approximately 30 Mb in size, and each amplicon consists of two large inverted repeats delimited by a primary replication initiation site. Our results suggest that these segments represent a higher order replication unit (megareplicon) of the centromeric region of mouse chromosomes. Analysis of the replication of the megareplicons indicates that the pericentric heterochromatin and the centromere of mouse chromosomes begin to replicate early, and that their replication continues through approximately three-quarters of the S-phase. We suggest that a replication-directed mechanism may account for the initiation of large-scale amplification in the centromeric regions of mouse chromosomes, and may also explain the formation of new, stable chromosome segments and chromosomes.